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Overview of the flexible design of different expression constructs used in this work.: Legend: LeuDH: leucine dehydrogenase; FDH: formate dehydrogenase; P: IPTG-inducible promoter; B0034, B0032 and B0030: RBS; B0015 and B0016: terminator.
Source publication
Systems that can regulate and coordinate the expression of multiple enzymes for metabolic regulation and synthesis of important drug intermediates are poorly explored. In this work, a strategy for constructing a tunable multi-enzyme-coordinate expression system for biosynthesis of chiral drug intermediates was developed and evaluated by connecting...
Citations
... Expression of multi-protein complexes or multiple enzymes can be influenced by plasmid copy number, ribosome binding site (RBS) sequence, and rare codon usage. [42][43][44][45] Controlled co-expression of up to four enzymes in E. coli BL21(DE3) cells has been achieved using T7 promoters and terminators for each gene in tandem sequence. [46] Gene position in the plasmid and plasmid copy number can also affect multiprotein expression. ...
Cyanobacteriochrome (CBCR) cGMP‐specific phosphodiesterase, adenylyl cyclase, and FhlA (GAF) domains bind bilin cofactors to confer sensory wavelengths important for various cyanobacterial photosensory processes. Many isolated GAF domains autocatalytically bind bilins, including the third GAF domain of CBCR Slr1393 from Synechocystis sp. PCC6803, which binds phycoerythrobilin (PEB) to yield a bright orange fluorescent protein. Compared to green fluorescent proteins, the smaller size and lack of an oxygen requirement for fluorescence make Slr1393g3 a promising platform for new genetically encoded fluorescent tools. Slr1393g3, however, shows low PEB binding efficiency (chromophorylation) at ~3 % compared to total Slr1393g3 expressed in E. coli. Here we used site‐directed mutagenesis and plasmid redesign methods to improve Slr1393g3‐PEB binding and demonstrate its utility as a fluorescent marker in live cells. Mutation at a single site, Trp496, tuned the emission over ~30 nm, likely by shifting autoisomerization of PEB to phycourobilin (PUB). Plasmid modifications for tuning relative expression of Slr1393g3 and PEB synthesis enzymes also improved chromophorylation and moving from a dual to single plasmid system facilitated exploration of a range of mutants via site saturation mutagenesis and sequence truncation. Collectively, the PEB/PUB chromophorylation was raised up to a total of 23 % with combined sequence truncation and W496H mutation.
... The advantage of this method is that it is appropriate for modifying enzymes without a clear crystal structure or catalytic mechanism (Xu et al. 2020;Pang et al. 2023;Wang et al. 2021). For multi-enzyme cascade reaction, numerous methods have been proposed for coordinating the production of multiple enzymes in a single cell, including the use of suitable plasmids with various copies, adjusting the strength of ribosome binding sites and changing the number of gene copies (Hou et al. 2017;Jiang and Fang 2016). These approaches are often combined to improve overall efficiency. ...
2’-deoxyguanosine is a key medicinal intermediate that could be used to synthesize anti-cancer drug and biomarker in type 2 diabetes. In this study, an enzymatic cascade using thymidine phosphorylase from Escherichia coli (EcTP) and purine nucleoside phosphorylase from Brevibacterium acetylicum (BaPNP) in a one-pot whole cell catalysis was proposed for the efficient synthesis of 2’-deoxyguanosine. BaPNP was semi-rationally designed to improve its activity, yielding the best triple variant BaPNP-Mu3 (E57A/T189S/L243I), with a 5.6-fold higher production of 2’-deoxyguanosine than that of wild-type BaPNP (BaPNP-Mu0). Molecular dynamics simulation revealed that the engineering of BaPNP-Mu3 resulted in a larger and more flexible substrate entrance channel, which might contribute to its catalytic efficiency. Furthermore, by coordinating the expression of BaPNP-Mu3 and EcTP, a robust whole cell catalyst W05 was created, capable of producing 14.8 mM 2’-deoxyguanosine (74.0% conversion rate) with a high time-space yield (1.32 g/L/h) and therefore being very competitive for industrial applications.
... The current central problem in the cascade catalytic system is the imbalance caused by different intermediate specific activities or expression levels of the multiple heterologous enzymes, resulting in substrate accumulation and/or decreased production (Muschiol et al. 2015;Yi et al. 2021). Currently, the expression levels are controlled by adjusting the strength of promoters and ribosome-binding sites (RBSs) (Jiang and Fang 2016), altering the number of gene copies (Yuan et al. 2018), and using compatible plasmids with different copies (Hou et al. 2017). In our previous studies, a combination of these approaches was successfully used to improve the catalytic abilities of an E. coli whole-cell catalyst with metabolic engineering of FAD/FADH 2 supply and its regeneration for the production of α-keto acids. ...
Efficient FAD/FADH2 regeneration is vital for enzymatic biocatalysis and metabolic pathway optimization. Here, we constructed an efficient and simple FAD/FADH2 regeneration system through a combination of l-amino acid deaminase (l-AAD) and halogenase (CombiAADHa), which was applied for catalyzing the conversion of an l-amino acid to halide and an α-keto acid. For cell-free biotransformation, the optimal activity ratio of l-AAD and halogenase was set between 1:50 and 1:60. Within 6 h, 170 mg/L of 7-chloro-tryptophan (7-Cl-Trp) and 193 mg/L of indole pyruvic acid (IPA) were synthesized in the selected mono-amino acid system. For whole-cell biotransformation, 7-Cl-Trp and IPA synthesis was enhanced by 15% (from 96 to 110 mg/L) and 12% (from 115 to 129 mg/L), respectively, through expression fine-tuning and the strengthening of FAD/FADH2 supply. Finally, ultrasound treatment was applied to improve membrane permeability and adjust the activity ratio, resulting in 1.6-and 1.4-fold higher 7-Cl-Trp and IPA yields. The products were then purified. This system could also be applied to the synthesis of other halides and α-keto acids.
Key points
• In this study, a whole cell FAD/FADH2 regeneration system co-expressing l-AAD and halogenase was constructed
• This study found that the activity and ratio of enzyme and the concentration of cofactors had a significant effect on the catalytic process for the efficient co-production of 7-chlorotryptophan and indole pyruvate
... Semi-rational design is an e cient strategy for improved catalytic activities of enzymes by site-directed saturation mutagenesis of residues lining the catalytic center, which is suitable for engineering enzyme without accurate structure or mechanism (Xu et al, 2020;Pang et al, 2023;Wang et al, 2021). For coordinating multienzyme expression in a single whole-cell, many strategies have been proposed, including using compatible plasmids with different copies, adjusting the strength of ribosome binding sites and altering the number of gene copies (Hou et al, 2017;Jiang and Fang, 2016). These approaches are often combined to further improve the overall e ciency. ...
2’-deoxyguanosine is a key medical intermediate which could be applied for the synthesis of anti-cancer drug and biomarker in type 2 diabetes. In present study, an enzymatic cascade for the efficient synthesis of 2’-deoxyguanosine was proposed by employing thymidine phosphorylase from Escherichia coli (EcTP) and purine nucleoside phosphorylase from Brevibacterium acetylicum (BaPNP) in a one-pot whole cell catalysis. Semi-rational design of BaPNP was performed to enhance its activity, resulting a best triple variant BaPNP-Mu3 (E57A/T189S/L243I), with an overall 5.6-fold higher yield of 2'-deoxyguanosine as compared with BaPNP-Mu0. Molecular dynamics simulation revealed that the engineering of BaPNP-Mu3 led to a larger and more flexible substrate entrance channel, which might contribute to its catalytic performance. Furthermore, by coordinating the expression of BaPNP-M3 and EcTP, a robust whole cell catalyst W05 was constructed, which could produce 14.8 mM 2’-deoxyguanosine with a high time-space yield (1.32 g/L/h) and therefore was very competitive for industrial applications.
... Expression of multi-protein complexes or multiple enzymes can be influenced by plasmid copy number, ribosome binding site (RBS) sequence, and rare codon usage. [36][37][38][39] Controlled co-expression of up to four enzymes in E. coli BL21(DE3) cells has been achieved using T7 promoters and terminators for each gene in tandem sequence. 40 Gene position in the plasmid and plasmid copy number can also affect multi-protein expression. ...
Cyanobacteriochrome (CBCR) GAF domains bind bilin cofactors to confer sensory wavelengths important for various cyanobacterial photosensory processes. Many isolated GAF domains autocatalytically bind bilins, becoming fluorescent. The third GAF domain of CBCR Slr1393 from Synechocystis sp. PCC6803 binds phycocyanobilin (PCB) natively, yielding red/green photoswitching properties but also binds phycoerythrobilin (PEB). GAF3-PCB has low quantum yields but non-photoswitching GAF3-PEB is brighter, making it a promising platform for new genetically encoded fluorescent tools. GAF3, however, shows low PEB binding efficiency (chromophorylation) at ~3% compared to total protein expressed in E. coli . Here we explored site-directed mutagenesis and plasmid-based methods to improve GAF3-PEB binding and demonstrate its utility as a fluorescent marker in live cells. We found that a single mutation improved chromophorylation while tuning the emission over ~30 nm, likely by shifting autoisomerization of PEB to phycourobilin (PUB). Plasmid modifications also improved chromophorylation and moving from a dual to single plasmid system facilitated exploration of a range of mutants via site saturation mutagenesis and sequence truncation. Collectively, the PEB/PUB chromophorylation was raised by ~7-fold. Moreover, we show that protein-chromophore interactions can tune autoisomerization of PEB to PUB in a GAF domain, which will facilitate future engineering of similar GAF domain-derived fluorescent proteins.
... In industry, L-Tle is mainly synthesized by chemical or enzymatic methods. Unluckily, many deficiencies, including tedious processes, harsh reaction conditions, severe pollution, poor enantioselectivity, and low yield in traditional chemical methods result in reduced competitiveness [9]. Alternatively, the green biomanufacturing of L-Tle via enzymatic catalysis became more popular owing to its high enantioselectivity and environmental friendliness [10]. ...
... Considering that the cofactor NADH is expensive, the in situ NADH regeneration system was deemed as the ideal strategy to reduce production costs. Hence, glucose dehydrogenase (GDH) or formate dehydrogenase (FDH), which use glucose or formate as co-substrates, are widely applied to drive the cofactor cycle by coupling with LeuDHs, thus improving the yield of L-Tle [4,9,15,16,28]. ...
... Considering that the cofactor NADH is expensive, the in situ NADH regeneration system was deemed as the ideal strategy to reduce production costs. Hence, glucose dehydrogenase (GDH) or formate dehydrogenase (FDH), which use glucose or formate as co-substrates, are widely applied to drive the cofactor cycle by coupling with LeuDHs, thus improving the yield of L-Tle [4,9,15,16,28]. In the present study, a whole-cell biocatalyst of E. coli (pET28a-Pbleudh-Bmgdh) carrying the genes of Pbleudh and Bmgdh (GenBank ID: ADE67871.1) from Bacillus megaterium were constructed for the co-expression of both enzymes, and a ribosomal binding site (RBS) of GTTAAAAAGGAGATATA was designed at the upstream of the Bmgdh gene. ...
Leucine dehydrogenase (LeuDH) has emerged as the most promising biocatalyst for L-tert-leucine (L-Tle) production via asymmetric reduction in trimethylpyruvate (TMP). In this study, a new LeuDH named PbLeuDH from marine Pseudomonas balearica was heterologously over-expressed in Escherichia coli, followed by purification and characterization. PbLeuDH possessed a broad substrate scope, displaying activities toward numerous L-amino acids and α-keto acids. Notably, compared with those reported LeuDHs, PbLeuDH exhibited excellent catalytic efficiency for TMP with a Km value of 4.92 mM and a kcat/Km value of 24.49 s−1 mM−1. Subsequently, L-Tle efficient production was implemented from TMP by whole-cell biocatalysis using recombinant E. coli as a catalyst, which co-expressed PbLeuDH and glucose dehydrogenase (GDH). Ultimately, using a fed-batch feeding strategy, 273 mM (35.8 g L−1) L-Tle was achieved with a 96.1% yield and 2.39 g L−1 h−1 productivity. In summary, our research provides a competitive biocatalyst for L-Tle green biosynthesis and lays a solid foundation for the realization of large-scale L-Tle industrial production.
... Precise control of the expression levels of multiple genes is critical in co-expression biocatalysis (Chen et al., 2017), and one strategy is to use different types of vectors or coexpress multiple genes through one or more vectors to avoid low expression of rate-limiting enzymes and overexpression of non-rate-limiting enzymes (Liu et al., 2019a;Yang et al., 2021;Hu et al., 2022). Other strategies are generally possible to precisely control certain enzyme expression levels by using different promoters and ribosomal binding sites (RBSs) (Jiang and Fang, 2016;Zhang et al., 2019). For example, researchers have found that using the RBS-optimized strain, the conversion rate of L-aspartate biotransformed from maleate was nearly 100%, with no intermediates or byproducts (Liu et al., 2019b). ...
Cis-3-hydroxypipecolic acid (cis-3-HyPip), a key structural component of tetrapeptide antibiotic GE81112, which has attracted substantial attention for its broad antimicrobial properties and unique ability to inhibit bacterial translation initiation. In this study, a combined strategy to increase the productivity of cis-3-HyPip was investigated. First, combinatorial optimization of the ribosomal binding site (RBS) sequence was performed to tune the gene expression translation rates of the pathway enzymes. Next, in order to reduce the addition of the co-substrate α-ketoglutarate (2-OG), the major engineering strategy was to reconstitute the tricarboxylic acid (TCA) cycle of Escherichia coli to force the metabolic flux to go through GetF catalyzed reaction for 2-OG to succinate conversion, a series of engineered strains were constructed by the deletion of the relevant genes. In addition, the metabolic flux (gltA and icd) was improved and glucose concentrations were optimized to enhance the supply and catalytic efficiency of continuous 2-OG supply powered by glucose. Finally, under optimal conditions, the cis-3-HyPip titer of the best strain catalysis reached 33 mM, which was remarkably higher than previously reported.
... Until recently, an efficient approach for (R)-or (S)-2-HBA production from L-threonine (L-Thr) was developed using recombinant Escherichia coli cells expressing separately or co-expressing L-threonine deaminase (TD), formate dehydrogenase (FDH) and lactate dehydrogenase (LDH) [20]. However, the mismatch in conversion rates for different steps in the multi-enzyme catalysis reactions often present a challenge to efficient bioconversion, hence requiring further optimization/ regulation of the different catalytic steps to get a dynamic equilibrium [21]. Thus, the development of reliable and efficient alternative routes to produce enantiopure 2-HBA is attractive. ...
Racemize 2-hydroxybutyric acid is usually synthesized by organic methods and needs additional deracemization to obtain optically pure enantiomers for industrial application. Here we present a cascade biocatalysis system in Escherichia coli BL21 which employed L-threonine deaminase (TD), NAD-dependent L-lactate dehydrogenase (LDH) and alcohol dehydrogenase (ADH) for producing optically pure (S)-2-hydroxybutyric acid ((S)-2-HBA) from bulk chemical L-threonine. To solve the mismatch in the conversion rate and the consumption rate of intermediate 2-oxobutyric acid (2-OBA) formed in the multi-enzyme catalysis reaction, ribosome binding site regulation strategy was explored to control TD expression levels, achieving an eightfold alteration in the conversion rate of 2-OBA. With the optimized activity ratio of the three enzymes and using ADH for NADH regeneration, the recombinant strain ADH-r53 showed increased production of (S)-2-HBA with the highest titer of 129 g/L and molar yield of 93% within 24 h, which is approximately 1.65 times that of the highest yield reported so far. Moreover, (S)-2-HBA could easily be purified by distillation, making it have great potential for industrial application. Additionally, our results indicated that constructing a tunable multi-enzyme-coordinate expression system in single cell had great significance in biocatalysis of hydroxyl acids.
... As a powerful tool for the heterologous expression of various proteins, Escherichia coli is an ideal host for the development of whole-cell catalysts [17,24,25]. Multiple enzymes have been co-expressed in E. coli from one vector (polycistronic vector), from multiple vectors, or from a mixture [26]. A polycistronic vector generally consists of a cluster of genes under the control of a single strong promoter (e.g., T7 promoter), whereas each gene has its own ribosome-binding site (RBS) and a stop codon [21]. ...
Background
d -Amino acids are increasingly used as building blocks to produce pharmaceuticals and fine chemicals. However, establishing a universal biocatalyst for the general synthesis of d -amino acids from cheap and readily available precursors with few by-products is challenging. In this study, we developed an efficient in vivo biocatalysis system for the synthesis of d -amino acids from l -amino acids by the co-expression of membrane-associated l -amino acid deaminase obtained from Proteus mirabilis (LAAD), meso -diaminopimelate dehydrogenases obtained from Symbiobacterium thermophilum (DAPDH), and formate dehydrogenase obtained from Burkholderia stabilis (FDH), in recombinant Escherichia coli .
Results
To generate the in vivo cascade system, three strategies were evaluated to regulate enzyme expression levels, including single-plasmid co-expression, double-plasmid co-expression, and double-plasmid MBP-fused co-expression. The double-plasmid MBP-fused co-expression strain Escherichia coli pET-21b-MBP- laad /pET-28a- dapdh - fdh , exhibiting high catalytic efficiency, was selected. Under optimal conditions, 75 mg/mL of E. coli pET-21b-MBP- laad /pET-28a- dapdh - fdh whole-cell biocatalyst asymmetrically catalyzed the stereoinversion of 150 mM l -Phe to d -Phe, with quantitative yields of over 99% ee in 24 h, by the addition of 15 mM NADP ⁺ and 300 mM ammonium formate. In addition, the whole-cell biocatalyst was used to successfully stereoinvert a variety of aromatic and aliphatic l -amino acids to their corresponding d -amino acids.
Conclusions
The newly constructed in vivo cascade biocatalysis system was effective for the highly selective synthesis of d -amino acids via stereoinversion.
... 168 Recent developments include the use of LeuDHs from other organisms and replacement of formate dehydrogenase-catalysed co-factor recycling by the more efficient glucose dehydrogenase system. [169][170][171][172][173] Directed evolution of the LeuDH from L. aphaericus resulted in a two-fold increase in specific activity as compared to the wild-type enzyme, which was met with a concomitant increase in space-time yield from 666 to 1170 g (L d) À1 . 174 Complementary to the use of LeuDH, several transaminases were also found capable of converting trimethylpyruvate (101) to L-tert-leucine (100). ...
... As shown by Chung and co-workers, production of 1,2,4-butanetriol could also be achieved from renewable resources through the use of an engineered E. coli strain capable of fermenting xylose (Scheme 37). 238 Introduction of heterologous genes encoding for Caulobacter crescentus xylose dehydrogenase (XDH) allowed for the oxidation of xylose (170) to xylonic acid (171). C3-dehydration by E. coli's native D-xylonic acid dehydratase (DHT) followed by decarboxylation with benzoylformate decarboxylase (mdlC) from Pseudomonas putida afforded 3,4-dihydroxybutanal (173). ...
With recent outbreaks of COVID-19 and Ebola, health and healthcare have once more shown to be heavily burdened by the lack of generally effective anti-viral therapies. Initial scientific ventures towards finding anti-viral agents are soon to be followed by challenges regarding their mass production. Biocatalysis offers mild, highly selective, and environmentally benign synthetic strategies for the production of pharmaceuticals in a sustainable fashion. Here we summarise biocatalytic methods that have been applied to the production of FDA-approved anti-viral drugs and their intermediates. Exemplary are the enzymatic asymmetric synthesis of amino acid components, the fermentative production of structurally complex intermediates of anti-influenza drugs and the fully enzymatic, large-scale synthesis of a potential block-buster HIV drug. With many enzyme classes being uncharted with regards to the synthesis of anti-viral agents, there is still a large unopened toolbox waiting to be unlocked. Additionally, by discussing biocatalytic strategies towards potential anti-viral agents against SARS-CoV-2, we hope to contribute to the development of novel synthetic routes to aid in the mass production of a future treatment of COVID-19.
