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Research Article
An Engineered Constitutive Promoter Set with Broad Activity Range for Cupriavidus necator H16Click to copy article linkArticle link copied!
- Abayomi Oluwanbe JohnsonAbayomi Oluwanbe JohnsonDepartment of Chemical & Biological Engineering and Advanced Biomanufacturing Centre, University of Sheffield, Sir Robert Hadfield Building, Mappin Street, Sheffield S1 3JD, United KingdomMore by Abayomi Oluwanbe Johnson
- Miriam Gonzalez-VillanuevaMiriam Gonzalez-VillanuevaDepartment of Chemical & Biological Engineering and Advanced Biomanufacturing Centre, University of Sheffield, Sir Robert Hadfield Building, Mappin Street, Sheffield S1 3JD, United KingdomMore by Miriam Gonzalez-Villanueva
- Kang Lan Tee*Kang Lan Tee*K.L.T.: Tel, +44 (0)114 222 7591; Fax, +44 (0)114 222 7501; E-mail, [email protected]Department of Chemical & Biological Engineering and Advanced Biomanufacturing Centre, University of Sheffield, Sir Robert Hadfield Building, Mappin Street, Sheffield S1 3JD, United KingdomMore by Kang Lan Tee
- Tuck Seng Wong*Tuck Seng Wong*T.S.W.: Tel, +44 (0)114 222 7591; Fax, +44 (0)114 222 7501; E-mail, [email protected]Department of Chemical & Biological Engineering and Advanced Biomanufacturing Centre, University of Sheffield, Sir Robert Hadfield Building, Mappin Street, Sheffield S1 3JD, United KingdomMore by Tuck Seng Wong
Abstract
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Well-characterized promoters with variable strength form the foundation of heterologous pathway optimization. It is also a key element that bolsters the success of microbial engineering and facilitates the development of biological tools like biosensors. In comparison to microbial hosts such as Escherichia coli and Saccharomyces cerevisiae, the promoter repertoire of Cupriavidus necator H16 is highly limited. This limited number of characterized promoters poses a significant challenge during the engineering of C. necator H16 for biomanufacturing and biotechnological applications. In this article, we first examined the architecture and genetic elements of the four most widely used constitutive promoters of C. necator H16 (i.e., PphaC1, PrrsC, Pj5, and Pg25) and established a narrow 6-fold difference in their promoter activities. Next, using these four promoters as starting points and applying a range of genetic modifications (including point mutation, length alteration, incorporation of regulatory genetic element, promoter hybridization, and configuration alteration), we created a library of 42 constitutive promoters, all of which are functional in C. necator H16. Although these promoters are also functional in E. coli, they show different promoter strength and hierarchical rank of promoter activity. Subsequently, the activity of each promoter was individually characterized, using l-arabinose-inducible PBAD promoter as a benchmark. This study has extended the range of constitutive promoter activities to 137-fold, with some promoter variants exceeding the l-arabinose-inducible range of PBAD promoter. Not only has the work enhanced our flexibility in engineering C. necator H16, it presented novel strategies in adjusting promoter activity in C. necator H16 and highlighted similarities and differences in transcriptional activity between this organism and E. coli.
Copyright © 2018 American Chemical Society
Cupriavidus necator H16 (or Ralstonia eutropha H16) is a chemolithoautotrophic soil bacterium, most widely known for its ability to accumulate polyhydroxyalkanoates (PHA). (1) This metabolically versatile organism is capable of utilizing a wide range of energy and carbon sources (including H2 and CO2) to support growth and achieving high cell density. (2) These intrinsic properties have cemented its potential applications in biological CO2 capture and utilization (3,4) as well as commercial-scale production of diverse bioproducts (5) including polymers, (6−9) hydrocarbons, (10−14) and amino acids. (15) Its potential will rapidly come into fruition, aided by the development of molecular tools. These include genome engineering methods to permanently alter its metabolic phenotype, (16) expression vectors to assemble heterologous pathways, (8,17) transposon-based random mutagenesis, (18) and transformation method to introduce recombinant plasmids. (19)
Maximal product yield and titer are key requirements in biomanufacturing. To this end, metabolic pathway optimization is vital in eliminating metabolic bottlenecks that compromise cellular productivity and metabolic phenotypes that are detrimental to cell viability. Proven strategies of tuning gene expression of a metabolic pathway include varying plasmid copy number, gene dosage, and promoter strength, among others. Leveraging on promoter strength for pathway optimization is the most straightforward strategy, which involves tuning promoter activity at both transcriptional and translational levels.
l-Arabinose-inducible PBAD promoter and anhydrotetracycline-inducible Ptet promoter are most widely applied to tune expression of genes, gene clusters, or operons in C. necator H16. (20−23) With a PBAD promoter, high inducer concentration (up to 1 g/L) is required to achieve high expression yield. The leaky Ptet promoter, on the other hand, is induced by a weak antibiotic that is undesirable and its promoter strength is comparatively weaker. These factors greatly limit the use of these two promoters for large-scale fermentation. The more recently developed 3-hydroxypropionic acid-inducible systems (24) and the p-cumate- and IPTG-inducible Pj5 promoters (25) are promising alternatives. Nonetheless, achieving scalable and tunable gene expression of a multigene pathway by solely relying on inducible expression systems is severely limited by the poor modularity of inducer-based systems (or potential risk of unwanted inducer crosstalk) and a limited number of C. necator H16-compatible inducible promoters. Further, the use of inducers such as l-arabinose or anhydrotetracycline on a large scale is commercially uneconomical.
In this regard, engineering constitutive promoters with a broad range of activities is a more facile means to modularly adjust gene expressions of a multigene pathway to the desired levels or ratios. In addition to facilitating static metabolic control, constitutive promoters are used to engineer more efficient inducible promoters (23) and to construct metabolite-sensing genetic circuits that in turn facilitate dynamic metabolic control in microorganisms. (26) Examples of constitutive promoters for use in C. necator H16 include Plac and Ptac promoters, native C. necator H16 promoters such as PphaC1 promoter, and coliphage T5 promoter and its variants such as Pj5, Pg25, Pn25, and Pn26 promoters. (17,21,27) Despite these precedent studies, there are knowledge gaps that hinder constitutive promoter utilization and engineering for C. necator H16, which are: (1) lack of a universal definition of promoter architecture, (2) lack of a universal reference scale for hierarchical ranking of constitutive promoter activities, and (3) limited examples of rational promoter engineering. The latter is in stark contrast to promoter engineering reported for E. coli and yeast.
In this study, we first examined the architecture of four notable C. necator H16-compatible constitutive promoters: the native PphaC1 promoter, a semisynthetic PrrsC promoter, and two coliphage T5 promoters, Pj5 and Pg25. We then evaluated their activities using in vivo fluorescence measurement of red fluorescent protein (RFP) expression to establish an understanding of the relationship between promoter architecture and activity. Guided by these structure–function relationships, we next proceeded to rational engineering of these four parental promoters. Our engineering strategies include combinations of point mutation, length alteration, incorporation of regulatory genetic element, promoter hybridization, and configuration alteration. This resulted in a collection of 42 promoters displaying a range of promoter activities. Of these, there are composite promoter variants that are stronger than the Pj5 promoter; the latter has previously been acclaimed to be the strongest known constitutive promoter for gene expression in C. necator H16. (17) This new promoter library is envisaged to further propel the biotechnological applications of C. necator H16.
Results and Discussion
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Defining a Promoter and Quantifying Its Activity
Standardizing the definition of a promoter is deemed necessary and particularly relevant to this study for four obvious reasons: (1) to objectively benchmark the activities of wildtype and engineered promoters, (2) to critically assess promoter structure–function relationships, (3) to measure the effectiveness of various promoter engineering strategies, and (4) to compare promoters reported by various research groups. In this study, we describe a promoter as a constellation of three distinct genetic elements as shown in Figure 1A: (part 1) a core promoter sequence comprising −35 box, −10 box (or the Pribnow box), +1 transcriptional start, spacer of 16–18 bp between the −35, and the −10 boxes as well as the spacer between the −10 box and +1 site, (part 2) an upstream element (UP) that refers to the entire DNA sequence upstream of the core promoter sequence, and (part 3) a downstream element spanning the nucleotide after the +1 transcriptional start and the nucleotide before the translation initiation codon. Therefore, the 5′-untranslated region (5′-UTR) consists of the +1 transcriptional start and the downstream element, with the latter typically containing cis-acting regulatory elements such as the ribosome binding site (RBS). Putting it simply, a promoter is a defined stretch of sequence upstream of the translational start. This definition is not uncommon in genome annotation, particularly when the promoter boundary is unclear or ambiguous. This definition also presupposes that the functional characteristics of a given promoter are composite effect of all genetic elements within the predefined promoter architecture. To quantify promoter activity in C. necator H16, RFP was used as a reporter protein (Figure 1B). Briefly, DNA fragments corresponding to a promoter and rfp gene were cloned, in tandem, into a broad host range pBBR1MCS plasmid backbone harboring a chloramphenicol resistance gene (Figure 1B and Supporting Information, Figure S1).
Figure 1
Figure 1. (A) Promoter definition used in this study. (B) High-throughput characterization of engineered promoters using a fluorescence-based assay (5′-UTR, 5′-untranslated region; bp, base pair; CamR, chloramphenicol resistance gene; Rep, replication gene; RFP, red fluorescent protein).
Defining the Boundaries and Architectures of Four Parental Promoters
Because the four parental promoters, PphaC1, PrrsC, Pj5, and Pg25, form the basis of this entire study, clearly defining their boundaries and examining their architectures are essential such that all subsequently engineered promoters can be compared against these four “standards”. All four parental promoters contain −35 box and −10 box that are almost identical to the hexameric promoter consensus sequences recognized by the E. coli housekeeping sigma factor σ70 (Figure 2A,B). (28) PphaC1 is known to be a relatively strong native promoter. (21,23,27) On the basis of the data reported by Fukui et al., the strength of PphaC1 is ∼58% that of Ptac. (21) It has been widely studied and applied for improved PHA-based biopolymer production in C. necator H16. (21,27) However, PphaC1 promoters of different lengths are used in various studies, making objective comparison impossible. In this study, PphaC1 promoter is defined as a 466-bp DNA sequence upstream of the translation start of the phaC1 gene. Previous studies of this promoter affirmed the presence of a 7-bp “AGAGAGA” Shine–Dalgarno (SD) sequence within its 5′-UTR. This native RBS is located 11 bp upstream of the translational start. (27) The PrrsC promoter used in this study is a combination of a 210-bp DNA sequence upstream of the +1 transcriptional start of the native rrsC gene, the first 5-bp of the native 5′-UTR and a 26-bp synthetic genetic element. The latter comprises a 20-bp RBS found in the pBBR1c-RFP PBAD promoter (see Supporting Information) flanked by an upstream 6-bp BglII restriction site. The synthetic RBS contains a purine-rich 5-bp “AGGAG” SD sequence known to markedly improve translation efficiency. This PrrsC is almost identical to the one used in Li and Liao, (23) with only minor difference in the downstream element that contains an RBS. Finally, the Pg25 and Pj5 promoters used in this study are both 75-bp DNA sequences, identical to those previously reported by Gentz and Bujard. (29)
Figure 2
Figure 2. (A) Boundaries and architectures of the four parental promoters, PphaC1, PrrsC, Pj5, and Pg25, used in this study. (B) Comparison of the four parental promoters to an Escherichia coli σ70 promoter. (C) Activities of the four parental promoters.
Narrow Range of Promoter Activities between the Four Parental Promoters
Fluorescence measurement of RFP expression revealed a narrow range of promoter activities (defined as relative fluorescence unit normalized by optical density; see Methods and Materials) between the four parental promoters (Figure 2C). The ratio of promoter activities between the strongest (Pg25) and the weakest (PphaC1) is only 6.3. The promoter activity of PrrsC is 1.7-fold higher than that of PphaC1. This difference may be attributed to the synthetic RBS in PrrsC being more effective in promoting translation compared to the native RBS in PphaC1. The coliphage T5 promoters, Pj5 (75 bp) and Pg25 (75 bp), are much shorter in length compared to PphaC1 (466 bp) and PrrsC (241 bp). They have previously been reported as some of the strongest constitutive promoters in E. coli. (29) The strong transcriptional activity was also verified in C. necator H16. (17) The A/T rich sequence of many coliphage T5 promoters, particularly in their upstream elements, has been implicated in accounting for their high transcriptional activity. (29) Indeed, the upstream elements of both Pj5 and Pg25 promoters show high A/T contents (65% for Pj5 and 85% for Pg25), and the difference in the A/T content may partly be responsible for the higher activity of Pg25 relative to Pj5 (Figure 2B). In addition, Pg25 possesses within its 5′-UTR the purine-rich 5-bp SD sequence used in most commercially available pET and pBAD vectors, which is lacking in Pj5 (Figure 2A). Important to point out, Gruber et al. had an opposite observation that the strength of Pj5 was stronger than that of Pg25. (17) The promoter sequences used by Gruber et al. were different compared to those used in this study, although the core promoter sequences remain identical (Supporting Information, Figure S2). Promoters in Gruber et al. were essentially a core promoter sequence flanked by NotI and EcoRI restriction sites. Further, they both contain an RBS of T7 gene 10, downstream to the core promoter sequence. These differences could explain the discrepancy between the two studies. It further emphasizes the necessity to define a promoter for objective comparison of promoters used, created or engineered by various researchers.
Our initial examination of the four parental promoters highlighted two critical points which guided our subsequent promoter engineering: (1) conservation of −35 and −10 boxes is important for maintaining high transcriptional activity in C. necator H16, and (2) promoter length or A/T content or cis-acting genetic regulatory element (such as a synthetic RBS) could potentially influence transcriptional activity in C. necator H16 significantly.
Expanding the Range of Promoter Activities through Rational Engineering
Informed by our understanding of the four “standard” promoters, we proceed to increase the range of promoter activities beyond the existing 6.3-folds between PphaC1, PrrsC, Pj5, and Pg25. Our objectives are threefold: (1) creating both weaker (“tuning down”) and stronger (“tuning up”) promoter variants to further expand the promoter activity range, (2) generating promoter variants that exceed or at least cover the entire l-arabinose inducible range of PBAD promoter, and (3) developing a set of promoters with gradual increase in activity (i.e., having promoters with activities evenly distributed across the entire promoter activity scale).
To this end, we applied a range of rational engineering approaches. These strategies, summarized in Supporting Information, Table S1 and Figure S3, can be loosely classified into five categories: (A) point mutation, (B) length alteration, (C) incorporation of regulatory genetic element, (D) promoter hybridization, and (E) configuration alteration. Category A, point mutation, includes base substitution, single-base insertion, and single-base deletion. Category B, length alteration, refers to truncation or extension of a promoter from either terminus and insertion or deletion of a stretch of random DNA sequence. Incorporating cis-acting translational regulatory elements such as T7 stem-loop and RBS are grouped within category C. Category D, promoter hybridization, encompasses both creating hybrid promoters and incorporating cis-acting transcriptional regulatory element such as an operator. Category E, configuration alteration, involves transcriptional amplification using a secondary promoter that is placed in divergent configuration to a primary promoter. In other words, composite promoters are placed in category E. Each category is further divided into subcategories (e.g., C1 for T7 stem-loop and C2 for RBS in category C) and subsub-categories (e.g., B1a for truncation of 25 bp upstream of −35 box and B1b for truncation of 50 bp upstream of −35 box in category B) to pinpoint specific modification made. Using a combination of the aforementioned strategies, we created in total 38 promoter variants as summarized in Table 1.
Table 1. Summary of 42 Parental Promoters and Their Variants Engineered Using a Combination of Promoter Engineering Strategies (A = Point Mutation, B = Length Alteration, C = Incorporation of Regulatory Genetic Element, D = Promoter Hybridization, and E = Configuration Alteration)a
a
Numbers in bracket represent promoter digital identifier, in the format of [activity level – relative activity to PphaC1[A1] – promoter length].
Promoter Nomenclature
On the basis of the classification of our promoter engineering strategies, we devised a promoter nomenclature system to systematically name all 38 engineered promoters (Table 1). The system is designed to provide three pieces of key information: parental promoter, modifications made, and promoter architecture, using the standard format of Pparent[M1M2M3···]. In this nomenclature system, capital P signifies a promoter. Italic subscript “parent” indicates the parental promoter from which the engineered promoter is derived. All modifications made are summarized in bracketed subscript “[M1M2M3···],” with the modifications arranged in sequence of appearance to reflect the engineered promoter architecture. As an example, Pj5[C1C2] is a promoter variant engineered from the parental promoter Pj5 by inserting a T7-stem loop (denoted by C1) as well as an RBS (denoted by C2) in its 5′-UTR. The T7-stem loop was placed upstream of the RBS, as indicated by C1 that comes before C2.
Mutations in −35 Box Tuned Transcriptional Activity down
Li and Liao previously reported PphaC1-G3 promoter, in which its −35 box was mutated from “TTGACA” to “TTCGGC” (Figure 3A). (23) This promoter variant was shown to retain 15% of the activity of its PphaC1 parent when characterized using enhanced green fluorescent protein (eGFP) as a reporter. (23) To validate our initial hypothesis that conserved −35 and −10 boxes are necessary in maintaining high transcriptional activity in C. necator H16, we recreated PphaC1-G3, and this variant was named PphaC1[A1] in our nomenclature system. RFP fluorescence measurement ascertained that PphaC1[A1] retains 16% of the activity of PphaC1 (Figure 3B). This data also indicated that both reporter proteins, RFP and eGFP, give similar outcome in transcriptional activity quantification. RFP and eGFP are commonly used in synthetic biology for characterization of biological parts (e.g., promoter, terminator). (30,31) Li and Liao also created a promoter library with mutated −35 box (TTNNNN). All promoter variants screened showed lower activity in comparison to the wildtype promoter. (23) Therefore, mutations in −35 box likely tune the transcriptional activity down.
Figure 3
Figure 3. (A) Architectures of parental promoters and their engineered variants. (B) Activities of parental promoters and their engineered variants.
A Minimal PphaC1 Promoter with Enhanced Activity
PphaC1 (466 bp) is the longest promoter among the four parental promoters. To find the minimal functional sequence, we created 5′→3′ truncated variants of PphaC1: 25 bp truncation in PphaC1[B1a], 50 bp in PphaC1[B1b], 100 bp in PphaC1[B1c], and 124 bp in PphaC1[B 1d] (Figure 3A). Interestingly, removing the entire upstream element (124 bp) in PphaC1[B 1d] resulted in highest promoter activity, which is 2-fold higher compared to that of its PphaC1 parent (Figure 3B). This suggested that cis-acting elements exist within the upstream element of PphaC1 and contribute to transcriptional suppression.
Synthetic RBS and RBS Repeat Increased Transcriptional Activity
Our initial study with the four parental promoters, along with a previous study conducted by Bi et al., (20) motivated us to further investigate the effects of cis-acting translational regulatory elements on promoter activity. We focused specifically on the 26-bp synthetic genetic element derived from PrrsC (containing a 20-bp RBS found in the pBBR1c-RFP PBAD promoter flanked by an upstream 6-bp BglII restriction site, herein denoted as synthetic RBS) and the 37-bp T7 stem-loop reported by Bi et al. (20) We observed a 4.7-fold increase in promoter activity when a synthetic RBS was added to Pj5 (the Pj5[C2] variant) (Figure 3B). On the contrary, there was no significant change in promoter activity when a T7 stem-loop was added to Pj5 (the Pj5[C1] variant). A drop in promoter activity was observed when a T7 stem-loop was added to Pj5[C2] (the Pj5[C1C2] variant). As such, synthetic RBS is an effective means to amplify promoter activity. We then added the same synthetic RBS to Pg25 and created a variant Pg25[C2] that also showed ∼50% increase in promoter activity. A smaller promoter activity increase in Pg25[C2] could be attributed to a pre-existing effective SD sequence (“AGGAG”) in Pg25.
Repeat of −35 and −10 Boxes Increased Transcriptional Activity
The −35 and −10 boxes are highly conserved regions in prokaryotic promoters, essential for the binding of RNA polymerases. To test if creating more binding sites for RNA polymerase would further increase transcriptional activity, we created the Pg25[D1] variant. In this variant, we duplicated the DNA sequence spanning −35 box and −10 box. In essence, this hybrid promoter is a tandem repeat of two Pg25 promoters, and we observed ∼40% increase in promoter activity (Figure 3). Again, incorporating a synthetic RBS (Pg25[D1C2]) gave an additive effect, resulting in further increase in promoter activity.
Operator Insertion Reduced Transcriptional Activity Drastically
Hybrid promoters are crucial genetic elements in the construction of biosensors. Using a malonyl-CoA biosensor (26) as an example, we inserted the fapO operator sequence “TTAGTACCTGATACTAA” in PrrsC promoter to create two variants: PrrsC[D4] (fapO inserted between −35 and −10 boxes) and PrrsC[D6] (fapO inserted within the 5′-UTR) (Figure 3A). fapO operator is conserved in Gram-positive bacteria such as Bacillus subtilis and acts as a cis-regulatory unit for transcriptional regulation of fatty acid biosynthesis. For both promoter variants, we observed a drastic activity reduction to ∼10% of their PrrsC parent (Figure 3B). The position of operator and the copy number of operator could therefore significantly change the transcriptional activity of the resultant hybrid promoters. Our data corroborated a previous study by Li and Liao, where tetO operators were inserted to create PrrsC hybrid promoters. (23)
Divergent Promoters, Arranged in Back-to-Back, Increased Transcriptional Activity
The distance between and the relative transcriptional directions of adjacent genes are known to be important in some organisms. Neighboring genes arranged in head-to-head (HH) orientation, for instance, could be coregulated and this has been proven experimentally. (32) For gene pairs in HH arrangement, promoters that effect divergent transcription can be organized in three possible ways: back-to-back, overlapping, or face-to-face (Figure 4A). (33) In a recent attempt to construct a malonyl-CoA biosensor for C. necator H16 (publication in preparation), we discovered the significance of divergent transcription in this organism. Therefore, we created 21 composite promoters to systematically investigate divergent transcription in C. necator H16. Each composite promoter is made up of two promoters arranged in back-to-back (or divergent to each other). The promoter driving the RFP expression is termed the primary promoter, while the counterpart is called the secondary promoter.
Figure 4
Figure 4. (A) For gene pairs in HH arrangement, promoters that effect divergent transcription can be organized in three possible ways: back-to-back, overlapping, or face-to-face. (B) Composite promoters engineered using Pg25 as parental promoter. (C) Composite promoters engineered using PrrsC as parental promoter. (D) Composite promoters engineered using Pj5 as parental promoter. (E) Composite promoters with PphaC1 as secondary promoter. (F) Composite promoters with Pg25 as secondary promoter. (G) Composite promoters with Pj5[A1C1C2] as secondary promoter. In graphs E and F, red, blue and orange symbols represent primary promoter activity, composite promoter activity, and fold change, respectively.
When PphaC1 (denoted as modification subscript E1), Pg25 (modification subscript E2), and Pj5[A1C1C2] (modification subscript E3) was applied individually as secondary promoters, they generally served as transcriptional amplifiers, increasing the transcriptional activity of the primary promoters (Figure 4B–G). The promoter activities of these three secondary promoters follow the order of Pj5[A1C1C2] > Pg25 > PphaC1 (eq E3 > E2 > E1). Interestingly, transcriptional amplification depends on the transcriptional activities of both the primary and the secondary promoters. For the same primary promoter, activity enhancement typically decreases with the increased activity of the secondary promoter (Figure 4B–D, herein described as secondary promoter effect). Also, for the same secondary promoter, activity enhancement decreases with the increased activity of the primary promoter (Figure 4E–G, herein described as primary promoter effect). We postulate that secondary promoter effect is attributed to the fact that weaker secondary promoter competes less with the primary promoter for transcriptional machinery or factors. If a secondary promoter of very high activity is used, the competition is so strong that it diminishes the activity of the primary promoter (data not shown). The primary promoter effect observed is perhaps more intuitive and easier to comprehend. If the primary promoter displays high activity, it is more difficult to further improve its activity using an amplifier. It is important to point out that transcriptional enhancement resulted from divergent promoters is not universal to all prokaryotic systems. Generally, we did not observe such behaviors when we tested our composite promoters in E. coli (data not shown).
Promoter Characterization using PBAD as Reference Scale
This study resulted in a set of 42 constitutive promoters, including the four parental promoters. The ratio of promoter activities between the strongest (Pj5[E1A3C2]) and the weakest (PphaC1[A1], eq PphaC1-G3 reported by Li and Liao (23)) is 137. These promoters showed incremental increase in activity across the entire scale (Figure 5A). To promote the widespread use of these promoters, we benchmarked each of them using l-arabinose inducible PBAD promoter as a reference scale. Supporting Information, Figure S4, illustrates the dose-dependent induction of PBAD promoter, using l-arabinose concentration from 0.001% (w/v) to 0.200% (w/v). Expression maxima was reached at 0.200% (w/v) l-arabinose. As depicted in Figure 5B, our engineered promoters covered the entire l-arabinose inducible range (indicated by scattered data points). We categorized all promoters into five activity levels to aid promoter selection: Level 1 (with promoter activity between 0–2000 au), level 2 (2000–4000 au), level 3 (4000–6000 au), level 4 (6000–8000 au), and level 5 (>8000 au). With a PBAD promoter, one could only achieve expression levels between 1 and 4. Through promoter engineering, we obtained seven level 5 variants (Pj5[A3C2], Pj5[C2], Pj5[E1C1C2], Pj5[E2C2], Pj5[E2A3C2], Pj5[E1C2], and Pj5[E1A3C2]) with promoter activities exceeding that of PBAD (Supporting Information, Table S2). For easy classification of all engineered promoters, we developed a numerical coding system (Table 1) and assigned a digital identifier to each promoter. This will allow us to develop a C. necator H16-specific promoter database (work in progress). Each promoter code is in the format of [X-Y-Z], with X representing activity level, Y representing relative activity to PphaC1[A1], and Z representing promoter length. While conceptualizing our engineered promoter nomenclature and coding systems, we have endeavored to make them universal such that they can be applied to other promoters yet to be developed.
Figure 5
Figure 5. (A) Hierarchical ranking of all 42 constitutive promoters reported in this study. (B) The range of promoter activity was expanded from 6-fold to 137-fold after applying combination of promoter engineering strategies (A = point mutation, B = length alteration, C = incorporation of regulatory genetic element, D = promoter hybridization and E = configuration alteration). Promoters derived from PphaC1, PrrsC, Pj5, and Pg25 were colored in blue, green, pink, and red, respectively. Promoters were categorized into five activity levels: Level 1 (with promoter activity between 0–2000 au), level 2 (2000–4000 au), level 3 (4000–6000 au), level 4 (6000–8000 au) and level 5 (>8000 au). Each promoter was benchmarked against PBAD promoter, induced using various concentrations of l-arabinose, from 0.001% (w/v) to 0.200% (w/v).
Summary of Rational Promoter Engineering for C. necator H16
Figure 6 provides an overview of the rational promoter engineering strategies discussed in this article. It shows the effect of a specific modification by looking at the promoter activity difference before and after that particular modification. Creating mutation(s) within −35 box and inserting operator sequence(s) resulted in drastic reduction in promoter activity (represented by red data points), while inserting a T7 stem-loop caused almost no change in promoter activity. On the contrary, inserting a synthetic RBS and applying a transcriptional amplifier (specifically PphaC1 or Pj5[A1C1C2]) gave the highest increase in promoter activity (>100%). In fact, those promoters that are stronger than PBAD promoter (indicated as blue data points) were mostly created using either one of these strategies or combination of them. All the other strategies provided marginal promoter activity increase (from 10% to 50%). Also clearly reflected in Figure 5B, creating divergent promoters is the most effective way of broadening the range of promoter activity. It is worthy of note that relative promoter activity change is dependent on the parental promoter, judging on the work presented in this article.
Figure 6
Figure 6. Relative promoter activity change upon application of promoter engineering strategies (A = point mutation, B = length alteration, C = incorporation of regulatory genetic element, D = promoter hybridization, and E = configuration alteration). Modifications that resulted in loss of promoter activity were indicated as red data points. Promoters with activities higher than PBAD promoter were indicated as blue data points.
The Use of Engineered Constitutive Promoters in C. necator H16
The use of strong constitutive promoters could potentially result in (a) bacterial growth impairment due to high metabolic burden and/or (b) protein excretion/leakage due to high protein expression level. To study these effects, we selected representative promoters from each activity level (Supporting Information, Table S3) and conducted further characterization. We observed similar growth for most of the strains (Figure 7), with growth rates (μmax) falling between 0.21 and 0.24 h–1 (Supporting Information, Table S3). For promoter Pj5[E2C2], which is a level 5 promoter, we noticed a slight drop in growth rate with μmax of 0.18 h–1 (Supporting Information, Table S3). Comparing fluorescence of cell culture and of spent medium (Figure 8) confirmed that there was no protein excretion/leakage. Fluorescence of spent medium was maintained at the level of ∼1000 au throughout the bacterial cultivation. This value was almost identical to that of the control (C. necator H16 harboring pBBR1MCS-1). To study the time-dependent increase in fluorescence signal, we fitted the cell culture fluorescence vs time data to a four-parameter dose–response curve (Supporting Information, Figure S5 and Table S4) for all promoters from level 2 and above. The fluorescence increase was mainly caused by bacterial growth. If we divided the fluorescence measured (Figure 8) by the OD600 (Figure 7), the ratio was kept almost constant at cultivation times above 12 h (Figure S6), further verifying our approach in promoter activity quantification in 96-well plate by taking the RFU/OD600 at t = 48 h.
Figure 7
Figure 7. Growth curves of C. necator H16 harboring either pBBR1MCS-1 (control; black line) or plasmids containing various engineered constitutive promoters [Pg25 (red line), PphaC1[B 1d] (blue line), PrrsC[E1D4] (brown line), Pg25[E3] (green line), Pg25[D1C2] (pink line), Pj5[E1A1C1C2] (orange line), and Pj5[E2C2] (purple line)].
Figure 8
Figure 8. Fluorescence of cell culture (black columns) and of spent medium (gray columns) of C. necator H16 harboring either pBBR1MCS-1 (control) or plasmids containing various engineered constitutive promoters (Pg25, PphaC1[B1d], PrrsC[E1D4], Pg25[E3], Pg25[D1C2], Pj5[E1A1C1C2], and Pj5[E2C2]).
Conclusion
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This article (1) reported and characterized a set of 42 constitutive promoters with a broad range of promoter activity, which are derived from the four most widely used constitutive promoters for C. necator H16 (PphaC1, PrrsC, Pj5, and Pg25), (2) introduced a nomenclature system and a coding system for engineered promoters, (3) sketched out the relationship between promoter architecture and its resultant activity, (4) highlighted similarities (conservation of −35 and −10 boxes) and differences (composite promoters) in transcriptional activity between C. necator H16 and E. coli, and (5) provided guidelines for rational promoter engineering. We strongly believe our constitutive promoter toolbox that exceeds the activity range of the inducible PBAD promoter will serve the biotechnology community working on C. necator H16, be it strain engineering for industrial biomanufacturing or developing advanced molecular biology tools for this organism.
Materials and Methods
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Materials
All DNA modifying enzymes were purchased from either New England Biolabs (Hitchin, UK) or Agilent (Craven Arms, UK). Nucleic acid purification kits were purchased from Qiagen (Manchester, UK). All oligonucleotides were synthesized by Eurofins (Ebersberg, Germany).
Strains
Escherichia coli DH5α was used for all molecular cloning, plasmid propagation, and maintenance. Cupriavidus necator H16 (DSM-428, purchased from DSMZ, Braunschweig, Germany) was used for all experiments described in this article.
Promoter Engineering and Sequences
All plasmids were derived from pBBR1c-RFP (see Supporting Information) and constructed using standard molecular biology techniques. All engineered promoters were verified by restrictive analysis and/or DNA sequencing, and their sequences were provided in the Supporting Information.
Bacterial Cultivation and Transformation
C. necator H16 was cultivated at 30 °C in nutrient broth (NB: 5 g/L peptone, 1 g/L beef extract, 2 g/L yeast extract, 5 g/L NaCl; pH 7.0 ± 0.2 @ 25 °C) supplemented with 10 μg/mL of gentamicin. Cells were transformed with plasmids using the electroporation protocol described by Tee et al., (19) plated on NB agar supplemented with 10 μg/mL of gentamicin and 25 μg/mL of chloramphenicol, and incubated at 30 °C for 40–60 h. E. coli DH5α was transformed with plasmids using the standard CaCl2 method, plated on TYE agar (10 g/L tryptone, 5 g/L yeast extract, 8 g/L NaCl, 15 g/L agar) supplemented with 25 μg/mL of chloramphenicol, and incubated overnight at 37 °C.
Promoter Activity Quantification Using Fluorescence Assay
Transformants of C. necator H16, carrying either an RFP-null or an RFP-expressing vector, were precultured in 96-well microtiter plate containing 200 μL/well of NB supplemented with 10 μg/mL of gentamicin and 25 μg/mL of chloramphenicol at 30 °C for 40 h. This preculture was used to inoculate a fresh clear-bottom 96-well microtiter plate [Greiner Bio-One (Stonehouse, UK)] containing 200 μL/well of NB supplemented with 10 μg/mL of gentamicin, 25 μg/mL of chloramphenicol as well as 0–0.2% (w/v) l-arabinose (when required) to induce RFP expression. The plate was cultivated at 30 °C for a total of 48 h. OD600 and fluorescence (Ex 584 nm, Em 607 nm; bottom read) were measured using SpectraMax M2e microplate/cuvette reader [Molecular Devices (Wokingham, UK)] after 12 h of cultivation and repeated at 6 h intervals. Relative fluorescence unit (RFU) was calculated by normalizing fluorescence value with the fluorescence value of C. necator H16 carrying an RFP-null vector (negative control). The RFU value therefore represents the fluorescence fold increase owing to RFP expression. RFU/OD600 value was then calculated as the ratio of RFU and OD600 value of the respective strain. The ratio was used to account for potential metabolic burden due to high protein expression level, affecting bacterial growth. Promoter activity (PA) was defined as the RFU/OD600 value after 48 h of cultivation. The ratio of RFU/OD600 was more or less a constant at cultivation time more than 12 h. All experiments were done in triplicate.
Fold Change and Relative Promoter Activity Change
Fold change and relative promoter activity change were calculated by using the formulas:
Effects of Engineered Constitutive Promoters on Bacterial Growth and Protein Excretion
Selected plasmids were freshly transformed into C. necator H16, and single colonies were picked to prepare overnight cultures. Falcon tubes, containing 6 mL of fresh mineral salts medium (MSM) (34) supplemented with 10 g/L sodium gluconate (carbon source), 10 μg/mL gentamicin, and 25 μg/mL chloramphenicol, were inoculated at a starting OD600 of 0.2. Cells were cultivated at 30 °C and sampled at regular time intervals. OD600 of each sample was measured using BioPhotometer Plus [Eppendorf (Stevenage, UK)]. For all samples collected, the fluorescence (Ex 584 nm, Em 607 nm; bottom read) of the cell culture (90 μL) and of the spent medium (90 μL) was measured using SpectraMax M2e microplate/cuvette reader [Molecular Devices (Wokingham, UK)].
Supporting Information
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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssynbio.8b00136.
Experimental procedures; plasmid map of pBBR1c-RFP; alignment of Pj5 and Pg25 promoters used in this study and in Gruber et al. (2014); graphical representation of rational promoter engineering strategies; l-arabinose-dose dependent induction of PBAD promoter; curve fitting of cell culture fluorescence vs time data; fluorescence of cell culture normalized by OD600 value; promoter sequences; subcategories of each promoter engineering strategy; promoter activity table; specific growth rates of C. necator H16 carrying plasmids containing various promoters (PDF)
Terms & Conditions
Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.
Author Information
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- Tuck Seng Wong - Department of Chemical & Biological Engineering and Advanced Biomanufacturing Centre, University of Sheffield, Sir Robert Hadfield Building, Mappin Street, Sheffield S1 3JD, United Kingdom;
http://orcid.org/0000-0001-7689-9057;
T.S.W. and K.L.T. conceived and supervised the project. A.O.J. and M.G. performed the experiments and analyzed the data. T.S.W., K.L.T., and A.O.J. wrote the manuscript. All authors discussed the content of the manuscript and provided critical revisions on the manuscript.
Acknowledgments
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We thank the Department of Chemical and Biological Engineering, ChELSI and EPSRC (EP/E036252/1), for financial support. A.O.J. and M.G.V. are supported by the University of Sheffield and CONACYT (Mexico) scholarships, respectively.
References
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This article references 34 other publications.
- 1Pohlmann, A., Fricke, W. F., Reinecke, F., Kusian, B., Liesegang, H., Cramm, R., Eitinger, T., Ewering, C., Pötter, M., Schwartz, E., Strittmatter, A., Voss, I., Gottschalk, G., Steinbüchel, A., Friedrich, B., and Bowien, B. (2006) Genome sequence of the Bioplastic-producing ″Knallgas″ bacterium Ralstonia eutropha H16. Nat. Biotechnol. 24 (10), 1257– 62, DOI: 10.1038/nbt1244Google ScholarThere is no corresponding record for this reference.
- 2Volodina, E., Raberg, M., and Steinbüchel, A. (2016) Engineering the heterotrophic carbon sources utilization range of Ralstonia eutropha H16 for applications in biotechnology. Crit. Rev. Biotechnol. 36 (6), 978– 991, DOI: 10.3109/07388551.2015.1079698Google Scholar2Engineering the heterotrophic carbon sources utilization range of Ralstonia eutropha H16 for applications in biotechnologyVolodina, Elena; Raberg, Matthias; Steinbuchel, AlexanderCritical Reviews in Biotechnology (2016), 36 (6), 978-991CODEN: CRBTE5; ISSN:0738-8551. (Taylor & Francis Ltd.)Ralstonia eutropha H16 is an interesting candidate for the biotechnol. prodn. of polyesters consisting of hydroxy- and mercaptoalkanoates, and other compds. It provides all the necessary characteristics, which are required for a biotechnol. prodn. strain. Due to its metabolic versatility, it can convert a broad range of renewable heterotrophic resources into diverse valuable compds. High cell d. fermns. of the non-pathogenic R. eutropha can be easily performed. Furthermore, this bacterium is accessible to engineering of its metab. by genetic approaches having available a large repertoire of genetic tools. Since the complete genome sequence of R. eutropha H16 has become available, a variety of transcriptome, proteome and metabolome studies provided valuable data elucidating its complex metab. and allowing a systematic biol. approach. However, high prodn. costs for bacterial large-scale prodn. of biomass and biotechnol. valuable products are still an economic challenge. The application of inexpensive raw materials could significantly reduce the expenses. Therefore, the conversion of diverse substrates to polyhydroxyalkanoates by R. eutropha was steadily improved by optimization of cultivation conditions, mutagenesis and metabolic engineering. Industrial byproducts and residual compds. like glycerol, and substrates contg. high carbon content per wt. like palm, soybean, corn oils as well as raw sugar-rich materials like molasses, starch and lignocellulose, are the most promising renewable substrates and were intensively studied.
- 3Jajesniak, P., Omar Ali, H. E. M., and Wong, T. S. (2014) Carbon dioxide capture and utilization using biological systems: Opportunities and challenges. J. Bioprocess. Biotech. 4, 155, DOI: 10.4172/2155-9821.1000155Google ScholarThere is no corresponding record for this reference.
- 4Peplow, M. (2015) Industrial biotechs turn greenhouse gas into feedstock opportunity. Nat. Biotechnol. 33, 1123– 1125, DOI: 10.1038/nbt1115-1123Google ScholarThere is no corresponding record for this reference.
- 5Brigham, C. J., Zhila, N., Shishatskaya, E., Volova, T. G., and Sinskey, A. J. (2012) Manipulation of Ralstonia eutropha carbon storage pathways to produce useful bio-based products. Subcell. Biochem. 64, 343– 66, DOI: 10.1007/978-94-007-5055-5_17Google Scholar5Manipulation of Ralstonia eutropha carbon storage pathways to produce useful bio-based productsBrigham, Christopher J.; Zhila, Natalia; Shishatskaya, Ekaterina; Volova, Tatiana G.; Sinskey, Anthony J.Subcellular Biochemistry (2012), 64 (Reprogramming Microbial Metabolic Pathways), 343-366CODEN: SBCBAG; ISSN:0306-0225. (Springer)A review. Ralstonia eutropha is a Gram-neg. betaproteobacterium found natively in soils that can utilize a wide array of carbon sources for growth, and can store carbon intracellularly in the form of polyhydroxyalkanoate. Many aspects of R. eutropha make it a good candidate for use in biotechnol. prodn. of polyhydroxyalkanoate and other bio-based, value added compds. Manipulation of the organism's carbon flux is a cornerstone to success in developing it as a biotechnol. relevant organism. Here, we examine the methods of controlling and adapting the flow of carbon in R. eutropha metab. and the wide range of compds. that can be synthesized as a result. The presence of many different carbon utilization pathways and the custom genetic toolkit for manipulation of those pathways gives R. eutropha a versatility that allows it to be a biotechnol. important organism.
- 6Lutke-Eversloh, T. and Steinbüchel, A. (2003) Novel precursor substrates for polythioesters (PTE) and limits of PTE biosynthesis in Ralstonia eutropha. FEMS Microbiol. Lett. 221, 191– 196, DOI: 10.1016/S0378-1097(03)00185-XGoogle ScholarThere is no corresponding record for this reference.
- 7Steinbüchel, A. and Pieper, U. (1992) Production of a copolyester of 3-hydroxybutyric acid and 3-hydroxyvaleric acid from single unrelated carbon sources by a mutant of Alcaligenes eutrophus. Appl. Microbiol. Biotechnol. 37, 1– 6, DOI: 10.1007/BF00174193Google ScholarThere is no corresponding record for this reference.
- 8Voss, I. and Steinbüchel, A. (2006) Application of a KDPG-aldolase gene-dependent addiction system for enhanced production of cyanophycin in Ralstonia eutropha strain H16. Metab. Eng. 8 (1), 66– 78, DOI: 10.1016/j.ymben.2005.09.003Google Scholar8Application of a KDPG-aldolase gene-dependent addiction system for enhanced production of cyanophycin in Ralstonia eutropha strain H16Voss, Ingo; Steinbuechel, AlexanderMetabolic Engineering (2006), 8 (1), 66-78CODEN: MEENFM; ISSN:1096-7176. (Elsevier)Two different recombinant plasmids both contg. the cyanophycin synthetase gene (cphA) of Synechocystis sp. strain PCC6308 but differing concerning the resistance marker gene were tested for their suitability to produce high amts. of cyanophycin in recombinant strains of Ralstonia eutropha. Various cultivation expts. at the 30-L scale revealed very low cyanophycin contents of the cells ranging from 4.6% to 6.2% (wt./wt.) of cellular dry wt. (CDW) only, most probably because most cells had lost the corresponding plasmid during cultivation. To establish a cost effective and high efficient system for prodn. of cyanophycin at larger scales using recombinant strains of R. eutropha, we applied two strategies: First, we integrated cphA into the dispensable chromosomal -lactate dehydrogenase gene (ldh) of R. eutropha. Depending on the cultivation conditions used, relatively low cyanophycin contents between 2.2% and 7.7% (wt./wt.) of CDW were reproducibly detected, which might be due to weak expression or low gene dosage in the single cphA copy strain of R. eutropha. In a second strategy we constructed a KDPG-aldolase gene (eda)-dependent addiction system, which combined features of a multi-copy plasmid with stabilized expression of cphA. Flasks expts. revealed that the cells accumulated extraordinarily high amts. of cyanophycin between 26.9% and 40.0% (wt./wt.) of CDW even under cultivation conditions lacking cyanophycin precursor substrates or plasmid stabilizing antibiotics. Cyanophycin contents of up to 40.0% (wt./wt.) of CDW were also obtained at a 30-L scale or a 500-L pilot-plant scale under such non-selective conditions. This demonstrates impressively that the stabilizing effect of the constructed eda-dependent addiction system can be used for prodn. of enhanced amts. of cyanophycin at a larger scale in recombinant strains of R. eutropha.
- 9Valentin, H. E., Zwingmann, G., Schönebaum, A., and Steinbüchel, A. (1995) Metabolic pathway for biosynthesis of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) from 4-hydroxybutyrate by Alcaligenes eutrophus. Eur. J. Biochem. 227 (1–2), 43– 60, DOI: 10.1111/j.1432-1033.1995.tb20358.xGoogle Scholar9Metabolic pathway for biosynthesis of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) from 4-hydroxybutyrate by Alcaligenes eutrophusValentin, Henry E.; Zwingmann, Gundula; Schoenebaum, Andreas; Steinbuechel, AlexanderEuropean Journal of Biochemistry (1995), 227 (1/2), 43-60CODEN: EJBCAI; ISSN:0014-2956. (Springer)Various aerobic gram-neg. bacteria have been examd. for their ability to use 4-hydroxybutyrate and 1,4-butanediol as carbon source for growth. Alcaligenes eutrophus strains H16, HF39, PHB-4 and Pseudomonas denitrificans 'Morris' were not able to grow with 1,4-butanediol or 4-hydroxybutyrate. From A. eutrophus HF39 spontaneous primary mutants (e.g. SK4040) were isolated which grew on 4-hydroxybutyrate with doubling times of approx. 3 h. Tn5::mob mutagenesis of mutant SK4040 led to the islation of two phenotypically different classes of secondary mutants which were affected in the utilization of 4-hydroxybutyrate. Mutants exhibiting the phenotype 4-hydroxybutyrate-neg. did not grow with 4-hydroxybutyrate, and mutants exhibiting the phenotype 4-hydroxybutyrate-leaky grew at a significantly lower rate with 4-hydroxybutyrate. Hybridization expts. led to the identification of a 10-kbp genomic EcoRI fragment of A. eutrophus SK4040, which was altered in mutants with the phenotype 4-hydroxybutyrate-neg., and of two 1-kbp and 4.5-kbp genomic EcoRI fragments, which were altered in mutants with the phenotype 4-hydroxybutyrate-leaky. This 10-kbp EcoRI fragment was cloned from A. eutrophus SK4040, and conjugative transfer of a pVDZ'2 hybrid plasmid to A. eutrophus H16 conferred the ability to grow with 4-hydroxybutyrate to the wild type. DNA-sequence anal. of this fragment, enzymic anal. of the wild type and of mutants of A. eutrophus as well as of recombinant strains of Escherichia coli led to the identification of a structural gene encoding for a 4-hydroxybutyrate dehydrogenase which was affected by transposon mutagenesis in five of six available 4-hydroxybutyrate-neg. mutants. Enzymic studies also provided evidence for the presence of an active succinate-semialdehyde dehydrogenase in 4-hydroxybutyrate-grown cells. This indicated that degrdn. of 4-hydroxybutyrate occurs via succinate semialdehyde and succinate and that the latter is degraded by the citric acid cycle. NMR studies of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) accumulated from 4-hydroxy [1-13C]butyrate or 4-hydroxy[2-13C]butyrate as substrate gave no evidence for a direct conversion of 4-hydroxybutyrate into 3-hydroxybutyrate and therefore supported the results of enzymic anal.
- 10Chen, J. S., Colon, B., Dusel, B., Ziesack, M., Way, J. C., and Torella, J. P. (2015) Production of fatty acids in Ralstonia eutropha H16 by engineering beta-oxidation and carbon storage. PeerJ 3, e1468, DOI: 10.7717/peerj.1468Google ScholarThere is no corresponding record for this reference.
- 11Crépin, L., Lombard, E., and Guillouet, S. E. (2016) Metabolic engineering of Cupriavidus necator for heterotrophic and autotrophic alka(e)ne production. Metab. Eng. 37, 92– 101, DOI: 10.1016/j.ymben.2016.05.002Google Scholar11Metabolic engineering of Cupriavidus necator for heterotrophic and autotrophic alka(e)ne productionCrepin, Lucie; Lombard, Eric; Guillouet, Stephane E.Metabolic Engineering (2016), 37 (), 92-101CODEN: MEENFM; ISSN:1096-7176. (Elsevier B. V.)Alkanes of defined carbon chain lengths can serve as alternatives to petroleum-based fuels. Recently, microbial pathways of alkane biosynthesis have been identified and enabled the prodn. of alkanes in non-native producing microorganisms using metabolic engineering strategies. The chemoautotrophic bacterium Cupriavidus necator has great potential for producing chems. from CO2: it is known to have one of the highest growth rate among natural autotrophic bacteria and under nutrient imbalance it directs most of its carbon flux to the synthesis of the acetyl-CoA derived polymer, polyhydroxybutyrate (PHB), (up to 80% of intracellular content). Alkane synthesis pathway from Synechococcus elongatus (2 genes coding an acyl-ACP reductase and an aldehyde deformylating oxygenase) was heterologously expressed in a C. necator mutant strain deficient in the PHB synthesis pathway. Under heterotrophic condition on fructose we showed that under nitrogen limitation, in presence of an org. phase (decane), the strain produced up to 670 mg/L total hydrocarbons contg. 435 mg/l of alkanes consisting of 286 mg/l of pentadecane, 131 mg/l of heptadecene, 18 mg/l of heptadecane, and 236 mg/l of hexadecanal. We report here the highest level of alka(e)nes prodn. by an engineered C. necator to date. We also demonstrated the first reported alka(e)nes prodn. by a non-native alkane producer from CO2 as the sole carbon source.
- 12Marc, J., Grousseau, E., Lombard, E., Sinskey, A. J., Gorret, N., and Guillouet, S. E. (2017) Over expression of GroESL in Cupriavidus necator for heterotrophic and autotrophic isopropanol production. Metab. Eng. 42, 74– 84, DOI: 10.1016/j.ymben.2017.05.007Google Scholar12Over expression of GroESL in Cupriavidus necator for heterotrophic and autotrophic isopropanol productionMarc, Jillian; Grousseau, Estelle; Lombard, Eric; Sinskey, Anthony J.; Gorret, Nathalie; Guillouet, Stephane E.Metabolic Engineering (2017), 42 (), 74-84CODEN: MEENFM; ISSN:1096-7176. (Elsevier B.V.)We previously reported a metabolic engineering strategy to develop an isopropanol producing strain of Cupriavidus necator leading to prodn. of 3.4 g L-1 isopropanol. To reach higher titers, isopropanol toxicity to the cells has to be considered. A toxic effect of isopropanol on the growth of C. necator has been indeed obsd. above a crit. value of 15 g L-1. GroESL chaperones were first searched and identified in the genome of C. necator. Native groEL and groES genes from C. necator were over-expressed in a strain deleted for PHA synthesis. We demonstrated that over-expressing groESL genes led to a better tolerance of the strain towards exogenous isopropanol. GroESL genes were then over-expressed within the best engineered isopropanol producing strain. A final isopropanol concn. of 9.8 g L-1 was achieved in fed-batch culture on fructose as the sole carbon source (equiv. to 16 g L-1 after taking into account evapn.). Cell viability was slightly improved by the chaperone over-expression, particularly at the end of the fermn. when the isopropanol concn. was the highest. Moreover, the strain over-expressing the chaperones showed higher enzyme activity levels of the 2 heterologous enzymes (acetoacetate carboxylase and alc. dehydrogenase) of the isopropanol synthetic operon, translating to a higher specific prodn. rate of isopropanol at the expense of the specific prodn. rate of acetone. Over-expressing the native chaperones led to a 9-18% increase in the isopropanol yield on fructose.
- 13Müller, J., MacEachran, D., Burd, H., Sathitsuksanoh, N., Bi, C., Yeh, Y. C., Lee, T. S., Hillson, N. J., Chhabra, S. R., Singer, S. W., and Beller, H. R. (2013) Engineering of Ralstonia eutropha H16 for autotrophic and heterotrophic production of methyl ketones. Appl. Environ. Microbiol. 79 (14), 4433– 9, DOI: 10.1128/AEM.00973-13Google ScholarThere is no corresponding record for this reference.
- 14Lu, J., Brigham, C. J., Gai, C. S., and Sinskey, A. J. (2012) Studies on the production of branched-chain alcohols in engineered Ralstonia eutropha. Appl. Microbiol. Biotechnol. 96 (1), 283– 97, DOI: 10.1007/s00253-012-4320-9Google Scholar14Studies on the production of branched-chain alcohols in engineered Ralstonia eutrophaLu, Jingnan; Brigham, Christopher J.; Gai, Claudia S.; Sinskey, Anthony J.Applied Microbiology and Biotechnology (2012), 96 (1), 283-297CODEN: AMBIDG; ISSN:0175-7598. (Springer)Wild-type Ralstonia eutropha H16 produces polyhydroxybutyrate (PHB) as an intracellular carbon storage material during nutrient stress in the presence of excess carbon. In this study, the excess carbon was redirected in engineered strains from PHB storage to the prodn. of isobutanol and 3-methyl-1-butanol (branched-chain higher alcs.). These branched-chain higher alcs. can directly substitute for fossil-based fuels and be employed within the current infrastructure. Various mutant strains of R. eutropha with isobutyraldehyde dehydrogenase activity, in combination with the overexpression of plasmid-borne, native branched-chain amino acid biosynthesis pathway genes and the overexpression of heterologous ketoisovalerate decarboxylase gene, were employed for the biosynthesis of isobutanol and 3-methyl-1-butanol. Prodn. of these branched-chain alcs. was initiated during nitrogen or phosphorus limitation in the engineered R. eutropha. One mutant strain not only produced over 180 mg/L branched-chain alcs. in flask culture, but also was significantly more tolerant of isobutanol toxicity than wild-type R. eutropha. After the elimination of genes encoding three potential carbon sinks (ilvE, bkdAB, and aceE), the prodn. titer improved to 270 mg/L isobutanol and 40 mg/L 3-methyl-1-butanol. Semicontinuous flask cultivation was utilized to minimize the toxicity caused by isobutanol while supplying cells with sufficient nutrients. Under this semicontinuous flask cultivation, the R. eutropha mutant grew and produced more than 14 g/L branched-chain alcs. over the duration of 50 days. These results demonstrate that R. eutropha carbon flux can be redirected from PHB to branched-chain alcs. and that engineered R. eutropha can be cultivated over prolonged periods of time for product biosynthesis.
- 15Lütte, S., Pohlmann, A., Zaychikov, E., Schwartz, E., Becher, J. R., Heumann, H., and Friedrich, B. (2012) Autotrophic production of stable-isotope-labeled arginine in Ralstonia eutropha strain H16. Appl. Environ. Microbiol. 78 (22), 7884– 90, DOI: 10.1128/AEM.01972-12Google ScholarThere is no corresponding record for this reference.
- 16Park, J. M., Jang, Y. S., Kim, T. Y., and Lee, S. Y. (2010) Development of a gene knockout system for Ralstonia eutropha H16 based on the broad-host-range vector expressing a mobile group II intron. FEMS Microbiol. Lett. 309, 193– 200, DOI: 10.1111/j.1574-6968.2010.02041.xGoogle ScholarThere is no corresponding record for this reference.
- 17Gruber, S., Hagen, J., Schwab, H., and Koefinger, P. (2014) Versatile and stable vectors for efficient gene expression in Ralstonia eutropha H16. J. Biotechnol. 186, 74– 82, DOI: 10.1016/j.jbiotec.2014.06.030Google Scholar17Versatile and stable vectors for efficient gene expression in Ralstonia eutropha H16Gruber, Steffen; Hagen, Jeremias; Schwab, Helmut; Koefinger, PetraJournal of Biotechnology (2014), 186 (), 74-82CODEN: JBITD4; ISSN:0168-1656. (Elsevier B.V.)The Gram-neg. β-proteobacterium Ralstonia eutropha H16 is primarily known for polyhydroxybutyrate (PHB) prodn. and its ability to grow chemolithoautotrophically by using CO2 and H2 as sole carbon and energy sources. The majority of metabolic engineering and heterologous expression studies conducted so far rely on a small no. of suitable expression systems. Particularly the plasmid based expression systems already developed for the use in R. eutropha H16 suffer from high segregational instability and plasmid loss after a short time of fermn. In order to develop efficient and highly stable plasmid expression vectors for the use in R. eutropha H16, a new plasmid design was created including the RP4 partitioning system, as well as various promoters and origins of replication. The application of minireplicons derived from broad-host-range plasmids RSF1010, pBBR1, RP4 and pSa for the construction of expression vectors and the use of numerous, versatile promoters extend the range of feasible expression levels considerably. In particular, the use of promoters derived from the bacteriophage T5 was described for the first time in this work, characterizing the j5 promoter as the strongest promoter yet to be applied in R. eutropha H16. Moreover, the implementation of the RP4 partition sequence in plasmid design increased plasmid stability significantly and enables fermns. with marginal plasmid loss of recombinant R. eutropha H16 for at least 96 h. The utility of the new vector family in R. eutropha H16 is demonstrated by providing expression data with different model proteins and consequently further raises the value of this organism as cell factory for biotechnol. applications including protein and metabolite prodn.
- 18Raberg, M., Heinrich, D., and Steinbüchel, A. (2015) Analysis of PHB Metabolism Applying Tn5Mutagenesis in Ralstonia eutropha. In Hydrocarbon and Lipid Microbiology Protocols; Springer Protocols Handbooks (McGenity, T., Timmis, K., and Nogales, B., Eds.) pp 120– 148, Springer, Berlin, Heidelberg.Google ScholarThere is no corresponding record for this reference.
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- 20Bi, C., Su, P., Müller, J., Yeh, Y. C., Chhabra, S. R., Beller, H. R., Singer, S. W., and Hillson, N. J. (2013) Development of a broad-host synthetic biology toolbox for Ralstonia eutropha and its application to engineering hydrocarbon biofuel production. Microb. Cell Fact. 12, 107, DOI: 10.1186/1475-2859-12-107Google Scholar20Development of a broad-host synthetic biology toolbox for Ralstonia eutropha and its application to engineering hydrocarbon biofuel productionBi, Changhao; Su, Peter; Muller, Jana; Yeh, Yi-Chun; Chhabra, Swapnil R.; Beller, Harry R.; Singer, Steven W.; Hilson, Nathan J.Microbial Cell Factories (2013), 12 (), 107/1-107/10, 10 pp.CODEN: MCFICT; ISSN:1475-2859. (BioMed Central Ltd.)Background: The chemoautotrophic bacterium Ralstonia eutropha can utilize H2/CO2 for growth under aerobic conditions. While this microbial host has great potential to be engineered to produce desired compds. (beyond polyhydroxybutyrate) directly from CO2, little work has been done to develop genetic part libraries to enable such endeavors. Results: We report the development of a toolbox for the metabolic engineering of Ralstonia eutropha H16. We have constructed a set of broad-host-range plasmids bearing a variety of origins of replication, promoters, 5' mRNA stem-loop structures, and ribosomal binding sites. Specifically, we analyzed the origins of replication pCM62 (IncP), pBBR1, pKT (IncQ), and their variants. We tested the promoters PBAD, T7, Pxyls/PM, PlacUV5, and variants thereof for inducible expression. We also evaluated a T7 mRNA stem-loop structure sequence and compared a set of ribosomal binding site (RBS) sequences derived from Escherichia coli, R. eutropha, and a computational RBS design tool. Finally, we employed the toolbox to optimize hydrocarbon prodn. in R. eutropha and demonstrated a 6-fold titer improvement using the appropriate combination of parts. Conclusion: We constructed and evaluated a versatile synthetic biol. toolbox for Ralstonia eutropha metabolic engineering that could apply to other microbial hosts as well.
- 21Fukui, T., Ohsawa, K., Mifune, J., Orita, I., and Nakamura, S. (2011) Evaluation of promoters for gene expression in polyhydroxyalkanoate-producing Cupriavidus necator H16. Appl. Microbiol. Biotechnol. 89 (5), 1527– 36, DOI: 10.1007/s00253-011-3100-2Google ScholarThere is no corresponding record for this reference.
- 22Guzman, L. M., Belin, D., Carson, M. J., and Beckwith, J. (1995) Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J. Bacteriol. 177 (14), 4121– 30, DOI: 10.1128/jb.177.14.4121-4130.1995Google Scholar22Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoterGuzman, Luz-Maria; Belin, Dominique; Carson, Michael J.; Beckwith, JonJournal of Bacteriology (1995), 177 (14), 4121-30CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)We have constructed a series of plasmid vectors (pBAD vectors) contg. the PBAD promoter of the araBAD (arabinose) operon and the gene encoding the pos. and neg. regulator of this promoter, araC. Using the phoA gene and phoA fusions to monitor expression in these vectors, we show that the ratio of induction/repression can be 1,200-fold, compared with 50-fold for PTAC-based vectors. PhoA expression can be modulated over a wide range of inducer (arabinose) concns. and reduced to extremely low levels by the presence of glucose, which represses expression. Also, the kinetics of induction and repression are very rapid and significantly affected by the ara allele in the host strain. Thus, the use of this system which can be efficiently and rapidly turned on and off allows the study of important aspects of bacterial physiol. in a very simple manner and without changes of temp. We have exploited the tight regulation of the PBAD promoter to study the phenotypes of null mutations of essential genes and explored the use of pBAD vectors as an expression system.
- 23Li, H. and Liao, J. C. (2015) A synthetic anhydrotetracycline-controllable gene expression system in Ralstonia eutropha H16. ACS Synth. Biol. 4 (2), 101– 6, DOI: 10.1021/sb4001189Google Scholar23A synthetic anhydrotetracycline-controllable gene expression system in Ralstonia eutropha H16Li, Han; Liao, James C.ACS Synthetic Biology (2015), 4 (2), 101-106CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)Controllable gene expression systems that are orthogonal to the host's native gene regulation network are invaluable tools for synthetic biol. In Ralstonia eutropha H16, such systems are extremely limited despite the importance of this organism in microbiol. research and biotechnol. application. Here the authors develop an anhydrotetracycline (aTc)-inducible gene expression system, which is composed of a synthetic promoter contg. the operator tetO, the repressor TetR, and the inducer aTc. Using a reporter-activity based promoter library screen, the authors first identified the active hybrids between the tetO operators and the R. eutropha native rrsC promoter (PrrsC). Next, the authors showed that the hybrid promoters are repressable by TetR. To optimize the dynamic range of the system, a high-throughput screening of 300 mutants of R. eutropha phaC1 promoter was conducted to identify suitable promoters to tune the tetR expression level. The final controllable expression system contains the modified PrrsC with two copies of the tetO1 operator integrated and the tetR driven by the mutated PphaC1. The system has decreased basal expression level and can be tuned by different aTc concns. with greater than 10-fold dynamic range. The system was used to alleviate cellular toxicity caused by AlsS overexpression, which impeded the authors' metabolic engineering work on isobutanol and 3-methyl-1-butanol prodn. in R. eutropha H16.
- 24Hanko, E. K. R., Minton, N. P., and Malys, N. (2017) Characterisation of a 3-hydroxypropionic acid-inducible system from Pseudomonas putida for orthogonal gene expression control in Escherichia coli and Cupriavidus necator. Sci. Rep. 7, 1724, DOI: 10.1038/s41598-017-01850-wGoogle Scholar24Characterisation of a 3-hydroxypropionic acid-inducible system from Pseudomonas putida for orthogonal gene expression control in Escherichia coli and Cupriavidus necatorHanko Erik K R; Minton Nigel P; Malys NaglisScientific reports (2017), 7 (1), 1724 ISSN:.3-hydroxypropionic acid (3-HP) is an important platform chemical used as a precursor for production of added-value compounds such as acrylic acid. Metabolically engineered yeast, Escherichia coli, cyanobacteria and other microorganisms have been developed for the biosynthesis of 3-HP. Attempts to overproduce this compound in recombinant Pseudomonas denitrificans revealed that 3-HP is consumed by this microorganism using the catabolic enzymes encoded by genes hpdH, hbdH and mmsA. 3-HP-inducible systems controlling the expression of these genes have been predicted in proteobacteria and actinobacteria. In this study, we identify and characterise 3-HP-inducible promoters and their corresponding LysR-type transcriptional regulators from Pseudomonas putida KT2440. A newly-developed modular reporter system proved possible to demonstrate that PpMmsR/P mmsA and PpHpdR/P hpdH are orthogonal and highly inducible by 3-HP in E. coli (12.3- and 23.3-fold, respectively) and Cupriavidus necator (51.5- and 516.6-fold, respectively). Bioinformatics and mutagenesis analyses revealed a conserved 40-nucleotide sequence in the hpdH promoter, which plays a key role in HpdR-mediated transcription activation. We investigate the kinetics and dynamics of the PpHpdR/P hpdH switchable system in response to 3-HP and show that it is also induced by both enantiomers of 3-hydroxybutyrate. These findings pave the way for use of the 3-HP-inducible system in synthetic biology and biotechnology applications.
- 25Gruber, S., Schwendenwein, D., Magomedova, Z., Thaler, E., Hagen, J., Schwab, H., and Heidinger, P. (2016) Design of inducible expression vectors for improved protein production in Ralstonia eutropha H16 derived host strains. J. Biotechnol. 235, 92– 9, DOI: 10.1016/j.jbiotec.2016.04.026Google Scholar25Design of inducible expression vectors for improved protein production in Ralstonia eutropha H16 derived host strainsGruber, Steffen; Schwendenwein, Daniel; Magomedova, Zalina; Thaler, Eva; Hagen, Jeremias; Schwab, Helmut; Heidinger, PetraJournal of Biotechnology (2016), 235 (), 92-99CODEN: JBITD4; ISSN:0168-1656. (Elsevier B.V.)A review. Ralstonia eutropha H16 (Cupriavidus necator H16) is a Gram-neg., facultative chemolithoautotrophic bacterium which can use H2 and CO2 as sole energy and carbon sources in the absence of org. substrates. The biotechnol. use of R. eutropha H16 on an industrial scale has already been established; however, only a small no. of tools promoting inducible gene expression is available. Within this study two systems promoting inducible expression were designed on the basis of the strong j5 promoter and the Escherichia coli lacI or the Pseudomonas putida cumate regulatory elements. Both expression vectors display desired regulatory features and further increase the no. of suitable inducible expression systems for the prodn. of metabolites and proteins with R. eutropha H16.
- 26Johnson, A. O., Gonzalez-Villanueva, M., Wong, L., Steinbüchel, A., Tee, K. L., Xu, P., and Wong, T. S. (2017) Design and application of genetically-encoded malonyl-CoA biosensors for metabolic engineering of microbial cell factories. Metab. Eng. 44, 253– 264, DOI: 10.1016/j.ymben.2017.10.011Google Scholar26Design and application of genetically-encoded malonyl-CoA biosensors for metabolic engineering of microbial cell factoriesJohnson, Abayomi Oluwanbe; Gonzalez-Villanueva, Miriam; Wong, Lynn; Steinbuchel, Alexander; Tee, Kang Lan; Xu, Peng; Wong, Tuck SengMetabolic Engineering (2017), 44 (), 253-264CODEN: MEENFM; ISSN:1096-7176. (Elsevier B.V.)A review. Malonyl-CoA is the basic building block for synthesizing a range of important compds. including fatty acids, phenylpropanoids, flavonoids and non-ribosomal polyketides. Centering around malonyl-CoA, we summarized here the various metabolic engineering strategies employed recently to regulate and control malonyl-CoA metab. and improve cellular productivity. Effective metabolic engineering of microorganisms requires the introduction of heterologous pathways and dynamically rerouting metabolic flux towards products of interest. Transcriptional factor-based biosensors translate an internal cellular signal to a transcriptional output and drive the expression of the designed genetic/biomol. circuits to compensate the activity loss of the engineered biosystem. Recent development of genetically-encoded malonyl-CoA sensor has stood out as a classical example to dynamically reprogram cell metab. for various biotechnol. applications. Here, we reviewed the design principles of constructing a transcriptional factor-based malonyl-CoA sensor with superior detection limit, high sensitivity and broad dynamic range. We discussed various synthetic biol. strategies to remove pathway bottleneck and how genetically-encoded metabolite sensor could be deployed to improve pathway efficiency. Particularly, we emphasized that integration of malonyl-CoA sensing capability with biocatalytic function would be crit. to engineer efficient microbial cell factory. Biosensors have also advanced beyond its classical function of a sensor actuator for in situ monitoring of intracellular metabolite concn. Applications of malonyl-CoA biosensors as a sensor-inventor for neg. feedback regulation of metabolic flux, a metabolic switch for oscillatory balancing of malonyl-CoA sink pathway and source pathway and a screening tool for engineering more efficient biocatalyst are also presented in this review. We envision the genetically-encoded malonyl-CoA sensor will be an indispensable tool to optimize cell metab. and cost-competitively manuf. malonyl-CoA-derived compds.
- 27Arikawa, H. and Matsumoto, K. (2016) Evaluation of gene expression cassettes and production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) with a fine modulated monomer composition by using it in Cupriavidus necator. Microb. Cell Fact. 15, 184, DOI: 10.1186/s12934-016-0583-7Google Scholar27Evaluation of gene expression cassettes and production of poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) with a fine modulated monomer composition by using it in Cupriavidus necatorArikawa, Hisashi; Matsumoto, KeijiMicrobial Cell Factories (2016), 15 (), 184/1-184/11CODEN: MCFICT; ISSN:1475-2859. (BioMed Central Ltd.)Background:Cupriavidus necator has attracted much attention as a platform for the prodn. of polyhydroxyalkanoate (PHA) and other useful materials. Therefore, an appropriate modulation of gene expression is needed for producing the desired materials effectively. However, there is insufficient information on the genetic engineering techniques required for this in C. necator. Results: We found that the disruption of a potential ribosome binding site (RBS) in the phaC1 gene in C. necator caused a small decrease in the PhaC1 expression level. We applied this result to finely regulate the expression of other genes. Several gene expression cassettes were constructed by combining three Escherichia coli derived promoters (PlacUV5, Ptrc and Ptrp) to the potential RBS of phaC1 or its disruptant, resp. Furthermore, they were used to finely regulate the (R)-3-hydroxyhexanoate (3HHx) monomer ratio in the prodn. of poly[(R)-3-hydroxybutyrate-co-3-hydroxyhexanoate] (PHBHHx) via R-specific enoyl-CoA hydratases (PhaJs). The 3HHx compn. in PHBHHx is crucial because it defines the thermal and mech. properties of the resulting plastic material. Conclusions: We constructed and evaluated several gene expression cassettes consisting of promoters and RBSs that finely regulate transcription and translation. These were then applied to finely modulate the monomer compn. in the prodn. of PHBHHx by recombinant C. necator.
- 28Kutuzova, G. I., Frank, G. K., Makeev, V., Esipova, N. G., and Polozov, R. V. (1997) [Fourier analysis of nucleotide sequences. Periodicity in E. coli promoter sequences]. Biofizika 42 (2), 354– 62Google ScholarThere is no corresponding record for this reference.
- 29Gentz, R. and Bujard, H. (1985) Promoters recognized by Escherichia coli RNA polymerase selected by function: highly efficient promoters from bacteriophage T5. J. Bacteriol. 164, 70– 7Google Scholar29Promoters recognized by Escherichia coli RNA polymerase selected by function: highly efficient promoters from bacteriophage T5Gentz, Reiner; Bujard, HermannJournal of Bacteriology (1985), 164 (1), 70-7CODEN: JOBAAY; ISSN:0021-9193.Highly efficient promoters of coliphage T5 were identified by selecting the functional properties. Eleven such promoters belonging to all 3 expression classes of the phage were analyzed. Their av. AT content was 75% and reached 83% in subregions of the sequences. Besides the well-known conserved sequences around -10 and -33, they exhibited homologies outside the region commonly considered to be essential for promoter function. The consensus hexamers around -10 (TAT AAT) and -35 (TTG ACA) were never found simultaneously within the sequence of highly efficient promoters. Several of these promoters compete extremely well for E. coli RNA polymerase [9014-24-8] and can be used for the efficient in vitro synthesis of defined RNA species. In addn., some of these promoters accept 7-mGpppA as the starting dinucleotide, thus producing capped mRNA in vitro which can be utilized in various eukaryotic translation systems.
- 30Guo, Y., Dong, J., Zhou, T., Auxillos, J., Li, T., Zhang, W., Wang, L., Shen, Y., Luo, Y., Zheng, Y., Lin, J., Chen, G. Q., Wu, Q., Cai, Y., and Dai, J. (2015) YeastFab: the design and construction of standard biological parts for metabolic engineering in Saccharomyces cerevisiae. Nucleic Acids Res. 43 (13), e88, DOI: 10.1093/nar/gkv464Google Scholar30YeastFab: the design and construction of standard biological parts for metabolic engineering in Saccharomyces cerevisiaeGuo, Yakun; Dong, Junkai; Zhou, Tong; Auxillos, Jamie; Li, Tianyi; Zhang, Weimin; Wang, Lihui; Shen, Yue; Luo, Yisha; Zheng, Yijing; Lin, Jiwei; Chen, Guo-Qiang; Wu, Qingyu; Cai, Yizhi; Dai, JunbiaoNucleic Acids Research (2015), 43 (13), e88/1-e88/14CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)It is a routine task in metabolic engineering to introduce multicomponent pathways into a heterologous host for prodn. of metabolites. However, this process sometimes may take weeks to months due to the lack of standardized genetic tools. Here, we present a method for the design and construction of biol. parts based on the native genes and regulatory elements in Saccharomyces cerevisiae. We have developed highly efficient protocols (termed YeastFab Assembly) to synthesize these genetic elements as standardized biol. parts, which can be used to assemble transcriptional units in a single-tube reaction. In addn., standardized characterization assays are developed using reporter constructs to calibrate the function of promoters. Furthermore, the assembled transcription units can be either assayed individually or applied to construct multi-gene metabolic pathways, which targets a genomic locus or a receiving plasmid effectively, through a simple in vitro reaction. Finally, using β-carotene biosynthesis pathway as an example, we demonstrate that our method allows us not only to construct and test a metabolic pathway in several days, but also to optimize the prodn. through combinatorial assembly of a pathway using hundreds of regulatory biol. parts.
- 31Phelan, R. M., Sachs, D., Petkiewicz, S. J., Barajas, J. F., Blake-Hedges, J. M., Thompson, M. G., Reider Apel, A., Rasor, B. J., Katz, L., and Keasling, J. D. (2017) Development of Next Generation Synthetic Biology Tools for Use in Streptomyces venezuelae. ACS Synth. Biol. 6 (1), 159– 166, DOI: 10.1021/acssynbio.6b00202Google Scholar31Development of Next Generation Synthetic Biology Tools for Use in Streptomyces venezuelaePhelan, Ryan M.; Sachs, Daniel; Petkiewicz, Shayne J.; Barajas, Jesus F.; Blake-Hedges4, Jacquelyn M.; Thompson, Mitchell G.; Apel, Amanda Reider; Rasor, Blake J.; Katz, Leonard; Keasling, Jay D.ACS Synthetic Biology (2017), 6 (1), 159-166CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)Streptomyces have a rich history as producers of important natural products and this genus of bacteria has recently garnered attention for its potential applications in the broader context of synthetic biol. However, the dearth of genetic tools available to control and monitor protein prodn. precludes rapid and predictable metabolic engineering that is possible in hosts such as Escherichia coli or Saccharomyces cerevisiae. In an effort to improve genetic tools for Streptomyces venezuelae, we developed a suite of standardized, orthogonal integration vectors and an improved method to monitor protein prodn. in this host. These tools were applied to characterize heterologous promoters and various attB chromosomal integration sites. A final study leveraged the characterized toolset to demonstrate its use in producing the biofuel precursor bisabolene using a chromosomally integrated expression system. These tools advance S. venezuelae to be a practical host for future metabolic engineering efforts.
- 32Gherman, A., Wang, R., and Avramopoulos, D. (2009) Orientation, distance, regulation and function of neighbouring genes. Hum. Genomics 3 (2), 143– 156, DOI: 10.1186/1479-7364-3-2-143Google Scholar32Orientation, distance, regulation and function of neighboring genesGherman, Adrian; Wang, Ruihua; Avramopoulos, DimitriosHuman Genomics (2009), 3 (2), 143-156CODEN: HGUEAT; ISSN:1479-7364. (Henry Stewart Publications)The sequencing of the human genome has allowed us to observe globally and in detail the arrangement of genes along the chromosomes. There are multiple lines of evidence that this arrangement is not random, both in terms of intergenic distances and orientation of neighboring genes. We have undertaken a systematic evaluation of the spatial distribution and orientation of known genes across the human genome. We used genome-level information, including phylogenetic conservation, single nucleotide polymorphism d. and correlation of gene expression to assess the importance of this distribution. In addn. to confirming and extending known properties of the genome, such as the significance of gene deserts and the importance of "head to head" orientation of gene pairs in proximity, we provide significant new observations that include a smaller av. size for intervals sepg. the 3' ends of neighboring genes, a correlation of gene expression across tissues for genes as far as 100 kilobases apart and signatures of increasing pos. selection with decreasing interval size surprisingly relaxing for intervals smaller than ∼500 base pairs. Further, we provide extensive graphical representations of the genome-wide data to allow for observations and comparisons beyond what we address.
- 33Beck, C. F. and Warren, R. A. (1988) Divergent promoters, a common form of gene organization. Microbiol. Rev. 52 (3), 318– 26Google Scholar33Divergent promoters, a common form of gene organizationBeck, C. F.; Warren, R. A. J.Microbiological Reviews (1988), 52 (3), 318-26CODEN: MBRED3; ISSN:0146-0749.A review with 144 refs. of the characteristics and occurrence of divergent promoters, advantages of regulatory units of divergent transcription, transcription from divergent promoters, vectors for detection and anal. of divergent promoters, and potential uses for them.
- 34Schlegel, H. G., Kaltwasser, H., and Gottschalk, G. (1961) [A submersion method for culture of hydrogen-oxidizing bacteria: growth physiological studies]. Arch. Microbiol. 38, 209– 22, DOI: 10.1007/BF00422356Google ScholarThere is no corresponding record for this reference.
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Abstract
Figure 1
Figure 1. (A) Promoter definition used in this study. (B) High-throughput characterization of engineered promoters using a fluorescence-based assay (5′-UTR, 5′-untranslated region; bp, base pair; CamR, chloramphenicol resistance gene; Rep, replication gene; RFP, red fluorescent protein).
Figure 2
Figure 2. (A) Boundaries and architectures of the four parental promoters, PphaC1, PrrsC, Pj5, and Pg25, used in this study. (B) Comparison of the four parental promoters to an Escherichia coli σ70 promoter. (C) Activities of the four parental promoters.
Figure 3
Figure 3. (A) Architectures of parental promoters and their engineered variants. (B) Activities of parental promoters and their engineered variants.
Figure 4
Figure 4. (A) For gene pairs in HH arrangement, promoters that effect divergent transcription can be organized in three possible ways: back-to-back, overlapping, or face-to-face. (B) Composite promoters engineered using Pg25 as parental promoter. (C) Composite promoters engineered using PrrsC as parental promoter. (D) Composite promoters engineered using Pj5 as parental promoter. (E) Composite promoters with PphaC1 as secondary promoter. (F) Composite promoters with Pg25 as secondary promoter. (G) Composite promoters with Pj5[A1C1C2] as secondary promoter. In graphs E and F, red, blue and orange symbols represent primary promoter activity, composite promoter activity, and fold change, respectively.
Figure 5
Figure 5. (A) Hierarchical ranking of all 42 constitutive promoters reported in this study. (B) The range of promoter activity was expanded from 6-fold to 137-fold after applying combination of promoter engineering strategies (A = point mutation, B = length alteration, C = incorporation of regulatory genetic element, D = promoter hybridization and E = configuration alteration). Promoters derived from PphaC1, PrrsC, Pj5, and Pg25 were colored in blue, green, pink, and red, respectively. Promoters were categorized into five activity levels: Level 1 (with promoter activity between 0–2000 au), level 2 (2000–4000 au), level 3 (4000–6000 au), level 4 (6000–8000 au) and level 5 (>8000 au). Each promoter was benchmarked against PBAD promoter, induced using various concentrations of l-arabinose, from 0.001% (w/v) to 0.200% (w/v).
Figure 6
Figure 6. Relative promoter activity change upon application of promoter engineering strategies (A = point mutation, B = length alteration, C = incorporation of regulatory genetic element, D = promoter hybridization, and E = configuration alteration). Modifications that resulted in loss of promoter activity were indicated as red data points. Promoters with activities higher than PBAD promoter were indicated as blue data points.
Figure 7
Figure 7. Growth curves of C. necator H16 harboring either pBBR1MCS-1 (control; black line) or plasmids containing various engineered constitutive promoters [Pg25 (red line), PphaC1[B 1d] (blue line), PrrsC[E1D4] (brown line), Pg25[E3] (green line), Pg25[D1C2] (pink line), Pj5[E1A1C1C2] (orange line), and Pj5[E2C2] (purple line)].
Figure 8
Figure 8. Fluorescence of cell culture (black columns) and of spent medium (gray columns) of C. necator H16 harboring either pBBR1MCS-1 (control) or plasmids containing various engineered constitutive promoters (Pg25, PphaC1[B1d], PrrsC[E1D4], Pg25[E3], Pg25[D1C2], Pj5[E1A1C1C2], and Pj5[E2C2]).
References
This article references 34 other publications.
- 1Pohlmann, A., Fricke, W. F., Reinecke, F., Kusian, B., Liesegang, H., Cramm, R., Eitinger, T., Ewering, C., Pötter, M., Schwartz, E., Strittmatter, A., Voss, I., Gottschalk, G., Steinbüchel, A., Friedrich, B., and Bowien, B. (2006) Genome sequence of the Bioplastic-producing ″Knallgas″ bacterium Ralstonia eutropha H16. Nat. Biotechnol. 24 (10), 1257– 62, DOI: 10.1038/nbt1244There is no corresponding record for this reference.
- 2Volodina, E., Raberg, M., and Steinbüchel, A. (2016) Engineering the heterotrophic carbon sources utilization range of Ralstonia eutropha H16 for applications in biotechnology. Crit. Rev. Biotechnol. 36 (6), 978– 991, DOI: 10.3109/07388551.2015.10796982Engineering the heterotrophic carbon sources utilization range of Ralstonia eutropha H16 for applications in biotechnologyVolodina, Elena; Raberg, Matthias; Steinbuchel, AlexanderCritical Reviews in Biotechnology (2016), 36 (6), 978-991CODEN: CRBTE5; ISSN:0738-8551. (Taylor & Francis Ltd.)Ralstonia eutropha H16 is an interesting candidate for the biotechnol. prodn. of polyesters consisting of hydroxy- and mercaptoalkanoates, and other compds. It provides all the necessary characteristics, which are required for a biotechnol. prodn. strain. Due to its metabolic versatility, it can convert a broad range of renewable heterotrophic resources into diverse valuable compds. High cell d. fermns. of the non-pathogenic R. eutropha can be easily performed. Furthermore, this bacterium is accessible to engineering of its metab. by genetic approaches having available a large repertoire of genetic tools. Since the complete genome sequence of R. eutropha H16 has become available, a variety of transcriptome, proteome and metabolome studies provided valuable data elucidating its complex metab. and allowing a systematic biol. approach. However, high prodn. costs for bacterial large-scale prodn. of biomass and biotechnol. valuable products are still an economic challenge. The application of inexpensive raw materials could significantly reduce the expenses. Therefore, the conversion of diverse substrates to polyhydroxyalkanoates by R. eutropha was steadily improved by optimization of cultivation conditions, mutagenesis and metabolic engineering. Industrial byproducts and residual compds. like glycerol, and substrates contg. high carbon content per wt. like palm, soybean, corn oils as well as raw sugar-rich materials like molasses, starch and lignocellulose, are the most promising renewable substrates and were intensively studied.
- 3Jajesniak, P., Omar Ali, H. E. M., and Wong, T. S. (2014) Carbon dioxide capture and utilization using biological systems: Opportunities and challenges. J. Bioprocess. Biotech. 4, 155, DOI: 10.4172/2155-9821.1000155There is no corresponding record for this reference.
- 4Peplow, M. (2015) Industrial biotechs turn greenhouse gas into feedstock opportunity. Nat. Biotechnol. 33, 1123– 1125, DOI: 10.1038/nbt1115-1123There is no corresponding record for this reference.
- 5Brigham, C. J., Zhila, N., Shishatskaya, E., Volova, T. G., and Sinskey, A. J. (2012) Manipulation of Ralstonia eutropha carbon storage pathways to produce useful bio-based products. Subcell. Biochem. 64, 343– 66, DOI: 10.1007/978-94-007-5055-5_175Manipulation of Ralstonia eutropha carbon storage pathways to produce useful bio-based productsBrigham, Christopher J.; Zhila, Natalia; Shishatskaya, Ekaterina; Volova, Tatiana G.; Sinskey, Anthony J.Subcellular Biochemistry (2012), 64 (Reprogramming Microbial Metabolic Pathways), 343-366CODEN: SBCBAG; ISSN:0306-0225. (Springer)A review. Ralstonia eutropha is a Gram-neg. betaproteobacterium found natively in soils that can utilize a wide array of carbon sources for growth, and can store carbon intracellularly in the form of polyhydroxyalkanoate. Many aspects of R. eutropha make it a good candidate for use in biotechnol. prodn. of polyhydroxyalkanoate and other bio-based, value added compds. Manipulation of the organism's carbon flux is a cornerstone to success in developing it as a biotechnol. relevant organism. Here, we examine the methods of controlling and adapting the flow of carbon in R. eutropha metab. and the wide range of compds. that can be synthesized as a result. The presence of many different carbon utilization pathways and the custom genetic toolkit for manipulation of those pathways gives R. eutropha a versatility that allows it to be a biotechnol. important organism.
- 6Lutke-Eversloh, T. and Steinbüchel, A. (2003) Novel precursor substrates for polythioesters (PTE) and limits of PTE biosynthesis in Ralstonia eutropha. FEMS Microbiol. Lett. 221, 191– 196, DOI: 10.1016/S0378-1097(03)00185-XThere is no corresponding record for this reference.
- 7Steinbüchel, A. and Pieper, U. (1992) Production of a copolyester of 3-hydroxybutyric acid and 3-hydroxyvaleric acid from single unrelated carbon sources by a mutant of Alcaligenes eutrophus. Appl. Microbiol. Biotechnol. 37, 1– 6, DOI: 10.1007/BF00174193There is no corresponding record for this reference.
- 8Voss, I. and Steinbüchel, A. (2006) Application of a KDPG-aldolase gene-dependent addiction system for enhanced production of cyanophycin in Ralstonia eutropha strain H16. Metab. Eng. 8 (1), 66– 78, DOI: 10.1016/j.ymben.2005.09.0038Application of a KDPG-aldolase gene-dependent addiction system for enhanced production of cyanophycin in Ralstonia eutropha strain H16Voss, Ingo; Steinbuechel, AlexanderMetabolic Engineering (2006), 8 (1), 66-78CODEN: MEENFM; ISSN:1096-7176. (Elsevier)Two different recombinant plasmids both contg. the cyanophycin synthetase gene (cphA) of Synechocystis sp. strain PCC6308 but differing concerning the resistance marker gene were tested for their suitability to produce high amts. of cyanophycin in recombinant strains of Ralstonia eutropha. Various cultivation expts. at the 30-L scale revealed very low cyanophycin contents of the cells ranging from 4.6% to 6.2% (wt./wt.) of cellular dry wt. (CDW) only, most probably because most cells had lost the corresponding plasmid during cultivation. To establish a cost effective and high efficient system for prodn. of cyanophycin at larger scales using recombinant strains of R. eutropha, we applied two strategies: First, we integrated cphA into the dispensable chromosomal -lactate dehydrogenase gene (ldh) of R. eutropha. Depending on the cultivation conditions used, relatively low cyanophycin contents between 2.2% and 7.7% (wt./wt.) of CDW were reproducibly detected, which might be due to weak expression or low gene dosage in the single cphA copy strain of R. eutropha. In a second strategy we constructed a KDPG-aldolase gene (eda)-dependent addiction system, which combined features of a multi-copy plasmid with stabilized expression of cphA. Flasks expts. revealed that the cells accumulated extraordinarily high amts. of cyanophycin between 26.9% and 40.0% (wt./wt.) of CDW even under cultivation conditions lacking cyanophycin precursor substrates or plasmid stabilizing antibiotics. Cyanophycin contents of up to 40.0% (wt./wt.) of CDW were also obtained at a 30-L scale or a 500-L pilot-plant scale under such non-selective conditions. This demonstrates impressively that the stabilizing effect of the constructed eda-dependent addiction system can be used for prodn. of enhanced amts. of cyanophycin at a larger scale in recombinant strains of R. eutropha.
- 9Valentin, H. E., Zwingmann, G., Schönebaum, A., and Steinbüchel, A. (1995) Metabolic pathway for biosynthesis of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) from 4-hydroxybutyrate by Alcaligenes eutrophus. Eur. J. Biochem. 227 (1–2), 43– 60, DOI: 10.1111/j.1432-1033.1995.tb20358.x9Metabolic pathway for biosynthesis of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) from 4-hydroxybutyrate by Alcaligenes eutrophusValentin, Henry E.; Zwingmann, Gundula; Schoenebaum, Andreas; Steinbuechel, AlexanderEuropean Journal of Biochemistry (1995), 227 (1/2), 43-60CODEN: EJBCAI; ISSN:0014-2956. (Springer)Various aerobic gram-neg. bacteria have been examd. for their ability to use 4-hydroxybutyrate and 1,4-butanediol as carbon source for growth. Alcaligenes eutrophus strains H16, HF39, PHB-4 and Pseudomonas denitrificans 'Morris' were not able to grow with 1,4-butanediol or 4-hydroxybutyrate. From A. eutrophus HF39 spontaneous primary mutants (e.g. SK4040) were isolated which grew on 4-hydroxybutyrate with doubling times of approx. 3 h. Tn5::mob mutagenesis of mutant SK4040 led to the islation of two phenotypically different classes of secondary mutants which were affected in the utilization of 4-hydroxybutyrate. Mutants exhibiting the phenotype 4-hydroxybutyrate-neg. did not grow with 4-hydroxybutyrate, and mutants exhibiting the phenotype 4-hydroxybutyrate-leaky grew at a significantly lower rate with 4-hydroxybutyrate. Hybridization expts. led to the identification of a 10-kbp genomic EcoRI fragment of A. eutrophus SK4040, which was altered in mutants with the phenotype 4-hydroxybutyrate-neg., and of two 1-kbp and 4.5-kbp genomic EcoRI fragments, which were altered in mutants with the phenotype 4-hydroxybutyrate-leaky. This 10-kbp EcoRI fragment was cloned from A. eutrophus SK4040, and conjugative transfer of a pVDZ'2 hybrid plasmid to A. eutrophus H16 conferred the ability to grow with 4-hydroxybutyrate to the wild type. DNA-sequence anal. of this fragment, enzymic anal. of the wild type and of mutants of A. eutrophus as well as of recombinant strains of Escherichia coli led to the identification of a structural gene encoding for a 4-hydroxybutyrate dehydrogenase which was affected by transposon mutagenesis in five of six available 4-hydroxybutyrate-neg. mutants. Enzymic studies also provided evidence for the presence of an active succinate-semialdehyde dehydrogenase in 4-hydroxybutyrate-grown cells. This indicated that degrdn. of 4-hydroxybutyrate occurs via succinate semialdehyde and succinate and that the latter is degraded by the citric acid cycle. NMR studies of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) accumulated from 4-hydroxy [1-13C]butyrate or 4-hydroxy[2-13C]butyrate as substrate gave no evidence for a direct conversion of 4-hydroxybutyrate into 3-hydroxybutyrate and therefore supported the results of enzymic anal.
- 10Chen, J. S., Colon, B., Dusel, B., Ziesack, M., Way, J. C., and Torella, J. P. (2015) Production of fatty acids in Ralstonia eutropha H16 by engineering beta-oxidation and carbon storage. PeerJ 3, e1468, DOI: 10.7717/peerj.1468There is no corresponding record for this reference.
- 11Crépin, L., Lombard, E., and Guillouet, S. E. (2016) Metabolic engineering of Cupriavidus necator for heterotrophic and autotrophic alka(e)ne production. Metab. Eng. 37, 92– 101, DOI: 10.1016/j.ymben.2016.05.00211Metabolic engineering of Cupriavidus necator for heterotrophic and autotrophic alka(e)ne productionCrepin, Lucie; Lombard, Eric; Guillouet, Stephane E.Metabolic Engineering (2016), 37 (), 92-101CODEN: MEENFM; ISSN:1096-7176. (Elsevier B. V.)Alkanes of defined carbon chain lengths can serve as alternatives to petroleum-based fuels. Recently, microbial pathways of alkane biosynthesis have been identified and enabled the prodn. of alkanes in non-native producing microorganisms using metabolic engineering strategies. The chemoautotrophic bacterium Cupriavidus necator has great potential for producing chems. from CO2: it is known to have one of the highest growth rate among natural autotrophic bacteria and under nutrient imbalance it directs most of its carbon flux to the synthesis of the acetyl-CoA derived polymer, polyhydroxybutyrate (PHB), (up to 80% of intracellular content). Alkane synthesis pathway from Synechococcus elongatus (2 genes coding an acyl-ACP reductase and an aldehyde deformylating oxygenase) was heterologously expressed in a C. necator mutant strain deficient in the PHB synthesis pathway. Under heterotrophic condition on fructose we showed that under nitrogen limitation, in presence of an org. phase (decane), the strain produced up to 670 mg/L total hydrocarbons contg. 435 mg/l of alkanes consisting of 286 mg/l of pentadecane, 131 mg/l of heptadecene, 18 mg/l of heptadecane, and 236 mg/l of hexadecanal. We report here the highest level of alka(e)nes prodn. by an engineered C. necator to date. We also demonstrated the first reported alka(e)nes prodn. by a non-native alkane producer from CO2 as the sole carbon source.
- 12Marc, J., Grousseau, E., Lombard, E., Sinskey, A. J., Gorret, N., and Guillouet, S. E. (2017) Over expression of GroESL in Cupriavidus necator for heterotrophic and autotrophic isopropanol production. Metab. Eng. 42, 74– 84, DOI: 10.1016/j.ymben.2017.05.00712Over expression of GroESL in Cupriavidus necator for heterotrophic and autotrophic isopropanol productionMarc, Jillian; Grousseau, Estelle; Lombard, Eric; Sinskey, Anthony J.; Gorret, Nathalie; Guillouet, Stephane E.Metabolic Engineering (2017), 42 (), 74-84CODEN: MEENFM; ISSN:1096-7176. (Elsevier B.V.)We previously reported a metabolic engineering strategy to develop an isopropanol producing strain of Cupriavidus necator leading to prodn. of 3.4 g L-1 isopropanol. To reach higher titers, isopropanol toxicity to the cells has to be considered. A toxic effect of isopropanol on the growth of C. necator has been indeed obsd. above a crit. value of 15 g L-1. GroESL chaperones were first searched and identified in the genome of C. necator. Native groEL and groES genes from C. necator were over-expressed in a strain deleted for PHA synthesis. We demonstrated that over-expressing groESL genes led to a better tolerance of the strain towards exogenous isopropanol. GroESL genes were then over-expressed within the best engineered isopropanol producing strain. A final isopropanol concn. of 9.8 g L-1 was achieved in fed-batch culture on fructose as the sole carbon source (equiv. to 16 g L-1 after taking into account evapn.). Cell viability was slightly improved by the chaperone over-expression, particularly at the end of the fermn. when the isopropanol concn. was the highest. Moreover, the strain over-expressing the chaperones showed higher enzyme activity levels of the 2 heterologous enzymes (acetoacetate carboxylase and alc. dehydrogenase) of the isopropanol synthetic operon, translating to a higher specific prodn. rate of isopropanol at the expense of the specific prodn. rate of acetone. Over-expressing the native chaperones led to a 9-18% increase in the isopropanol yield on fructose.
- 13Müller, J., MacEachran, D., Burd, H., Sathitsuksanoh, N., Bi, C., Yeh, Y. C., Lee, T. S., Hillson, N. J., Chhabra, S. R., Singer, S. W., and Beller, H. R. (2013) Engineering of Ralstonia eutropha H16 for autotrophic and heterotrophic production of methyl ketones. Appl. Environ. Microbiol. 79 (14), 4433– 9, DOI: 10.1128/AEM.00973-13There is no corresponding record for this reference.
- 14Lu, J., Brigham, C. J., Gai, C. S., and Sinskey, A. J. (2012) Studies on the production of branched-chain alcohols in engineered Ralstonia eutropha. Appl. Microbiol. Biotechnol. 96 (1), 283– 97, DOI: 10.1007/s00253-012-4320-914Studies on the production of branched-chain alcohols in engineered Ralstonia eutrophaLu, Jingnan; Brigham, Christopher J.; Gai, Claudia S.; Sinskey, Anthony J.Applied Microbiology and Biotechnology (2012), 96 (1), 283-297CODEN: AMBIDG; ISSN:0175-7598. (Springer)Wild-type Ralstonia eutropha H16 produces polyhydroxybutyrate (PHB) as an intracellular carbon storage material during nutrient stress in the presence of excess carbon. In this study, the excess carbon was redirected in engineered strains from PHB storage to the prodn. of isobutanol and 3-methyl-1-butanol (branched-chain higher alcs.). These branched-chain higher alcs. can directly substitute for fossil-based fuels and be employed within the current infrastructure. Various mutant strains of R. eutropha with isobutyraldehyde dehydrogenase activity, in combination with the overexpression of plasmid-borne, native branched-chain amino acid biosynthesis pathway genes and the overexpression of heterologous ketoisovalerate decarboxylase gene, were employed for the biosynthesis of isobutanol and 3-methyl-1-butanol. Prodn. of these branched-chain alcs. was initiated during nitrogen or phosphorus limitation in the engineered R. eutropha. One mutant strain not only produced over 180 mg/L branched-chain alcs. in flask culture, but also was significantly more tolerant of isobutanol toxicity than wild-type R. eutropha. After the elimination of genes encoding three potential carbon sinks (ilvE, bkdAB, and aceE), the prodn. titer improved to 270 mg/L isobutanol and 40 mg/L 3-methyl-1-butanol. Semicontinuous flask cultivation was utilized to minimize the toxicity caused by isobutanol while supplying cells with sufficient nutrients. Under this semicontinuous flask cultivation, the R. eutropha mutant grew and produced more than 14 g/L branched-chain alcs. over the duration of 50 days. These results demonstrate that R. eutropha carbon flux can be redirected from PHB to branched-chain alcs. and that engineered R. eutropha can be cultivated over prolonged periods of time for product biosynthesis.
- 15Lütte, S., Pohlmann, A., Zaychikov, E., Schwartz, E., Becher, J. R., Heumann, H., and Friedrich, B. (2012) Autotrophic production of stable-isotope-labeled arginine in Ralstonia eutropha strain H16. Appl. Environ. Microbiol. 78 (22), 7884– 90, DOI: 10.1128/AEM.01972-12There is no corresponding record for this reference.
- 16Park, J. M., Jang, Y. S., Kim, T. Y., and Lee, S. Y. (2010) Development of a gene knockout system for Ralstonia eutropha H16 based on the broad-host-range vector expressing a mobile group II intron. FEMS Microbiol. Lett. 309, 193– 200, DOI: 10.1111/j.1574-6968.2010.02041.xThere is no corresponding record for this reference.
- 17Gruber, S., Hagen, J., Schwab, H., and Koefinger, P. (2014) Versatile and stable vectors for efficient gene expression in Ralstonia eutropha H16. J. Biotechnol. 186, 74– 82, DOI: 10.1016/j.jbiotec.2014.06.03017Versatile and stable vectors for efficient gene expression in Ralstonia eutropha H16Gruber, Steffen; Hagen, Jeremias; Schwab, Helmut; Koefinger, PetraJournal of Biotechnology (2014), 186 (), 74-82CODEN: JBITD4; ISSN:0168-1656. (Elsevier B.V.)The Gram-neg. β-proteobacterium Ralstonia eutropha H16 is primarily known for polyhydroxybutyrate (PHB) prodn. and its ability to grow chemolithoautotrophically by using CO2 and H2 as sole carbon and energy sources. The majority of metabolic engineering and heterologous expression studies conducted so far rely on a small no. of suitable expression systems. Particularly the plasmid based expression systems already developed for the use in R. eutropha H16 suffer from high segregational instability and plasmid loss after a short time of fermn. In order to develop efficient and highly stable plasmid expression vectors for the use in R. eutropha H16, a new plasmid design was created including the RP4 partitioning system, as well as various promoters and origins of replication. The application of minireplicons derived from broad-host-range plasmids RSF1010, pBBR1, RP4 and pSa for the construction of expression vectors and the use of numerous, versatile promoters extend the range of feasible expression levels considerably. In particular, the use of promoters derived from the bacteriophage T5 was described for the first time in this work, characterizing the j5 promoter as the strongest promoter yet to be applied in R. eutropha H16. Moreover, the implementation of the RP4 partition sequence in plasmid design increased plasmid stability significantly and enables fermns. with marginal plasmid loss of recombinant R. eutropha H16 for at least 96 h. The utility of the new vector family in R. eutropha H16 is demonstrated by providing expression data with different model proteins and consequently further raises the value of this organism as cell factory for biotechnol. applications including protein and metabolite prodn.
- 18Raberg, M., Heinrich, D., and Steinbüchel, A. (2015) Analysis of PHB Metabolism Applying Tn5Mutagenesis in Ralstonia eutropha. In Hydrocarbon and Lipid Microbiology Protocols; Springer Protocols Handbooks (McGenity, T., Timmis, K., and Nogales, B., Eds.) pp 120– 148, Springer, Berlin, Heidelberg.There is no corresponding record for this reference.
- 19Tee, K. L., Grinham, J., Othusitse, A. M., Gonzalez-Villanueva, M., Johnson, A. O., and Wong, T. S. (2017) An Efficient Transformation Method for the Bioplastic-Producing ″Knallgas″ Bacterium Ralstonia eutropha H16. Biotechnol. J. 12, 1700081, DOI: 10.1002/biot.201700081There is no corresponding record for this reference.
- 20Bi, C., Su, P., Müller, J., Yeh, Y. C., Chhabra, S. R., Beller, H. R., Singer, S. W., and Hillson, N. J. (2013) Development of a broad-host synthetic biology toolbox for Ralstonia eutropha and its application to engineering hydrocarbon biofuel production. Microb. Cell Fact. 12, 107, DOI: 10.1186/1475-2859-12-10720Development of a broad-host synthetic biology toolbox for Ralstonia eutropha and its application to engineering hydrocarbon biofuel productionBi, Changhao; Su, Peter; Muller, Jana; Yeh, Yi-Chun; Chhabra, Swapnil R.; Beller, Harry R.; Singer, Steven W.; Hilson, Nathan J.Microbial Cell Factories (2013), 12 (), 107/1-107/10, 10 pp.CODEN: MCFICT; ISSN:1475-2859. (BioMed Central Ltd.)Background: The chemoautotrophic bacterium Ralstonia eutropha can utilize H2/CO2 for growth under aerobic conditions. While this microbial host has great potential to be engineered to produce desired compds. (beyond polyhydroxybutyrate) directly from CO2, little work has been done to develop genetic part libraries to enable such endeavors. Results: We report the development of a toolbox for the metabolic engineering of Ralstonia eutropha H16. We have constructed a set of broad-host-range plasmids bearing a variety of origins of replication, promoters, 5' mRNA stem-loop structures, and ribosomal binding sites. Specifically, we analyzed the origins of replication pCM62 (IncP), pBBR1, pKT (IncQ), and their variants. We tested the promoters PBAD, T7, Pxyls/PM, PlacUV5, and variants thereof for inducible expression. We also evaluated a T7 mRNA stem-loop structure sequence and compared a set of ribosomal binding site (RBS) sequences derived from Escherichia coli, R. eutropha, and a computational RBS design tool. Finally, we employed the toolbox to optimize hydrocarbon prodn. in R. eutropha and demonstrated a 6-fold titer improvement using the appropriate combination of parts. Conclusion: We constructed and evaluated a versatile synthetic biol. toolbox for Ralstonia eutropha metabolic engineering that could apply to other microbial hosts as well.
- 21Fukui, T., Ohsawa, K., Mifune, J., Orita, I., and Nakamura, S. (2011) Evaluation of promoters for gene expression in polyhydroxyalkanoate-producing Cupriavidus necator H16. Appl. Microbiol. Biotechnol. 89 (5), 1527– 36, DOI: 10.1007/s00253-011-3100-2There is no corresponding record for this reference.
- 22Guzman, L. M., Belin, D., Carson, M. J., and Beckwith, J. (1995) Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J. Bacteriol. 177 (14), 4121– 30, DOI: 10.1128/jb.177.14.4121-4130.199522Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoterGuzman, Luz-Maria; Belin, Dominique; Carson, Michael J.; Beckwith, JonJournal of Bacteriology (1995), 177 (14), 4121-30CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)We have constructed a series of plasmid vectors (pBAD vectors) contg. the PBAD promoter of the araBAD (arabinose) operon and the gene encoding the pos. and neg. regulator of this promoter, araC. Using the phoA gene and phoA fusions to monitor expression in these vectors, we show that the ratio of induction/repression can be 1,200-fold, compared with 50-fold for PTAC-based vectors. PhoA expression can be modulated over a wide range of inducer (arabinose) concns. and reduced to extremely low levels by the presence of glucose, which represses expression. Also, the kinetics of induction and repression are very rapid and significantly affected by the ara allele in the host strain. Thus, the use of this system which can be efficiently and rapidly turned on and off allows the study of important aspects of bacterial physiol. in a very simple manner and without changes of temp. We have exploited the tight regulation of the PBAD promoter to study the phenotypes of null mutations of essential genes and explored the use of pBAD vectors as an expression system.
- 23Li, H. and Liao, J. C. (2015) A synthetic anhydrotetracycline-controllable gene expression system in Ralstonia eutropha H16. ACS Synth. Biol. 4 (2), 101– 6, DOI: 10.1021/sb400118923A synthetic anhydrotetracycline-controllable gene expression system in Ralstonia eutropha H16Li, Han; Liao, James C.ACS Synthetic Biology (2015), 4 (2), 101-106CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)Controllable gene expression systems that are orthogonal to the host's native gene regulation network are invaluable tools for synthetic biol. In Ralstonia eutropha H16, such systems are extremely limited despite the importance of this organism in microbiol. research and biotechnol. application. Here the authors develop an anhydrotetracycline (aTc)-inducible gene expression system, which is composed of a synthetic promoter contg. the operator tetO, the repressor TetR, and the inducer aTc. Using a reporter-activity based promoter library screen, the authors first identified the active hybrids between the tetO operators and the R. eutropha native rrsC promoter (PrrsC). Next, the authors showed that the hybrid promoters are repressable by TetR. To optimize the dynamic range of the system, a high-throughput screening of 300 mutants of R. eutropha phaC1 promoter was conducted to identify suitable promoters to tune the tetR expression level. The final controllable expression system contains the modified PrrsC with two copies of the tetO1 operator integrated and the tetR driven by the mutated PphaC1. The system has decreased basal expression level and can be tuned by different aTc concns. with greater than 10-fold dynamic range. The system was used to alleviate cellular toxicity caused by AlsS overexpression, which impeded the authors' metabolic engineering work on isobutanol and 3-methyl-1-butanol prodn. in R. eutropha H16.
- 24Hanko, E. K. R., Minton, N. P., and Malys, N. (2017) Characterisation of a 3-hydroxypropionic acid-inducible system from Pseudomonas putida for orthogonal gene expression control in Escherichia coli and Cupriavidus necator. Sci. Rep. 7, 1724, DOI: 10.1038/s41598-017-01850-w24Characterisation of a 3-hydroxypropionic acid-inducible system from Pseudomonas putida for orthogonal gene expression control in Escherichia coli and Cupriavidus necatorHanko Erik K R; Minton Nigel P; Malys NaglisScientific reports (2017), 7 (1), 1724 ISSN:.3-hydroxypropionic acid (3-HP) is an important platform chemical used as a precursor for production of added-value compounds such as acrylic acid. Metabolically engineered yeast, Escherichia coli, cyanobacteria and other microorganisms have been developed for the biosynthesis of 3-HP. Attempts to overproduce this compound in recombinant Pseudomonas denitrificans revealed that 3-HP is consumed by this microorganism using the catabolic enzymes encoded by genes hpdH, hbdH and mmsA. 3-HP-inducible systems controlling the expression of these genes have been predicted in proteobacteria and actinobacteria. In this study, we identify and characterise 3-HP-inducible promoters and their corresponding LysR-type transcriptional regulators from Pseudomonas putida KT2440. A newly-developed modular reporter system proved possible to demonstrate that PpMmsR/P mmsA and PpHpdR/P hpdH are orthogonal and highly inducible by 3-HP in E. coli (12.3- and 23.3-fold, respectively) and Cupriavidus necator (51.5- and 516.6-fold, respectively). Bioinformatics and mutagenesis analyses revealed a conserved 40-nucleotide sequence in the hpdH promoter, which plays a key role in HpdR-mediated transcription activation. We investigate the kinetics and dynamics of the PpHpdR/P hpdH switchable system in response to 3-HP and show that it is also induced by both enantiomers of 3-hydroxybutyrate. These findings pave the way for use of the 3-HP-inducible system in synthetic biology and biotechnology applications.
- 25Gruber, S., Schwendenwein, D., Magomedova, Z., Thaler, E., Hagen, J., Schwab, H., and Heidinger, P. (2016) Design of inducible expression vectors for improved protein production in Ralstonia eutropha H16 derived host strains. J. Biotechnol. 235, 92– 9, DOI: 10.1016/j.jbiotec.2016.04.02625Design of inducible expression vectors for improved protein production in Ralstonia eutropha H16 derived host strainsGruber, Steffen; Schwendenwein, Daniel; Magomedova, Zalina; Thaler, Eva; Hagen, Jeremias; Schwab, Helmut; Heidinger, PetraJournal of Biotechnology (2016), 235 (), 92-99CODEN: JBITD4; ISSN:0168-1656. (Elsevier B.V.)A review. Ralstonia eutropha H16 (Cupriavidus necator H16) is a Gram-neg., facultative chemolithoautotrophic bacterium which can use H2 and CO2 as sole energy and carbon sources in the absence of org. substrates. The biotechnol. use of R. eutropha H16 on an industrial scale has already been established; however, only a small no. of tools promoting inducible gene expression is available. Within this study two systems promoting inducible expression were designed on the basis of the strong j5 promoter and the Escherichia coli lacI or the Pseudomonas putida cumate regulatory elements. Both expression vectors display desired regulatory features and further increase the no. of suitable inducible expression systems for the prodn. of metabolites and proteins with R. eutropha H16.
- 26Johnson, A. O., Gonzalez-Villanueva, M., Wong, L., Steinbüchel, A., Tee, K. L., Xu, P., and Wong, T. S. (2017) Design and application of genetically-encoded malonyl-CoA biosensors for metabolic engineering of microbial cell factories. Metab. Eng. 44, 253– 264, DOI: 10.1016/j.ymben.2017.10.01126Design and application of genetically-encoded malonyl-CoA biosensors for metabolic engineering of microbial cell factoriesJohnson, Abayomi Oluwanbe; Gonzalez-Villanueva, Miriam; Wong, Lynn; Steinbuchel, Alexander; Tee, Kang Lan; Xu, Peng; Wong, Tuck SengMetabolic Engineering (2017), 44 (), 253-264CODEN: MEENFM; ISSN:1096-7176. (Elsevier B.V.)A review. Malonyl-CoA is the basic building block for synthesizing a range of important compds. including fatty acids, phenylpropanoids, flavonoids and non-ribosomal polyketides. Centering around malonyl-CoA, we summarized here the various metabolic engineering strategies employed recently to regulate and control malonyl-CoA metab. and improve cellular productivity. Effective metabolic engineering of microorganisms requires the introduction of heterologous pathways and dynamically rerouting metabolic flux towards products of interest. Transcriptional factor-based biosensors translate an internal cellular signal to a transcriptional output and drive the expression of the designed genetic/biomol. circuits to compensate the activity loss of the engineered biosystem. Recent development of genetically-encoded malonyl-CoA sensor has stood out as a classical example to dynamically reprogram cell metab. for various biotechnol. applications. Here, we reviewed the design principles of constructing a transcriptional factor-based malonyl-CoA sensor with superior detection limit, high sensitivity and broad dynamic range. We discussed various synthetic biol. strategies to remove pathway bottleneck and how genetically-encoded metabolite sensor could be deployed to improve pathway efficiency. Particularly, we emphasized that integration of malonyl-CoA sensing capability with biocatalytic function would be crit. to engineer efficient microbial cell factory. Biosensors have also advanced beyond its classical function of a sensor actuator for in situ monitoring of intracellular metabolite concn. Applications of malonyl-CoA biosensors as a sensor-inventor for neg. feedback regulation of metabolic flux, a metabolic switch for oscillatory balancing of malonyl-CoA sink pathway and source pathway and a screening tool for engineering more efficient biocatalyst are also presented in this review. We envision the genetically-encoded malonyl-CoA sensor will be an indispensable tool to optimize cell metab. and cost-competitively manuf. malonyl-CoA-derived compds.
- 27Arikawa, H. and Matsumoto, K. (2016) Evaluation of gene expression cassettes and production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) with a fine modulated monomer composition by using it in Cupriavidus necator. Microb. Cell Fact. 15, 184, DOI: 10.1186/s12934-016-0583-727Evaluation of gene expression cassettes and production of poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) with a fine modulated monomer composition by using it in Cupriavidus necatorArikawa, Hisashi; Matsumoto, KeijiMicrobial Cell Factories (2016), 15 (), 184/1-184/11CODEN: MCFICT; ISSN:1475-2859. (BioMed Central Ltd.)Background:Cupriavidus necator has attracted much attention as a platform for the prodn. of polyhydroxyalkanoate (PHA) and other useful materials. Therefore, an appropriate modulation of gene expression is needed for producing the desired materials effectively. However, there is insufficient information on the genetic engineering techniques required for this in C. necator. Results: We found that the disruption of a potential ribosome binding site (RBS) in the phaC1 gene in C. necator caused a small decrease in the PhaC1 expression level. We applied this result to finely regulate the expression of other genes. Several gene expression cassettes were constructed by combining three Escherichia coli derived promoters (PlacUV5, Ptrc and Ptrp) to the potential RBS of phaC1 or its disruptant, resp. Furthermore, they were used to finely regulate the (R)-3-hydroxyhexanoate (3HHx) monomer ratio in the prodn. of poly[(R)-3-hydroxybutyrate-co-3-hydroxyhexanoate] (PHBHHx) via R-specific enoyl-CoA hydratases (PhaJs). The 3HHx compn. in PHBHHx is crucial because it defines the thermal and mech. properties of the resulting plastic material. Conclusions: We constructed and evaluated several gene expression cassettes consisting of promoters and RBSs that finely regulate transcription and translation. These were then applied to finely modulate the monomer compn. in the prodn. of PHBHHx by recombinant C. necator.
- 28Kutuzova, G. I., Frank, G. K., Makeev, V., Esipova, N. G., and Polozov, R. V. (1997) [Fourier analysis of nucleotide sequences. Periodicity in E. coli promoter sequences]. Biofizika 42 (2), 354– 62There is no corresponding record for this reference.
- 29Gentz, R. and Bujard, H. (1985) Promoters recognized by Escherichia coli RNA polymerase selected by function: highly efficient promoters from bacteriophage T5. J. Bacteriol. 164, 70– 729Promoters recognized by Escherichia coli RNA polymerase selected by function: highly efficient promoters from bacteriophage T5Gentz, Reiner; Bujard, HermannJournal of Bacteriology (1985), 164 (1), 70-7CODEN: JOBAAY; ISSN:0021-9193.Highly efficient promoters of coliphage T5 were identified by selecting the functional properties. Eleven such promoters belonging to all 3 expression classes of the phage were analyzed. Their av. AT content was 75% and reached 83% in subregions of the sequences. Besides the well-known conserved sequences around -10 and -33, they exhibited homologies outside the region commonly considered to be essential for promoter function. The consensus hexamers around -10 (TAT AAT) and -35 (TTG ACA) were never found simultaneously within the sequence of highly efficient promoters. Several of these promoters compete extremely well for E. coli RNA polymerase [9014-24-8] and can be used for the efficient in vitro synthesis of defined RNA species. In addn., some of these promoters accept 7-mGpppA as the starting dinucleotide, thus producing capped mRNA in vitro which can be utilized in various eukaryotic translation systems.
- 30Guo, Y., Dong, J., Zhou, T., Auxillos, J., Li, T., Zhang, W., Wang, L., Shen, Y., Luo, Y., Zheng, Y., Lin, J., Chen, G. Q., Wu, Q., Cai, Y., and Dai, J. (2015) YeastFab: the design and construction of standard biological parts for metabolic engineering in Saccharomyces cerevisiae. Nucleic Acids Res. 43 (13), e88, DOI: 10.1093/nar/gkv46430YeastFab: the design and construction of standard biological parts for metabolic engineering in Saccharomyces cerevisiaeGuo, Yakun; Dong, Junkai; Zhou, Tong; Auxillos, Jamie; Li, Tianyi; Zhang, Weimin; Wang, Lihui; Shen, Yue; Luo, Yisha; Zheng, Yijing; Lin, Jiwei; Chen, Guo-Qiang; Wu, Qingyu; Cai, Yizhi; Dai, JunbiaoNucleic Acids Research (2015), 43 (13), e88/1-e88/14CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)It is a routine task in metabolic engineering to introduce multicomponent pathways into a heterologous host for prodn. of metabolites. However, this process sometimes may take weeks to months due to the lack of standardized genetic tools. Here, we present a method for the design and construction of biol. parts based on the native genes and regulatory elements in Saccharomyces cerevisiae. We have developed highly efficient protocols (termed YeastFab Assembly) to synthesize these genetic elements as standardized biol. parts, which can be used to assemble transcriptional units in a single-tube reaction. In addn., standardized characterization assays are developed using reporter constructs to calibrate the function of promoters. Furthermore, the assembled transcription units can be either assayed individually or applied to construct multi-gene metabolic pathways, which targets a genomic locus or a receiving plasmid effectively, through a simple in vitro reaction. Finally, using β-carotene biosynthesis pathway as an example, we demonstrate that our method allows us not only to construct and test a metabolic pathway in several days, but also to optimize the prodn. through combinatorial assembly of a pathway using hundreds of regulatory biol. parts.
- 31Phelan, R. M., Sachs, D., Petkiewicz, S. J., Barajas, J. F., Blake-Hedges, J. M., Thompson, M. G., Reider Apel, A., Rasor, B. J., Katz, L., and Keasling, J. D. (2017) Development of Next Generation Synthetic Biology Tools for Use in Streptomyces venezuelae. ACS Synth. Biol. 6 (1), 159– 166, DOI: 10.1021/acssynbio.6b0020231Development of Next Generation Synthetic Biology Tools for Use in Streptomyces venezuelaePhelan, Ryan M.; Sachs, Daniel; Petkiewicz, Shayne J.; Barajas, Jesus F.; Blake-Hedges4, Jacquelyn M.; Thompson, Mitchell G.; Apel, Amanda Reider; Rasor, Blake J.; Katz, Leonard; Keasling, Jay D.ACS Synthetic Biology (2017), 6 (1), 159-166CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)Streptomyces have a rich history as producers of important natural products and this genus of bacteria has recently garnered attention for its potential applications in the broader context of synthetic biol. However, the dearth of genetic tools available to control and monitor protein prodn. precludes rapid and predictable metabolic engineering that is possible in hosts such as Escherichia coli or Saccharomyces cerevisiae. In an effort to improve genetic tools for Streptomyces venezuelae, we developed a suite of standardized, orthogonal integration vectors and an improved method to monitor protein prodn. in this host. These tools were applied to characterize heterologous promoters and various attB chromosomal integration sites. A final study leveraged the characterized toolset to demonstrate its use in producing the biofuel precursor bisabolene using a chromosomally integrated expression system. These tools advance S. venezuelae to be a practical host for future metabolic engineering efforts.
- 32Gherman, A., Wang, R., and Avramopoulos, D. (2009) Orientation, distance, regulation and function of neighbouring genes. Hum. Genomics 3 (2), 143– 156, DOI: 10.1186/1479-7364-3-2-14332Orientation, distance, regulation and function of neighboring genesGherman, Adrian; Wang, Ruihua; Avramopoulos, DimitriosHuman Genomics (2009), 3 (2), 143-156CODEN: HGUEAT; ISSN:1479-7364. (Henry Stewart Publications)The sequencing of the human genome has allowed us to observe globally and in detail the arrangement of genes along the chromosomes. There are multiple lines of evidence that this arrangement is not random, both in terms of intergenic distances and orientation of neighboring genes. We have undertaken a systematic evaluation of the spatial distribution and orientation of known genes across the human genome. We used genome-level information, including phylogenetic conservation, single nucleotide polymorphism d. and correlation of gene expression to assess the importance of this distribution. In addn. to confirming and extending known properties of the genome, such as the significance of gene deserts and the importance of "head to head" orientation of gene pairs in proximity, we provide significant new observations that include a smaller av. size for intervals sepg. the 3' ends of neighboring genes, a correlation of gene expression across tissues for genes as far as 100 kilobases apart and signatures of increasing pos. selection with decreasing interval size surprisingly relaxing for intervals smaller than ∼500 base pairs. Further, we provide extensive graphical representations of the genome-wide data to allow for observations and comparisons beyond what we address.
- 33Beck, C. F. and Warren, R. A. (1988) Divergent promoters, a common form of gene organization. Microbiol. Rev. 52 (3), 318– 2633Divergent promoters, a common form of gene organizationBeck, C. F.; Warren, R. A. J.Microbiological Reviews (1988), 52 (3), 318-26CODEN: MBRED3; ISSN:0146-0749.A review with 144 refs. of the characteristics and occurrence of divergent promoters, advantages of regulatory units of divergent transcription, transcription from divergent promoters, vectors for detection and anal. of divergent promoters, and potential uses for them.
- 34Schlegel, H. G., Kaltwasser, H., and Gottschalk, G. (1961) [A submersion method for culture of hydrogen-oxidizing bacteria: growth physiological studies]. Arch. Microbiol. 38, 209– 22, DOI: 10.1007/BF00422356There is no corresponding record for this reference.
Supporting Information
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssynbio.8b00136.
Experimental procedures; plasmid map of pBBR1c-RFP; alignment of Pj5 and Pg25 promoters used in this study and in Gruber et al. (2014); graphical representation of rational promoter engineering strategies; l-arabinose-dose dependent induction of PBAD promoter; curve fitting of cell culture fluorescence vs time data; fluorescence of cell culture normalized by OD600 value; promoter sequences; subcategories of each promoter engineering strategy; promoter activity table; specific growth rates of C. necator H16 carrying plasmids containing various promoters (PDF)
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