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One-Pot Sustainable Enzymatic Cascade for Production of High-Value Succinyl-CoA and 5-Aminolevulinic Acid
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ACS Sustainable Chemistry & Engineering

Cite this: ACS Sustainable Chem. Eng. 2025, 13, 9, 3406–3412
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https://doi.org/10.1021/acssuschemeng.5c00001
Published February 25, 2025

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Abstract

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Succinyl-CoA, a vital metabolic intermediate, serves as a key cofactor in numerous biosynthetic pathways, including the synthesis of 5-aminolevulinic acid (5-ALA), which is extensively used in agriculture to improve crop growth and stress tolerance as well as in medicine for photodynamic therapy. This study demonstrates a sustainable biocatalytic approach for 5-ALA production from CoA using an in vitro enzymatic cascade relying on feedstocks of α-ketoglutarate and glycine. The green synthesis pathway integrates the 2-oxoglutarate dehydrogenase complex (SucA and SucB) with ALA synthase fromRhodobacter capsulatusin an atom-economical process. By coexpressing GroELS chaperone in the BD7G strain, we enhanced enzyme solubility over 90% and achieved 1.155 g of protein per liter. Process intensification through enzyme ratio optimization revealed that a 1:2 ratio of SucA to SucB maximized succinyl-CoA production of 0.5 mM, with NAD+ regeneration by a flavin reductase (FRE) and catalase (CAT) system under mild conditions. The integrated five-enzyme cascade achieved complete substrate conversion from α-ketoglutarate with 1 mM 5-ALA titer in one-pot. Notably, the process incorporates efficient CO2 capture strategies, with biocatalytic sequestration using carbonic anhydrase demonstrating superior sustainability metrics compared with chemical methods. This environmentally conscious enzymatic platform provides a novel strategy to produce high-value succinyl-CoA and an alternative to 5-ALA production.

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Synopsis

One-pot enzymatic cascade reaction for succinyl-CoA and 5-ALA synthesis toward low-carbon-footprint emission via using carbonic anhydrase.

Introduction

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Succinyl-CoA plays a crucial role in cellular metabolism, serving as an important intermediate in the tricarboxylic acid (TCA) cycle, a central pathway in energy production. The synthesis of succinyl-CoA is vital not only for generating ATP, the primary energy currency of the cell, but also for the synthesis of heme, an essential component of hemoglobin and certain enzymes. (1,2) The intrinsic chemical reactivity and thermodynamic instability of succinyl-CoA render it unsuitable for prolonged storage or industrial-scale production. Therefore, microbial fermentation presents a feasible method for succinyl-CoA biosynthesis. Through the heterologous expression of key enzymatic components in the TCA cycle, specifically the 2-oxoglutarate dehydrogenase complex and succinyl-CoA synthetase, intracellular accumulation of succinyl-CoA can be achieved during fermentation. (3,4) However, downstream processing remains limited by the molecule’s intrinsic instability, necessitating expedited isolation protocols and potential integration with 5-aminolevulinic acid (5-ALA) biosynthesis as a value-added coproduct to enhance economic feasibility.
5-ALA is a pivotal intermediate in the biosynthesis of tetrapyrroles, serving as a precursor to essential molecules, such as heme, chlorophyll, and vitamin B12. (5) Such molecules are fundamental to key biological processes, including oxygen transport, photosynthesis, and electron transfer. In humans, 5-ALA is critical for cellular respiration as a precursor to heme, (6) while in plants, it is vital for energy production and serves as a plant growth regulator through chlorophyll-driven photosynthesis. (7) Recently, 5-ALA has been used as a photosensitizer in photodynamic therapy (PDT) for cancer treatment. (8) The rising demand for 5-ALA, driven by its diverse applications, is reflected in the growing global market, which was valued at approximately USD 167.2 million in 2023 and is projected to reach USD 321.7 million by 2030. (9)
Conventional 5-ALA synthesis relies on the complex chemical pathway using precursors like levulinic acid and furfurylamine, (10) leading to low yields and high costs. (11) An alternative approach uses microbial fermentation, which originates from precursors in the TCA cycle via C4 and C5 metabolic pathways. The C4 pathway relies on ALA synthase-mediated condensation of succinyl-CoA and glycine, while the C5 pathway involves three consecutive enzymatic reactions, catalyzed by glutamyl-tRNA synthetase, glutamyl-tRNA reductase, and glutamate-1-semialdehyde aminotransferase (encoded by gltX, hemA, and hemL), converted from α-ketoglutarate to 5-ALA. (5,12)Escherichia coliandCorynebacterium glutamicumare the most prevalent microbial hosts to produce 5-ALA using C4 and C5 pathways. (13,14) However, the key bottleneck of 5-ALA production is fine-tuning the metabolic burden between key enzyme production and cell tolerance to 5-ALA.
In vitro synthesis offers notable advantages by isolating essential enzymatic steps for 5-ALA production, eliminating cellular metabolic constraints, and enabling high-percentage-yield synthesis with reduced waste. (15,16) Unlike in vivo production, where metabolic competition and regulatory constraints limit yields, cell-free enzymatic synthesis directs all resources toward the target reaction, significantly improving efficiency. Cell-free enzymatic systems allow precise control over reaction parameters, (17) preventing metabolic competition that typically reduces yields in fermentation systems. (18) This approach eliminates cell growth requirements, reducing production costs and waste, while adhering to green chemistry principles. The flexibility in pathway design enables optimization through various enzyme combinations, maximizing efficiency. (19−21) In addition, in vitro enzymatic synthesis eliminates the need for transition metal catalysts, avoids the use of organic solvents, and operates at mild temperatures, reducing energy consumption compared to traditional chemical synthesis. These features enhance the process efficiency while minimizing unwanted side reactions and simplifying downstream purification. Recent studies using ALAS fromLaceyella sacchariachieved 5.4 mM 5-ALA from succinate and coenzyme A in an ATP-dependent manner, showing the potential of optimized cell-free system. (22)
In this study, we first employed 2-oxoglutarate dehydrogenase complex (SucAB) to generate succinyl-CoA from α-ketoglutarate (23) without ATP supplementation, followed by ALAS-mediated conversion of succinyl-CoA and glycine into 5-ALA. We designed a five-enzyme cascade system for 5-ALA production using α-ketoglutarate and glycine as starting materials, with regenerated free NAD+ as a cofactor using FRE and CAT as oxide-reduction balancing. (24) The in vitro enzymatic cascade exhibited high thermodynamic feasibility with a favorable ΔrG° of −30.4 kJ/mol (detailed calculations in Table S1). To reduce the carbon footprint, we integrated an in situ CO2 capture system during the one-pot catalytic reaction and delivered a high-yield and environmentally conscious solution for sustainable chemical production.

Results and Discussion

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Designing the Best Combinations of Chassis for Enzyme Production

The first step was to optimize the expression of recombinant soluble proteins, i.e., the 2-oxoglutarate dehydrogenase complex of SucA (102.6 kDa) and SucB (44.9 kDa) under T5 (Figure 1A) and T7 (Figure 1B) promoters, using either coexpression (SucAB) or individual systems (Figure 1C,D). Both T5 and T7 promoters were evaluated to compare their effects on protein expression and solubility. The T5 promoter facilitated balanced expression, reducing misfolding and aggregation, whereas the T7 promoter drove higher expression but often led to solubility challenges. (25,26) This comparison provided insights into whether maximizing expression (T7) or optimizing solubility (T5) was more effective for SucA and SucB production.

Figure 1

Figure 1. Recombinant protein production of SucA and SucB. Schematic illustration of the coexpressing SucA and SucB (SucAB), or individual expression of SucA and SucB, which are regulated by (A) T5 promoter and (B) T7 promoter. SDS-PAGE image of recombinant SucA, SucB, and SucAB under (C) T5 promoter in E. coli W3110 and (D) T7 promoter in E. coli BD7G at 22 °C. (E) Confirmation of SucB expression via Western blot analysis. Heat diagram of protein expression levels at mg/L under (F) T5 promoter in BL21, EcN, MG1655, and W3110, or under (G) T7 promoter in BD, BD7G, WL, and WL7G. SucA: E1 component of 2-oxoglutarate dehydrogenase complex at 102.6 kDa; SucB: E2 component of 2-oxoglutarate dehydrogenase complex at 44.9 kDa. WC represents whole cell protein, while S represents soluble protein. BD: BL21(DE3); BD7G: BL21(DE3)::T7-GroELS; WL: W3110::T7 RNA polymerase; WL7G: WL::T7-GroELS.

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SDS-PAGE revealed SucB to have a molecular weight of 50–55 kDa, larger than expected; thus, we confirmed this result through Western blot (Figure 1E) and proteomic analysis (Table S2).The increased molecular weight of SucB was supposed to be ubiquitination, a post-translational modification involving the covalent attachment of ubiquitin (a 76-residue protein with a molecular mass of 8.6 kDa) to specific amino acid residues, including lysine, cysteine, serine, and threonine at the N-terminal region. (27)
Furthermore, we considered production in four different hosts (BL21, EcN, MG1655, and W3110; Figure 1F). The T5 promoter drove a balanced expression, particularly in the W3110, which achieved the highest solubility for coexpressed SucAB (60.5% and 88.0%), individual SucA (51.5%), and SucB (73.0%), with protein concentrations ranging from 440.5 to 815 mg/L (Figure 1F). BL21 and MG1655 showed an expression efficiency lower than that of W3110, while EcN exhibited the lowest protein expression among the four strains.
The T7 promoter, known for its strong transcriptional activity, drove significantly higher protein expression levels in cells. (28) However, the high expression levels often caused solubility bottlenecks due to the overwhelming demand on cellular folding machinery. (29) The integration of the GroELS chaperone into BL21(DE3), annotated as BD7G, greatly improved solubility up to 90% for SucAB and individual proteins of SucA and SucB (Figure 1G), with protein concentrations ranging from 923.1 to 1155.2 mg/L. Similarly, WL7G, which also incorporated GroELS into the T7 RNA polymerase-integrated W3110 (WL), (30) exhibited moderate expression levels while improving protein solubility by 38%- to 60%- compared to WL. However, its performance remained below that of BD7G. This result underscores the critical role of molecular chaperones in proper protein folding. (26)
The T7 promoter effectively drove high protein expression but often required GroELS for solubility. In contrast, the T5 promoter offered balanced expression with high solubility, especially in W3110, where chaperones were unnecessary. (25) Coexpression of SucAB generally showed lower solubility due to folding competition, but GroELS alleviated this, particularly for large proteins like SucA. Individual SucB expression consistently achieved higher solubility, reflecting its simpler folding requirements. Tailoring expression strategies to the target protein and host strain is crucial, with GroELS proving invaluable under the T7 promoter. (31)

Optimized Enzyme Synergy for Succinyl-CoA Production

Next, we evaluated all parameters of temperature, reaction time, substrate concentrations, and cofactor to optimize the one-pot succinyl-CoA synthesis from α-ketoglutarate (α-KG) and coenzyme A through the catalytic action of the enzyme complex SucAB. Regarding the temperature effect, reaction at 30 °C provided the highest conversion efficiency (19.64%), as higher temperatures (i.e., 42 °C) led to enzyme denaturation. The reaction showed maximum efficiency within 30 min, and the optimal NAD+ concentration was found to be 1 mM, achieving a 30% conversion rate, while higher concentrations of α-KG slightly reduced efficiency, possibly due to substrate competition. Under the optimal temperature of 30 °C, a reaction time of 30 min, 0.5 mM CoA, and 1 mM NAD+, succinyl-CoA reached a maximum conversion of 46% when α-KG was used at 1 mM (Figure 2A).

Figure 2

Figure 2. Optimization of one-pot succinyl-CoA synthesis. (A) Effects of reaction temperature and time, initial NAD+ concentration, and α-ketoglutarate (α-KG) concentration on the conversion (%) of coenzyme A to succinyl-CoA. Succinyl-CoA titer and conversion efficiency using (B) coexpression SucAB coupling SucA or SucB, and (C) using various combinations of SucA and SucB at different protein concentrations (mg/mL). (D) Scheme illustration of one-pot succinyl-CoA synthesis with NAD+ regeneration using flavin reductase (FRE), catalase (CAT), and FAD. (E) Succinyl-CoA production with (FRE/CAT) or without (Φ) NAD+ regeneration by FRE/CAT system. Conversion was calculated based on coenzyme A consumption. Error bars represent the standard deviation of three independent experiments (n = 3).

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To explore the synergistic effect between SucA and SucB, we found that only a low succinyl-CoA yield (3.88%) was obtained from SucAB, while adding SucA or SucB separately increased the yield significantly (Figure 2B). At higher concentration (1 mg/mL of SucAB and SucB), conversion reached 58%, with 0.29 mM succinyl-CoA produced. Consistently, findings from individually expressed SucA and SucB combinations demonstrated that increasing the ratio of SucB relative to SucA enhanced yields, achieving a maximum conversion of 60% at approximately 0.3 mM succinyl-CoA titer with a 1:2 ratio of SucA/SucB (Figure 2C). This suggests that SucB availability is the rate-limiting step in this pathway.
As NAD+ is consumed and NADH accumulates as a byproduct, this inhibits the reaction due to redox imbalance. (24) Efficient NAD+ regeneration is essential to sustain the reaction and mitigate the inhibitory effects of NADH buildup. (32) The FRE/CAT system efficiently regenerates NADH to NAD+ (Figure 2D). NADH-dependent flavin reductase (FRE) facilitates this process by converting NADH to NAD+ while reducing FAD to FADH2. Catalase (CAT) then converts the FADH2-generated H2O2 into water, ensuring continuous NAD+ recycling and maintaining the efficiency of succinyl-CoA synthesis. In reactions using the FRE/CAT system, no NADH accumulation was observed (as indicated by the absence of a 7.3 min peak in Figure S2). The succinyl-CoA yield increased by 68.9%, reaching 0.49 mM with a 97% conversion rate, while using only 0.5 mM NAD+ in the FRE/CAT system. This highlights the system’s efficiency in NAD+ recycling, significantly reducing costs and enhancing the economic and operational feasibility of the reaction (Figure 2E). The FRE/CAT system maintains NAD+ availability at sufficient levels, eliminating the bottleneck typically caused by NADH buildup and ensuring optimal catalytic efficiency.
Both coexpressed and individually expressed SucA and SucB systems achieved high conversion rates under optimized conditions. However, individual expression offered greater flexibility by allowing precise adjustments of enzyme ratios, which enhanced the catalytic efficiency. Coexpression encountered challenges like suboptimal enzyme ratios, reliance on additional SucB to maximize yields, and difficulties in maintaining balanced expression levels due to vector and host system limitations.
Further enhancement of succinyl-CoA production could be achieved by increasing the initial CoA concentration beyond 0.5 mM, as the reaction had reached full conversion at this level. Additionally, fine-tuning the SucA-to-SucB ratio beyond the tested 1:2 proportion might further optimize the yields. Improved NAD+ recycling strategies, such as alternative cofactor regeneration systems, could maintain redox balance and sustain higher reaction rates. (24) Moreover, optimizing reaction conditions, including buffer composition and pH, could enhance enzyme stability and efficiency. (18) Finally, enzyme engineering approaches, such as directed evolution or rational mutagenesis of SucAB, may improve the catalytic activity and thermostability, ultimately pushing succinyl-CoA production toward higher titers suitable for industrial applications.
Chemical synthesis of succinyl-CoA, while scalable, suffers from low yields, side reactions, and high environmental costs due to toxic reagents and waste. (33) One-pot bioconversion provides a sustainable alternative with high conversion efficiency (up to 97%) without the metabolic constraints of cellular systems. (34) This method allows precise optimization of reaction conditions to boost the yield. Unlike chemical synthesis, it minimizes harsh reagents and complex purification, offering a selective and efficient route to succinyl-CoA production.

One-Pot Biosynthesis of 5-ALA with Low-Carbon Emissions

Finally, we managed to synthesize 5-ALA using the one-pot succinyl-CoA via integrating all the optimized conditions mentioned above followed by ALAS-mediated reaction (Figure 3A). A highly active ALAS fromRhodobacter capsulatuswas selected to enhance reaction efficiency in producing 5-ALA via a C4 pathway. (35)

Figure 3

Figure 3. One-pot cascade synthesis of 5-ALA from α-ketoglutarate (α-KG) and CoA. (A) Schematic overview of 5-ALA synthesis via a five-enzyme cascade including SucA, SucB, FRE, and CAT for succinyl-CoA, followed by ALAS converting succinyl-CoA and glycine into 5-ALA. Optimization of 5-ALA synthesis by varying concentration of (B) cofactor PLP and (C) glycine. Time course of 5-ALA synthesis using 2 mg/mL ALAS in (D) two steps and (E) one step over 1–5 h. In situ CO2 capture during 5-ALA synthesis using human carbonic anhydrase II (hCAII) (F) illustration and (G) CO2 capture efficiency depends on 5-ALA production and hCAII concentration. Conversion was calculated based on α-KG consumption. Error bars represent the standard deviation of three independent experiments (n = 3).

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Key reaction parameters, including pyridoxal phosphate (PLP) and glycine concentrations, were optimized to maximize 5-ALA production. At an optimal PLP concentration of 0.25 mM, the 5-ALA titer was 0.23 mM, representing a 3-fold improvement compared to that of 0.05 mM PLP (Figure 3B). Increasing the PLP concentration to 1.0 mM led to a 55.2% decrease in 5-ALA titer, highlighting an inhibitory effect beyond 0.5 mM PLP. (36) Similarly, glycine optimization resulted in a maximum 5-ALA titer of 0.37 mM at 2 mM glycine (Figure 3C).
To further optimize 5-ALA production, two- and one-step reactions (Figure S3) were evaluated using the five-enzyme cascade. In the two-step reaction, succinyl-CoA was synthesized first for 30 min. Subsequently, glycine, PLP, and varying concentrations of ALAS (0.25 and 2 mg/mL) were added to initiate 5-ALA production (Figure S3A). In this setup, 5-ALA increased proportionally with ALAS concentration, peaking at approximately 0.5 mM with 2 mg/mL ALAS and achieving a maximum conversion rate of ∼50% (Figure S3B). In contrast, the one-step setup combined all substrates and enzymes simultaneously, resulting in a superior performance (Figure S3C). This setup reached approximately 0.6 mM at 2 mg/mL ALAS with a slightly improved conversion rate of 59% (Figure S3D).
A time-course analysis further highlighted the differences; in the two-step reaction, the 5-ALA titer reached about 0.79 mM after 5 h (Figure 3D), while in the one-step reaction, the titer reached 1 mM, achieving nearly 100% conversion from 1 mM α-KG (Figure 3E). The improvement in the one-pot setup can be attributed to the continuous and simultaneous presence of all components, facilitating a seamless reaction cascade. (37) Notably, CoA is regenerated in the one-step reaction, eliminating the need for its stoichiometric addition to obtain 5-ALA, thereby enhancing the cost efficiency and process sustainability. Moreover, the streamlined protocol reduces operational complexity, energy input, and waste, contributing to more sustainable production. (37−39)
To manage the CO2 byproduct in the reaction system, two capture strategies were assessed: a chemical approach using NaOH and an enzyme-based method employing human carbonic anhydrase II (hCAII) (Figure 3F). Increasing the NaOH concentration from 1 to 5 mM progressively improved the CO2 capture efficiency from 58% to 100% (Figure S4). Meanwhile, the enzyme-based approach, leveraging the high catalytic activity of hCAII, exhibited a more pronounced improvement in efficiency. (40) The CO2 capture increased from 15.5% with 0.125 mg/mL crude hCAII to complete capture at 0.5 mg/mL crude hCAII (Figure 3G). Carbonic anhydrase is a key enzyme in carbon capture and sequestration (CCS), catalyzing the rapid hydration of CO2 into bicarbonate ions for conversion into stable carbonates. (41) The enzyme-based approach aligns with sustainable industrial practices, reduces chemical usage, and supports a circular bioeconomy by enabling CO2 reutilization in other processes, enhancing overall sustainability. (42−44)
This study explores an enzymatic cascade for producing succinyl-CoA and 5-ALA, but sustainability remains a challenge due to costly starting materials and purified enzymes. Enzyme production via bacterial fermentation generates CO2 and requires expensive purification. While the one-pot system is advantageous over chemical synthesis, its sustainability hinges on optimizing enzyme production and cofactor recycling. Future improvements, such as using crude lysates or whole-cell biotransformation, could lower the costs. Enhancing enzyme stability, reducing purification needs, and improving cofactor regeneration are crucial for economic and environmental viability.

Conclusion

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We demonstrated a proof of concept for a sustainable biocatalytic strategy to produce 5-ALA from a high-value CoA through green process engineering, enzyme optimization, and integrated CO2 sequestration for the first time. The optimal quantity and quality of enzyme production relied on the T7 system within the GroELS-assisted folding strain BD7G. The integrated one-pot enzymatic cascade demonstrated superior atom economy and process intensification compared with sequential reactions, reaching approximately 0.5 mM succinyl-CoA and 1 mM 5-ALA with nearly 100% conversion. Notably, the incorporation of a biocatalytic CO2 capture system employing carbonic anhydrase exemplifies green chemistry principles by closing the carbon loop, thus supporting greener pharmaceutical and chemical manufacturing processes with a minimal carbon footprint. However, further optimization is required to enhance enzyme stability, reaction efficiency, and scalability to achieve sustainable production metrics.

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acssuschemeng.5c00001.

  • SDS-PAGE gel images of purified recombinant enzymes fromE. coliin this study, HPLC analysis of the one-pot reaction products, strategies for improving 5-ALA synthesis using sequential or one-pot reaction with varying ALAS amounts, thermodynamic calculation of the overall CoA synthesis reaction, mascot result of LC-MS/MS protein identification of SucB, strains, plasmids, and primers used in this study, sequence of coexpression SucA and SucB (SucAB), individual sequence of SucA, and individual sequence of SucB under T5 promoter, sequence of coexpression SucA and SucB (SucAB), individual sequence of SucA, and individual sequence of SucB under T7 promoter, list of recombinant proteins used in this study, and the price of coenzyme A, succinyl-CoA, and 5-ALA (PDF)

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Author Information

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  • Corresponding Author
  • Authors
    • Yu-Chieh Lin - Department of Chemical Engineering, National Cheng Kung University, Tainan 70101, Taiwan (R.O.C.)
    • Wan-Wen Ting - Department of Chemical Engineering, National Cheng Kung University, Tainan 70101, Taiwan (R.O.C.);  Orcidhttps://orcid.org/0000-0002-3018-4774
    • Po-Hsiang Wang - Graduate Institute of Environmental Engineering, National Central University, Taoyuan 32001, Taiwan (R.O.C.);  Department of Chemical and Materials Engineering, National Central University, Taoyuan 32001, Taiwan (R.O.C.);  Orcidhttps://orcid.org/0000-0001-9900-0972
  • Author Contributions

    Y.-C.L. and I.-S.N. conceived the study. Y.-C.L. performed most of the experiments and original draft investigation. W.-W.T. and I.-S.N. did methodology validation and supervised the experiments. I.-S.N. and P.-H.W. prepared, reviewed, and edited the manuscript.

  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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The authors are grateful for the financial support received from the Ministry of Science and Technology (NSTC 111-2221-E-006-012-MY3 and NSTC 112-2218-006-012) in Taiwan.

References

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    Abstr.: As a novel protein post-translational modification (PTM), lysine succinylation is widely involved in metab. regulation by altering the activity of catalytic enzymes. Inactivating succinyl-CoA synthetase in Saccharopolyspora erythraea HL3168 E3 was proved significantly inducing the global protein hypersuccinylation. To investigate the effects, succinylome of the mutant strain E3ΔsucC was identified by using a high-resoln. mass spectrometry-based proteomics approach. PTMomics analyses suggested the important roles of succinylation on protein biosynthesis, carbon metab., and antibiotics biosynthesis in S. erythraea. Enzymic expts. in vivo and in vitro were further conducted to det. the succinylation regulation in the TCA cycle. We found out that the activity of aconitase (SACE_3811) was significantly inhibited by succinylation in E3ΔsucC, which probably led to the extracellular accumulation of pyruvate and citrate during the fermn. Enzyme structural analyses indicated that the succinylation of K278 and K373, conservative lysine residues locating around the protein binding pocket, possibly affects the activity of aconitase. To alleviate the metab. changes caused by succinyl-CoA synthetase inactivation and protein hypersuccinylation, CRISPR interference (CRISPRi) was applied to mildly downregulate the transcription level of gene sucC in E3. The erythromycin titer of the CRISPRi mutant E3-sucC-sg1 was increased by 54.7% compared with E3, which was 1200.5 mg/L. Taken together, this work not only expands our knowledge of succinylation regulation in the TCA cycle, but also validates that CRISPRi is an efficient strategy on the metabolic engineering of S. erythraea. Key points: • We reported the first systematic profiling of the S. erythraea succinylome. • We found that the succinylation regulation on the activity of aconitase. • We enhanced the prodn. of erythromycin by using CRISPRi to regulate the transcription of gene sucC. Graphical abstr.: [graphic not available: see fulltext].
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    Jiang, M.; Hong, K.; Mao, Y.; Ma, H.; Chen, T.; Wang, Z. Natural 5-aminolevulinic acid: sources, biosynthesis, detection and applications. Front. Bioeng. Biotechnol. 2022, 10, 841443  DOI: 10.3389/fbioe.2022.841443
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    Natural 5-Aminolevulinic Acid: Sources, Biosynthesis, Detection and Applications
    Jiang Meiru; Hong Kunqiang; Chen Tao; Wang Zhiwen; Mao Yufeng; Ma Hongwu
    Frontiers in bioengineering and biotechnology (2022), 10 (), 841443 ISSN:2296-4185.
    5-Aminolevulinic acid (5-ALA) is the key precursor for the biosynthesis of tetrapyrrole compounds, with wide applications in medicine, agriculture and other burgeoning fields. Because of its potential applications and disadvantages of chemical synthesis, alternative biotechnological methods have drawn increasing attention. In this review, the recent progress in biosynthetic pathways and regulatory mechanisms of 5-ALA synthesis in biological hosts are summarized. The research progress on 5-ALA biosynthesis via the C4/C5 pathway in microbial cells is emphasized, and the corresponding biotechnological design strategies are highlighted and discussed in detail. In addition, the detection methods and applications of 5-ALA are also reviewed. Finally, perspectives on potential strategies for improving the biosynthesis of 5-ALA and understanding the related mechanisms to further promote its industrial application are conceived and proposed.
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    Sansaloni-Pastor, S.; Varesio, E.; Lange, N. Modulation and proteomic changes on the heme pathway following treatment with 5-aminolevulinic acid. J. Photochem. Photobiol., B 2022, 233, 112484  DOI: 10.1016/j.jphotobiol.2022.112484
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    Wu, Y.; Liao, W.; Dawuda, M. M.; Hu, L.; Yu, J. 5-Aminolevulinic acid (ALA) biosynthetic and metabolic pathways and its role in higher plants: a review. Plant Growth Regul. 2019, 87 (2), 357– 374,  DOI: 10.1007/s10725-018-0463-8
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    Ding, W.; Weng, H.; Du, G.; Chen, J.; Kang, Z. 5-aminolevulinic acid production from inexpensive glucose by engineering the C4 pathway in Escherichia coli. J. Ind. Microbiol. Biotechnol. 2017, 44 (8), 1127– 1135,  DOI: 10.1007/s10295-017-1940-1
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    5-Aminolevulinic acid production from inexpensive glucose by engineering the C4 pathway in Escherichia coli
    Ding, Wenwen; Weng, Huanjiao; Du, Guocheng; Chen, Jian; Kang, Zhen
    Journal of Industrial Microbiology & Biotechnology (2017), 44 (8), 1127-1135CODEN: JIMBFL; ISSN:1367-5435. (Springer)
    5-Aminolevulinic acid (ALA), the first committed intermediate for natural biosynthesis of tetrapyrrole compds., has recently drawn intensive attention due to its broad potential applications. In this study, we describe the construction of recombinant Escherichia coli strains for ALA prodn. from glucose via the C4 pathway. The hemA gene from Rhodobacter capsulatus was optimally overexpressed using a ribosome binding site engineering strategy, which enhanced ALA prodn. substantially from 20 to 689 mg/L. Following optimization of biosynthesis pathways towards CoA and precursor (glycine and succinyl-CoA), and downregulation of hemB expression, the prodn. of ALA was further increased to 2.81 g/L in batch-fermn.
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    Yang, P.; Liu, W.; Cheng, X.; Wang, J.; Wang, Q.; Qi, Q. A new strategy for production of 5-aminolevulinic acid in recombinant Corynebacterium glutamicum with high yield. Appl. Environ. Microbiol. 2016, 82 (9), 2709– 2717,  DOI: 10.1128/AEM.00224-16
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    Reetz, M. T. Biocatalysis in organic chemistry and biotechnology: Past, present, and future. J. Am. Chem. Soc. 2013, 135 (34), 12480– 12496,  DOI: 10.1021/ja405051f
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    Biocatalysis in Organic Chemistry and Biotechnology: Past, Present, and Future
    Reetz, Manfred T.
    Journal of the American Chemical Society (2013), 135 (34), 12480-12496CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    A review. Enzymes as catalysts in synthetic org. chem. gained importance in the latter half of the 20th century, but nevertheless suffered from two major limitations. First, many enzymes were not accessible in large enough quantities for practical applications. The advent of recombinant DNA technol. changed this dramatically in the late 1970s. Second, many enzymes showed a narrow substrate scope, often poor stereo- and/or regioselectivity and/or insufficient stability under operating conditions. With the development of directed evolution beginning in the 1990s and continuing to the present day, all of these problems can be addressed and generally solved. The present Perspective focuses on these and other developments which have popularized enzymes as part of the toolkit of synthetic org. chemists and biotechnologists. Included is a discussion of the scope and limitation of cascade reactions using enzyme mixts. in vitro and of metabolic engineering of pathways in cells as factories for the prodn. of simple compds. such as biofuels and complex natural products. Future trends and problems are also highlighted, as is the discussion concerning biocatalysis vs. nonbiol. catalysis in synthetic org. chem. This Perspective does not constitute a comprehensive review, and therefore the author apologizes to those researchers whose work is not specifically treated here.
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    Wang, Z.; Sundara Sekar, B.; Li, Z. Recent advances in artificial enzyme cascades for the production of value-added chemicals. Bioresour. Technol. 2021, 323, 124551  DOI: 10.1016/j.biortech.2020.124551
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    Recent advances in artificial enzyme cascades for the production of value-added chemicals
    Wang, Zilong; Sundara Sekar, Balaji; Li, Zhi
    Bioresource Technology (2021), 323 (), 124551CODEN: BIRTEB; ISSN:0960-8524. (Elsevier Ltd.)
    A review. Enzyme cascades are efficient tools to perform multi-step synthesis in one-pot in a green and sustainable manner, enabling non-natural synthesis of valuable chems. from easily available substrates by artificially combining two or more enzymes. Bioprodn. of many high-value chems. such as chiral and highly functionalised mols. have been achieved by developing new enzyme cascades. This review summarizes recent advances on engineering and application of enzyme cascades to produce high-value chems. (alcs., aldehydes, ketones, amines, carboxylic acids, etc) from simple starting materials. While 2-step enzyme cascades are developed for versatile enantioselective synthesis, multi-step enzyme cascades are engineered to functionalise basic chems., such as styrenes, cyclic alkanes, and arom. compds. New cascade reactions have also been developed for producing valuable chems. from bio-based substrates, such as L-phenylalanine, and renewable feedstocks such as glucose and glycerol. The challenges in current process and future outlooks in the development of enzyme cascades are also addressed.
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    Straathof, A. J. J. Transformation of biomass into commodity chemicals using enzymes or cells. Chem. Rev. 2014, 114 (3), 1871– 1908,  DOI: 10.1021/cr400309c
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    Transformation of Biomass into Commodity Chemicals Using Enzymes or Cells
    Straathof, Adrie J. J.
    Chemical Reviews (Washington, DC, United States) (2014), 114 (3), 1871-1908CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
    A review on the prodn. of commodity chems. from biomass using enzymes or cells. The biochem. formation of commodity chems. from biomass has been reviewed before,but the current review covers a wider range of chems. and the most recent literature.
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    Siedentop, R.; Claaßen, C.; Rother, D.; Lütz, S.; Rosenthal, K. Getting the most out of enzyme cascades: Strategies to optimize in vitro multi-enzymatic reactions. Catalysts 2021, 11 (10), 1183,  DOI: 10.3390/catal11101183
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    Wu, S.; Snajdrova, R.; Moore, J. C.; Baldenius, K.; Bornscheuer, U. T. Biocatalysis: Enzymatic synthesis for industrial applications. Angew. Chem., Int. Ed. Engl. 2021, 60 (1), 88– 119,  DOI: 10.1002/anie.202006648
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    Biocatalysis: Enzymatic Synthesis for Industrial Applications
    Wu Shuke; Bornscheuer Uwe T; Snajdrova Radka; Moore Jeffrey C; Baldenius Kai
    Angewandte Chemie (International ed. in English) (2021), 60 (1), 88-119 ISSN:.
    Biocatalysis has found numerous applications in various fields as an alternative to chemical catalysis. The use of enzymes in organic synthesis, especially to make chiral compounds for pharmaceuticals as well for the flavors and fragrance industry, are the most prominent examples. In addition, biocatalysts are used on a large scale to make specialty and even bulk chemicals. This review intends to give illustrative examples in this field with a special focus on scalable chemical production using enzymes. It also discusses the opportunities and limitations of enzymatic syntheses using distinct examples and provides an outlook on emerging enzyme classes.
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    Wu, S.; Li, Z. Whole-cell cascade biotransformations for one-pot multistep organic synthesis. Chem. Catal. Chem. 2018, 10 (10), 2164– 2178,  DOI: 10.1002/cctc.201701669
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    Walsh, C. T.; Moore, B. S. Enzymatic cascade reactions in biosynthesis. Angew. Chem., Int. Ed. Engl. 2019, 58 (21), 6846,  DOI: 10.1002/anie.201807844
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    Meng, Q.; Zhang, Y.; Ju, X.; Ma, C.; Ma, H.; Chen, J.; Zheng, P.; Sun, J.; Zhu, J.; Ma, Y.; Zhao, X.; Chen, T. Production of 5-aminolevulinic acid by cell free multi-enzyme catalysis. J. Biotechnol. 2016, 226, 8– 13,  DOI: 10.1016/j.jbiotec.2016.03.024
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    Production of 5-aminolevulinic acid by cell free multi-enzyme catalysis
    Meng, Qinglong; Zhang, Yanfei; Ju, Xiaozhi; Ma, Chunling; Ma, Hongwu; Chen, Jiuzhou; Zheng, Ping; Sun, Jibin; Zhu, Jun; Ma, Yanhe; Zhao, Xueming; Chen, Tao
    Journal of Biotechnology (2016), 226 (), 8-13CODEN: JBITD4; ISSN:0168-1656. (Elsevier B.V.)
    5-Aminolevulinic acid (ALA) is the precursor for the biosynthesis of tetrapyrroles and has broad agricultural and medical applications. Currently ALA is mainly produced by chem. synthesis and microbial fermn. Cell free multi-enzyme catalysis is a promising method for producing high value chems. Here we reported our work on developing a cell free process for ALA prodn. using thermostable enzymes. Cheap substrates (succinate and glycine) were used for ALA synthesis by two enzymes: 5-aminolevulinic acid synthase (ALAS) from Laceyella sacchari (LS-ALAS) and succinyl-CoA synthase (Suc) from Escherichia coli. ATP was regenerated by polyphosphate kinase (Ppk) using polyphosphate as the substrate. Succinate was added into the reaction system in a fed-batch mode to avoid its inhibition effect on Suc. After reaction for 160 min, ALA concn. was increased to 5.4 mM. This is the first reported work on developing the cell free process for ALA prodn. Through further process and enzyme optimization the cell free process could be an effective and economic way for ALA prodn.
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    Li, X.; Wu, F.; Beard, D. A. Identification of the kinetic mechanism of succinyl-CoA synthetase. Biosci. Rep. 2013, 33 (1), 145– 163,  DOI: 10.1042/BSR20120069
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    Hsiang, C. C.; Tan, S. I.; Chen, Y. C.; Ng, I. S. Genetic design of co-Expressing a novel aconitase with cis-aconitate decarboxylase and chaperone GroELS for high-level itaconic acid production. Process Biochem. 2023, 129, 133– 139,  DOI: 10.1016/j.procbio.2023.03.021
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    Genetic design of co-expressing a novel aconitase with cis-aconitate decarboxylase and chaperone GroELS for high-level itaconic acid production
    Hsiang, Chuan-Chieh; Tan, Shih-I.; Chen, Yeong-Chang; Ng, I-Son
    Process Biochemistry (Oxford, United Kingdom) (2023), 129 (), 133-139CODEN: PBCHE5; ISSN:1359-5113. (Elsevier Ltd.)
    Itaconic acid (IA) is regarded as one of the 12 important building blocks as recognized by US Department of Energy. Thus, the prodn. of IA is crucial and urgent. In this study, we attempted to explore a new aconitase from an acidophilic E. coli Nissle 1917, denoted as ENAcnA, and was compared to Corynebacterium glutamicum for the first time. The native ENAcnA has 10 amino acid mutations and the supposed crit. mutations at N397S, Q409L, S522G, and G826E in the aconitase family domain contributed to the high hydratase activity at the low pH condition. Co-expression of ENAcnA and cis-aconitate decarboxylase from Aspergillus terreus in the recombinant E. coli BL21(DE3) resulted in IA titer and productivity of 80.4 g/L and 10.1 g/L/h, resp. Integration of GroELS chaperon to chromosome of BL21(DE3) for generating BD::7G strain even enhanced IA titer by 13%. The cold treatment of whole-cell biocatalyst substantially assisted the mass transfer of substrate through the cell membrane, and consequently achieved the max. IA productivity. The discovery of ENAcnA, chaperon assistance in host, and cold treatment of whole cell biocatalysts contributed to high-level IA prodn. up to 95.5 g/L for the industrial scale.
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    The preparation of S-succinyl coenzyme A
    Simon, Eric J.; Shemin, David
    Journal of the American Chemical Society (1953), 75 (), 2520CODEN: JACSAT; ISSN:0002-7863.
    cf. C.A. 47, 740h. The succinyl intermediate (I) in the formation of protoporphyrin, probably S-succinyl coenzyme A, is readily formed by treating coenzyme A with succinic anhydride as shown by the formation of a hydroxamic acid, the disappearance of the SH group, and an increased light absorption of 232 mμ. When I is warmed a few min. on the steam bath, the SH group reappears, there is no reaction with HONH2, and light absorption at 232 mμ decreases. Synthetic I behaves as S-succinyl in enzymic systems.
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    The prospective and potential of carbonic anhydrase for carbon dioxide sequestration: A critical review
    Effendi, Sefli Sri Wahyu; Ng, I-Son
    Process Biochemistry (Oxford, United Kingdom) (2019), 87 (), 55-65CODEN: PBCHE5; ISSN:1359-5113. (Elsevier Ltd.)
    A review. From 1990 to 2000, carbon dioxide emissions increased dramatically by 13% and are estd. to increase by 30-50% until 2050, which has already caused global warming and affected the environment and human health care. Moreover, carbon dioxide has dramatically increased up to 20 times in atm., and the temp. of earth surface also raised 1°C from the past fifty years. Although novel and globally technologies integrated carbon capture utilization and storage (CCUS) is already considered, there are still significant challenges of cost, economic barriers, and uncertainties on environmental impacts. One promising way to mediate those issues is to use carbonic anhydrase (CA) as enzyme based is eco-friendly and no secondary pollutant will emerge. In order to propose CAs for carbon fixation, high stable CAs on the extreme and harsh environment is essential and urgent. This review aims to present advanced developments in CA, efficient strategies engineering to improve the productivity and stability, immobilization techniques towards an industrial operating system, and also highlight recent challenges in the CAs area along with perspective has also conducted.
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  • Abstract

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    Figure 1

    Figure 1. Recombinant protein production of SucA and SucB. Schematic illustration of the coexpressing SucA and SucB (SucAB), or individual expression of SucA and SucB, which are regulated by (A) T5 promoter and (B) T7 promoter. SDS-PAGE image of recombinant SucA, SucB, and SucAB under (C) T5 promoter in E. coli W3110 and (D) T7 promoter in E. coli BD7G at 22 °C. (E) Confirmation of SucB expression via Western blot analysis. Heat diagram of protein expression levels at mg/L under (F) T5 promoter in BL21, EcN, MG1655, and W3110, or under (G) T7 promoter in BD, BD7G, WL, and WL7G. SucA: E1 component of 2-oxoglutarate dehydrogenase complex at 102.6 kDa; SucB: E2 component of 2-oxoglutarate dehydrogenase complex at 44.9 kDa. WC represents whole cell protein, while S represents soluble protein. BD: BL21(DE3); BD7G: BL21(DE3)::T7-GroELS; WL: W3110::T7 RNA polymerase; WL7G: WL::T7-GroELS.

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    Figure 2

    Figure 2. Optimization of one-pot succinyl-CoA synthesis. (A) Effects of reaction temperature and time, initial NAD+ concentration, and α-ketoglutarate (α-KG) concentration on the conversion (%) of coenzyme A to succinyl-CoA. Succinyl-CoA titer and conversion efficiency using (B) coexpression SucAB coupling SucA or SucB, and (C) using various combinations of SucA and SucB at different protein concentrations (mg/mL). (D) Scheme illustration of one-pot succinyl-CoA synthesis with NAD+ regeneration using flavin reductase (FRE), catalase (CAT), and FAD. (E) Succinyl-CoA production with (FRE/CAT) or without (Φ) NAD+ regeneration by FRE/CAT system. Conversion was calculated based on coenzyme A consumption. Error bars represent the standard deviation of three independent experiments (n = 3).

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    Figure 3

    Figure 3. One-pot cascade synthesis of 5-ALA from α-ketoglutarate (α-KG) and CoA. (A) Schematic overview of 5-ALA synthesis via a five-enzyme cascade including SucA, SucB, FRE, and CAT for succinyl-CoA, followed by ALAS converting succinyl-CoA and glycine into 5-ALA. Optimization of 5-ALA synthesis by varying concentration of (B) cofactor PLP and (C) glycine. Time course of 5-ALA synthesis using 2 mg/mL ALAS in (D) two steps and (E) one step over 1–5 h. In situ CO2 capture during 5-ALA synthesis using human carbonic anhydrase II (hCAII) (F) illustration and (G) CO2 capture efficiency depends on 5-ALA production and hCAII concentration. Conversion was calculated based on α-KG consumption. Error bars represent the standard deviation of three independent experiments (n = 3).

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      Ke, Xiang; Jiang, Xing; Huang, Mingzhi; Tian, Xiwei; Chu, Ju
      Applied Microbiology and Biotechnology (2022), 106 (13-16), 5153-5165CODEN: AMBIDG; ISSN:0175-7598. (Springer International Publishing AG)
      Abstr.: As a novel protein post-translational modification (PTM), lysine succinylation is widely involved in metab. regulation by altering the activity of catalytic enzymes. Inactivating succinyl-CoA synthetase in Saccharopolyspora erythraea HL3168 E3 was proved significantly inducing the global protein hypersuccinylation. To investigate the effects, succinylome of the mutant strain E3ΔsucC was identified by using a high-resoln. mass spectrometry-based proteomics approach. PTMomics analyses suggested the important roles of succinylation on protein biosynthesis, carbon metab., and antibiotics biosynthesis in S. erythraea. Enzymic expts. in vivo and in vitro were further conducted to det. the succinylation regulation in the TCA cycle. We found out that the activity of aconitase (SACE_3811) was significantly inhibited by succinylation in E3ΔsucC, which probably led to the extracellular accumulation of pyruvate and citrate during the fermn. Enzyme structural analyses indicated that the succinylation of K278 and K373, conservative lysine residues locating around the protein binding pocket, possibly affects the activity of aconitase. To alleviate the metab. changes caused by succinyl-CoA synthetase inactivation and protein hypersuccinylation, CRISPR interference (CRISPRi) was applied to mildly downregulate the transcription level of gene sucC in E3. The erythromycin titer of the CRISPRi mutant E3-sucC-sg1 was increased by 54.7% compared with E3, which was 1200.5 mg/L. Taken together, this work not only expands our knowledge of succinylation regulation in the TCA cycle, but also validates that CRISPRi is an efficient strategy on the metabolic engineering of S. erythraea. Key points: • We reported the first systematic profiling of the S. erythraea succinylome. • We found that the succinylation regulation on the activity of aconitase. • We enhanced the prodn. of erythromycin by using CRISPRi to regulate the transcription of gene sucC. Graphical abstr.: [graphic not available: see fulltext].
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      Production of 5-aminolevulinic acid by cell free multi-enzyme catalysis
      Meng, Qinglong; Zhang, Yanfei; Ju, Xiaozhi; Ma, Chunling; Ma, Hongwu; Chen, Jiuzhou; Zheng, Ping; Sun, Jibin; Zhu, Jun; Ma, Yanhe; Zhao, Xueming; Chen, Tao
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      5-Aminolevulinic acid (ALA) is the precursor for the biosynthesis of tetrapyrroles and has broad agricultural and medical applications. Currently ALA is mainly produced by chem. synthesis and microbial fermn. Cell free multi-enzyme catalysis is a promising method for producing high value chems. Here we reported our work on developing a cell free process for ALA prodn. using thermostable enzymes. Cheap substrates (succinate and glycine) were used for ALA synthesis by two enzymes: 5-aminolevulinic acid synthase (ALAS) from Laceyella sacchari (LS-ALAS) and succinyl-CoA synthase (Suc) from Escherichia coli. ATP was regenerated by polyphosphate kinase (Ppk) using polyphosphate as the substrate. Succinate was added into the reaction system in a fed-batch mode to avoid its inhibition effect on Suc. After reaction for 160 min, ALA concn. was increased to 5.4 mM. This is the first reported work on developing the cell free process for ALA prodn. Through further process and enzyme optimization the cell free process could be an effective and economic way for ALA prodn.
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      Hsiang, C. C.; Tan, S. I.; Chen, Y. C.; Ng, I. S. Genetic design of co-Expressing a novel aconitase with cis-aconitate decarboxylase and chaperone GroELS for high-level itaconic acid production. Process Biochem. 2023, 129, 133– 139,  DOI: 10.1016/j.procbio.2023.03.021
      26
      Genetic design of co-expressing a novel aconitase with cis-aconitate decarboxylase and chaperone GroELS for high-level itaconic acid production
      Hsiang, Chuan-Chieh; Tan, Shih-I.; Chen, Yeong-Chang; Ng, I-Son
      Process Biochemistry (Oxford, United Kingdom) (2023), 129 (), 133-139CODEN: PBCHE5; ISSN:1359-5113. (Elsevier Ltd.)
      Itaconic acid (IA) is regarded as one of the 12 important building blocks as recognized by US Department of Energy. Thus, the prodn. of IA is crucial and urgent. In this study, we attempted to explore a new aconitase from an acidophilic E. coli Nissle 1917, denoted as ENAcnA, and was compared to Corynebacterium glutamicum for the first time. The native ENAcnA has 10 amino acid mutations and the supposed crit. mutations at N397S, Q409L, S522G, and G826E in the aconitase family domain contributed to the high hydratase activity at the low pH condition. Co-expression of ENAcnA and cis-aconitate decarboxylase from Aspergillus terreus in the recombinant E. coli BL21(DE3) resulted in IA titer and productivity of 80.4 g/L and 10.1 g/L/h, resp. Integration of GroELS chaperon to chromosome of BL21(DE3) for generating BD::7G strain even enhanced IA titer by 13%. The cold treatment of whole-cell biocatalyst substantially assisted the mass transfer of substrate through the cell membrane, and consequently achieved the max. IA productivity. The discovery of ENAcnA, chaperon assistance in host, and cold treatment of whole cell biocatalysts contributed to high-level IA prodn. up to 95.5 g/L for the industrial scale.
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      cf. C.A. 47, 740h. The succinyl intermediate (I) in the formation of protoporphyrin, probably S-succinyl coenzyme A, is readily formed by treating coenzyme A with succinic anhydride as shown by the formation of a hydroxamic acid, the disappearance of the SH group, and an increased light absorption of 232 mμ. When I is warmed a few min. on the steam bath, the SH group reappears, there is no reaction with HONH2, and light absorption at 232 mμ decreases. Synthetic I behaves as S-succinyl in enzymic systems.
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      The prospective and potential of carbonic anhydrase for carbon dioxide sequestration: A critical review
      Effendi, Sefli Sri Wahyu; Ng, I-Son
      Process Biochemistry (Oxford, United Kingdom) (2019), 87 (), 55-65CODEN: PBCHE5; ISSN:1359-5113. (Elsevier Ltd.)
      A review. From 1990 to 2000, carbon dioxide emissions increased dramatically by 13% and are estd. to increase by 30-50% until 2050, which has already caused global warming and affected the environment and human health care. Moreover, carbon dioxide has dramatically increased up to 20 times in atm., and the temp. of earth surface also raised 1°C from the past fifty years. Although novel and globally technologies integrated carbon capture utilization and storage (CCUS) is already considered, there are still significant challenges of cost, economic barriers, and uncertainties on environmental impacts. One promising way to mediate those issues is to use carbonic anhydrase (CA) as enzyme based is eco-friendly and no secondary pollutant will emerge. In order to propose CAs for carbon fixation, high stable CAs on the extreme and harsh environment is essential and urgent. This review aims to present advanced developments in CA, efficient strategies engineering to improve the productivity and stability, immobilization techniques towards an industrial operating system, and also highlight recent challenges in the CAs area along with perspective has also conducted.
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  • Supporting Information

    Supporting Information

    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acssuschemeng.5c00001.

    • SDS-PAGE gel images of purified recombinant enzymes fromE. coliin this study, HPLC analysis of the one-pot reaction products, strategies for improving 5-ALA synthesis using sequential or one-pot reaction with varying ALAS amounts, thermodynamic calculation of the overall CoA synthesis reaction, mascot result of LC-MS/MS protein identification of SucB, strains, plasmids, and primers used in this study, sequence of coexpression SucA and SucB (SucAB), individual sequence of SucA, and individual sequence of SucB under T5 promoter, sequence of coexpression SucA and SucB (SucAB), individual sequence of SucA, and individual sequence of SucB under T7 promoter, list of recombinant proteins used in this study, and the price of coenzyme A, succinyl-CoA, and 5-ALA (PDF)


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