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β‐Alanine is the only β‐amino acid in nature and one of the most important three‐carbon chemicals. This work was aimed to construct a non‐inducible β‐alanine producer with enhanced metabolic flux towards β‐alanine biosynthesis in Escherichia coli. First of all, the assembled E. coli endogenous promoters and 5′‐untranslated regions (PUTR) were screened to finely regulate the combinatorial expression of genes panDBS and aspBCG for an optimal flux match between two key pathways. Subsequently, additional copies of key genes (panDBSK104S and ppc) were chromosomally introduced into the host A1. On these bases, dynamical regulation of the gene thrA was performed to reduce the carbon flux directed in the competitive pathway. Finally, the β‐alanine titer reached 10.25 g/L by strain A14‐R15, 361.7% higher than that of the original strain. Under fed‐batch fermentation in a 5‐L fermentor, a titer of 57.13 g/L β‐alanine was achieved at 80 h. This is the highest titer of β‐alanine production ever reported using non‐inducible engineered E. coli. This metabolic modification strategy for optimal carbon flux distribution developed in this work could also be used for the production of various metabolic products. [ABSTRACT FROM AUTHOR]

D‐amino acid oxidase (DAAO)‐catalyzed selective oxidative deamination is a very promising process for synthesizing l‐amino acids including l‐phosphinothricin (l‐PPT, a high‐efficiency and broad‐spectrum herbicide). However, the wild‐type DAAO's low activity toward unnatural substrates like d‐phosphinothricin (d‐PPT) hampers its application. Herein, a DAAO from Caenorhabditis elegans (CeDAAO) was screened and engineered to improve the catalytic potential on d‐PPT. First, we designed a novel growth selection system, taking into account the intricate relationship between the growth of Escherichia coli (E. coli) and the catalytic mechanism of DAAO. The developed system was used for high‐throughput screening of gene libraries, resulting in the discovery of a variant (M6) with significantly increased catalytic activity against d‐PPT. The variant displays different catalytic properties on substrates with varying hydrophobicity and hydrophilicity. Analysis using Alphafold2 modeling and molecular dynamic simulations showed that the reason for the enhanced activity was the substrate‐binding pocket with enlarged size and suitable charge distribution. Further QM/MM calculations revealed that the crucial factor for enhancing activity lies in reducing the initial energy barrier of the reductive half reaction. Finally, a comprehensive binding‐model index to predict the enhanced activity of DAAO toward d‐PPT, and an enzymatic deracemization approach was developed, enabling the efficient synthesis of l‐PPT with remarkable efficiency. [ABSTRACT FROM AUTHOR]

Carbonyl reductases are useful for producing optically active alcohols from their corresponding prochiral ketones. Herein, we applied a computer‐assisted strategy to increase the thermostability of a previously constructed carbonyl reductase, LsCRM4 (N101D/A117G/F147L/E145A), which showed an outstanding activity in the synthesis of the ticagrelor precursor (1S)‐2‐chloro‐1‐(3,4‐difluorophenyl)ethanol. The stability changes introduced by mutations at the flexible sites were predicted using the computational tools FoldX, I‐Mutant 3.0, and DeepDDG, which demonstrated that 12 virtually screened mutants could be thermally stable; 11 of these mutants exhibited increased thermostability. Then a superior mutant LsCRM4‐V99L/D150F was screened out from the library that was constructed by iteratively combining the beneficial sites, which showed a 78% increase in activity and a 17.4°C increase in melting temperature compared to LsCRM4. Our computer‐assisted design and combinatorial strategy dramatically increased the efficiency of thermostable enzyme production. [ABSTRACT FROM AUTHOR]

Aldo‐keto reductases (AKRs) are important biocatalysts that can be used to synthesize chiral pharmaceutical alcohols. In this study, the catalytic activity and stereoselectivity of a NADPH‐dependent AKR from Kluyveromyces dobzhanskii (KdAKR) toward t‐butyl 6‐chloro (5S)‐hydroxy‐3‐oxohexanoate ((5S)‐CHOH) were improved by mutating its residues in the loop regions around the substrate‐binding pocket. And the thermostability of KdAKR was improved by a consensus sequence method targeted on the flexible regions. The best mutant M6 (Y28A/L58I/I63L/G223P/Y296W/W297H) exhibited a 67‐fold higher catalytic efficiency compared to the wild‐type (WT) KdAKR, and improved R‐selectivity toward (5S)‐CHOH (dep value from 47.6% to >99.5%). Moreover, M6 exhibited a 6.3‐fold increase in half‐life (t1/2) at 40°C compared to WT. Under the optimal conditions, M6 completely converted 200 g/L (5S)‐CHOH to diastereomeric pure t‐butyl 6‐chloro‐(3R, 5S)‐dihydroxyhexanoate ((3R, 5S)‐CDHH) within 8.0 h, with a space‐time yield of 300.7 g/L/day. Our results deepen the understandings of the structure−function relationship of AKRs, providing a certain guidance for the modification of other AKRs. [ABSTRACT FROM AUTHOR]

Structural information can help engineer enzymes. Usually, specific amino acids in particular regions are targeted for functional reconstruction to enhance the catalytic performance, including activity, stereoselectivity, and thermostability. Appropriate selection of target sites is the key to structure‐based design, which requires elucidation of the structure–function relationships. Here, we summarize the mutations of residues in different specific regions, including active center, access tunnels, and flexible loops, on fine‐tuning the catalytic performance of enzymes, and discuss the effects of altering the local structural environment on the functions. In addition, we keep up with the recent progress of structure‐based approaches for enzyme engineering, aiming to provide some guidance on how to take advantage of the structural information. [ABSTRACT FROM AUTHOR]

Nitrilase‐mediated bioprocesses exhibited great potential in the production of value‐added carboxylic acids. However, poor thermostability of nitrilases usually restricts their industrial applications. Herein, the thermostability of nitrilase BaNITM0 was significantly improved by engineering the amino acid residues on the intersection of two dimers (C interface). Except for simultaneous enhancement of enantioselectivity and activity, the best variant V82L/M127I/L159M/F166Q/C237S/Q260H (BaNITM4) showed a 10.8‐fold increase in half‐life at 30°C compared with BaNITM0. Structural analysis demonstrated that additional hydrogen bonds were formed between the residues on the C interface, which strengthened the interactions of two symmetrical regions and spirals. Subsequently, the engineered nitrilase was immobilized onto epoxy resins LXTE‐603 and the immobilized nitrilase exhibited excellent stability over 12 repeated cycles, which indicated a great industrial potential for biosynthesis of Pregabalin precursor. [ABSTRACT FROM AUTHOR]

2‐oxo‐4‐[(hydroxy)(methyl)phosphinoyl]butyric acid (PPO) is the essential precursor keto acid for the asymmetric biosynthesis of herbicide l‐phosphinothricin (l‐PPT). Developing a biocatalytic cascade for PPO production with high efficiency and low cost is highly desired. Herein, a d‐amino acid aminotransferase from Bacillus sp. YM‐1 (Ym DAAT) with high activity (48.95 U/mg) and affinity (Km = 27.49 mM) toward d‐PPT was evaluated. To circumvent the inhibition of by‐product d‐glutamate (d‐Glu), an amino acceptor (α‐ketoglutarate) regeneration cascade was constructed as a recombinant Escherichia coli (E. coli D), by coupling Ymd‐AAT, d‐aspartate oxidase from Thermomyces dupontii (TdDDO) and catalase from Geobacillus sp. CHB1. Moreover, the regulation of the ribosome binding site was employed to overcome the limiting step of expression toxic protein TdDDO in E. coli BL21(DE3). The aminotransferase‐driven whole‐cell biocatalytic cascade (E. coli D) showed superior catalytic efficiency for the synthesis of PPO from d,l‐phosphinothricin (d,l‐PPT). It revealed the production of PPO exhibited high space–time yield (2.59 g L−1 h−1) with complete conversion of d‐PPT to PPO at high substrate concentration (600 mM d,l‐PPT) in 1.5 L reaction system. This study first provides the synthesis of PPO from d,l‐PPT employing an aminotransferase‐driven biocatalytic cascade. [ABSTRACT FROM AUTHOR]

Carbonyl reductase (CR)‐catalyzed bioreduction in the organic phase and the neat substrate reaction system is a lasting challenge, placing higher requirements on the performance of enzymes. Protein engineering is an effective method to enhance the properties of enzymes for industrial applications. In the present work, a single point mutation E145A on our previously constructed CR mutant LsCRM3, coevolved thermostability, and activity. Compared with LsCRM3, the catalytic efficiency kcat/KM of LsCRM3‐E145A (LsCRM4) was increased from 6.6 to 21.9 s−1 mM−1. Moreover, E145A prolonged the half‐life t1/2 at 40°C from 4.1 to 117 h, Tm ${T}_{m}$ was increased by 5°C, T5030 ${T}_{50}^{30}$ was increased by 14.6°C, and Topt was increased by 15°C. Only 1 g/L of lyophilized Escherichia coli cells expressing LsCRM4 completely reduced up to 600 g/L 2‐chloro‐1‐(3,4‐difluorophenyl)ethanone (CFPO) within 13 h at 45°C, yielding the corresponding (1S)‐2‐chloro‐1‐(3,4‐difluorophenyl)ethanol ((S)‐CFPL) in 99.5% eeP, with a space‐time yield of 1.0 kg/L d, the substrate to catalyst ratios (S/C) of 600 g/g. Compared with LsCRM3, the substrate loading was increased by 50%, with the S/C increased by 14 times. Compared with LsCRWT, the substrate loading was increased by 6.5 times. In contrast, LsCRM4 completely converted 600 g/L CFPO within 12 h in the neat substrate bioreaction system. [ABSTRACT FROM AUTHOR]

L‐Methionine is the unique sulfur‐containing amino acid essential for humans and animals, which is widely used in pharmaceutical, food and feed industries. Its green and efficient production has attracted a great deal of attention. Fermentation‐enzymatic coupling route is regarded to be promising for L‐methionine biosynthesis, while O‐succinyl‐L‐homoserine mercaptotransferase (MetZ) is the key biocatalyst. Exploring and engineering of MetZ with ideal catalytic properties is highly desired. Herein, the MetZ from Chromobacterium violaceum (CvMetZ) was screened and through in silico analyses, potential beneficial amino acid residues both at the access channel and around the oxygen anion holes were anchored for site‐saturation mutagenesis. The positive mutants were obtained with improved activity for L‐methionine biosynthesis using O‐succinyl‐L‐homoserine (OSH) and methyl mercaptan as substrate. The best mutant was further applied for L‐methionine production and supply of methyl mercaptan was optimized in a fed‐batch approach. The conversion of 100 g/L OSH reached 92% with L‐methionine yield of 62.6 g/L, which was well coupled with the OSH fermentation level. [ABSTRACT FROM AUTHOR]

Phosphatases are a class of enzymes catalyzing the cleavage of monophosphate ester bonds from the phosphorylated substrates. They have important applications in construction of in vitro multi‐enzymatic system for monosaccharides. However, the enzymes generally show substrate ambiguity, which has become a bottleneck for efficient biosynthesis of target products with high purity. In this study, semirational design was performed on phosphatase from Thermosipho atlanticus (Ta‐PST). The hotspot amino acid residues forming a "cap domain" were identified and selected for saturation mutagenesis. The mutant F179T and F179M showed improved substrate preference toward fructose‐6‐phosphate and mannose‐6‐phosphate, respectively. Coupling with other enzymes involved in the multi‐enzymatic system under optimized conditions, the application of F179T led to fructose yield of 80% from 10 g/L maltodextrin and the ratio between the target product and by‐product glucose was increased from 2:1 to 19:1. On the other hand, the application of F179M led to mannose yield of 59% with ratio of mannose to the by‐products glucose and fructose increased from 1:1:1 to 14:2:1. Moreover, the molecular understanding of the beneficial substitution was gained by structural analysis and molecular dynamic simulations, giving important guidance to regulate the enzyme's substrate preference. [ABSTRACT FROM AUTHOR]

Nitrilases are promising biocatalysts to produce high‐value‐added carboxylic acids through hydrolysis of nitriles. However, since the enzymes always show low activity and sometimes with poor reaction specificity toward 2‐chloronicotinonitrile (2‐CN), very few robust nitrilases have been reported for efficient production of 2‐chloronicotinic acid (2‐CA) from 2‐CN. Herein, a nitrilase from Paraburkholderia graminis (PgNIT) was engineered to improve its catalytic properties. We identified the beneficial residues via computational analysis and constructed the mutant library. The positive mutants were obtained and the activity of the "best" mutant F164G/I130L/N167Y/A55S/Q260C/T133I/R199Q toward 2‐CN was increased from 0.14 × 10−3 to 4.22 U/mg. Its reaction specificity was improved with elimination of hydration activity. Molecular docking and molecular dynamics simulation revealed that the conformational flexibility, the nucleophilic attack distance, as well as the interaction forces between the enzyme and substrate were the main reason alternating the catalytic properties of PgNIT. With the best mutant as biocatalyst, 150 g/L 2‐CN was completely converted, resulting in 2‐CA accumulated to 169.7 g/L. When the substrate concentration was increased to 200 g/L, 203.1 g/L 2‐CA was obtained with yield of 85.7%. The results laid the foundation for industrial production of 2‐CA with the nitrilase‐catalyzed route. [ABSTRACT FROM AUTHOR]

We report the discovery of an unusual halohydrin dehalogenase, HHDHamb, that can work under relatively low acidic conditions and extremely low temperatures for the bio‐nitration of epoxides using nitrite as a nitrating agent. The bio‐nitration strategy exhibits high chemo‐, regio‐, and enantioselectivity, catalyzing the kinetic resolution of various epoxides to enantiopure β‐nitroalcohols with nitro‐bearing stereocenters in up to 41 % isolated yield and >99 % enantiomeric excess (ee). Additionally, the bio‐nitration method displays a high reaction efficiency and can be performed on a gram scale. We also solved the crystal structure of HHDHamb to understand the possible structural determinants of chemoselectivity control in the bio‐nitration reaction. [ABSTRACT FROM AUTHOR]

Simultaneous evolution of multiple enzyme properties remains challenging in protein engineering. A chimeric nitrilase (BaNITM0) with high activity towards isobutylsuccinonitrile (IBSN) was previously constructed for biosynthesis of pregabalin precursor (S)‐3‐cyano‐5‐methylhexanoic acid ((S)‐CMHA). However, BaNITM0 also catalyzed the hydration of IBSN to produce by‐product (S)‐3‐cyano‐5‐methylhexanoic amide. To obtain industrial nitrilase with vintage performance, we carried out engineering of BaNITM0 for simultaneous evolution of reaction specificity, enantioselectivity, and catalytic activity. The best variant V82L/M127I/C237S (BaNITM2) displayed higher enantioselectivity (E = 515), increased enzyme activity (5.4‐fold) and reduced amide formation (from 15.8% to 1.9%) compared with BaNITM0. Structure analysis and molecular dynamics simulations indicated that mutation M127I and C237S restricted the movement of E66 in the catalytic triad, resulting in decreased amide formation. Mutation V82L was incorporated to induce the reconstruction of the substrate binding region in the enzyme catalytic pocket, engendering the improvement of stereoselectivity. Enantio‐ and regio‐selective hydrolysis of 150 g/L IBSN using 1.5 g/L Escherichia coli cells harboring BaNITM2 as biocatalyst afforded (S)‐CMHA with >99.0% ee and 45.9% conversion, which highlighted the robustness of BaNITM2 for efficient manufacturing of pregabalin. [ABSTRACT FROM AUTHOR]

Streptomyces nodosus is known as the main manufacturer of amphotericin B (AmB), which is an effective antifungal drug. It is verified that the optimization of fermentation conditions and key growth factors have a great impact on the yield of AmB. The AmB production of S. nodosus in cotton‐seed meal (CM) medium was 1.6 times than that of beef‐paste medium. The transcriptome analysis was used to analyze the effects of different nitrogen media and calcium on S. nodosus. Related genes of the EMP and TCA pathways, such as phosphofructokinase, pyruvate dehydrogenase, and citrate synthase, were upregulated in CM medium. The expression level of the PKS modules of the amphotericin synthesis gene cluster in beef‐paste medium was higher. Other functional genes, such as amphGH and amphRIV, have the advantage of expressing in CM medium. Ca2+ promoted the upregulation of genes in metabolic pathways such as EMP pathway (pyruvate dehydrogenase), TCA pathway (citrate synthase), and amphotericin synthesis genes (PKS modules). The expression of WhiB family genes SNOD_RS 13310 and SNOD_RS 17625 was positively correlated with Ca2+ concentration. In addition, in the presence of calcium, the expression level of Sec transport system genes of S. nodosus was lower. [ABSTRACT FROM AUTHOR]

Chiral amino acids are extensively applied in the pharmaceutical, food, cosmetic, and agricultural industries. As a representative example, l‐phosphinothricin (l‐PPT, a chiral non‐natural amino acid) is a broad‐spectrum herbicide. An NAD(H)‐driven biocatalytic system for the asymmetric synthesis of chiral amino acids (focused on l‐PPT) with high efficiency and low cost is highly desired. The key for the development of such biocatalytic system is to obtain an NADH‐dependent biocatalyst with high catalytic performance toward l‐PPT pro‐ketone PPO. Herein, an engineered glutamate dehydrogenase from Lysinibacillus composti (LcGluDH) with desired activity was obtained by gene mining and protein engineering. In silico analyses suggested that the volume of substrate‐binding pocket was substantially enlarged from 330.5 Å3 to 409.6 Å3. The stability of LcGluDH was increased (Tm value increased from 47.3 °C to 65.3 °C) by introducing positively charged amino acids or aromatic amino acids at position 375. The desired biocatalytic system was constructed by coupling the engineered LcGluDH and an NAD+‐dependent FDH. Through this biocatalytic system, the batch production of l‐PPT exhibited high space‐time yield (207.3 g ⋅ L−1 ⋅ day−1) with strict stereoselectivity (ee of l‐PPT>99%). Furthermore, eight other chiral amino acids were synthesised by the developed NAD(H)‐driven biocatalytic system with high ee values. [ABSTRACT FROM AUTHOR]

Biocatalysis in high‐concentration organic solvents (OSs) offers many advantages, but realizing this process remains a huge challenge. An R‐selective ω‐amine transaminase variant (AcATAM2) exhibited high activity toward 50 g/L pro‐sitagliptin ketone 1‐[1‐piperidinyl]‐4‐[2,4,5‐trifluorophenyl]‐1,3‐butanedione (PTfpB). However, AcATAM2 displayed unsatisfactory organic–cosolvent resistance against high‐concentration dimethyl sulfoxide (DMSO), which is required to enhance the solubility of the hydrophobic substrate PTfpB. Located in the substrate‐binding tunnel, enzyme gates are structural elements that undergo reversible conformational transitions, thus affecting the accessibility of the binding pocket to solvent molecules. Depending on the conformation of the enzyme gates, one can define an open or closed conformation on which the enzyme activity in OSs may depend. To enhance the DMSO resistance of AcATAM2, we identified the beneficial residues at the "enzyme gate" region via computational analysis, alanine scanning, and site‐saturation mutagenesis. Two beneficial variants, namely, AcATAM2F56D and AcATAM2F56V, not only displayed improved enzyme activity but also exhibited enhanced DMSO resistance (the half‐life value increased from 25.71 to 42.49 h under 60% DMSO). Molecular dynamic simulations revealed that the increase in DMSO resistance was mainly caused by the decrease in the number of DMSO molecules in the substrate‐binding pocket. Moreover, in the kilogram‐scale experiment, the conversion of 80 g/L substrate was increased from 50% (AcATAM2) to 85% (M2F56D in 40% DMSO) with a high e.e. of >99% within 24 h. [ABSTRACT FROM AUTHOR]

The Candida antarctica lipase B (CALB) was embedded in the metal‐organic framework, zeolitic imidazolate framework‐8 (ZIF‐8), and applied in the enzymatic synthesis of L‐ascorbic acid palmitate (ASP) for the first time. The obtained CALB@ZIF‐8 achieved the enzyme loading of 80 mg g−1 with 11.3 U g−1 (dry weight) unit activity, 59.8% activity recovery, and 92.7% immobilization yield. Under the optimal condition, ASP was synthesized with over 75.9% conversion of L‐ascorbic acid in a 10‐batch reaction. Continuous synthesis of ASP was subsequently performed in a packed bed bioreactor with an outstanding average space–time yield of 58.1 g L−1 h−1, which was higher than ever reported continuous ASP biosynthesis reactions. [ABSTRACT FROM AUTHOR]

d‐Pantothenic acid (D‐PA) is an essential vitamin widely used in food, feed, chemical, and pharmaceutical industries. An Escherichia coli platform was developed for the high‐level production of D‐PA from glucose through fed‐batch cultivation. Initially, the effects of different glucose feeding strategies D‐PA synthesis were studied. It was found that D‐PA production in glucose control (5 g/L) fed‐batch culture reached 24.3 g/L, which was 4.09 times that in the batch culture. Next, the effect of auxotrophic amino acid (isoleucine)‐limited feeding on D‐PA production was investigated. The results revealed that isoleucine feeding decreased the accumulation of by‐product acetic acid and promoted D‐PA production significantly. Furthermore, an isoleucine feeding embedded multistage glucose supply strategy was established, and a maximum titer of 39.1 g/L was achieved. To the best of our knowledge, this levels are the highest reported so far in engineered E. coli for the D‐PA production. The developed fed‐batch feeding strategy may be useful for the industrial D‐PA production by E. coli. [ABSTRACT FROM AUTHOR]

Enzyme engineering toward catalytic‐tetrad residues usually results in activity loss. Unexpectedly, we found that a directed evolution campaign yielded a beneficial residue A100 in KmCR (a carbonyl reductase from Kluyveromyces marxianus ZJB14056), which is a residue of catalytic tetrad and conserved according to multiple sequence alignment. Inspired by this finding, we performed saturation mutagenesis on all the four residues of catalytic tetrad of KmCR. A number of variants with improved enzymatic activities were obtained. Among them, the variant KmCR_A100S exhibited increased catalytic efficiency (kcat/KM = 47.3 s−1·mM−1), improved stereoselectivity (from moderate selectivity (deP = 66.7%) to strict (S)‐selectivity (deP > 99.5%)), and extended substrate scope, compared to those of KmCR_WT. In silico analysis showed that a relay system was rebuilt in KmCR via the beneficial residue S100. Furthermore, comparison of 11 protein engineering campaigns indicated that the beneficial position is easily overlooked due to the long distance (>10 Å) from ketone substrates. Since CRs share similar catalytic mechanism, the knowledge gained from this study has universal significance to CR engineering. [ABSTRACT FROM AUTHOR]

BACKGROUND: Non‐aqueous biocatalytic system has exhibited advantages including outstanding substrate solubility, facilitation for product purification, suppression of unwanted water‐dependent side reactions and rare wastewater discharge. However, limited by enzyme stability, selectivity and cofactor recycling, there has been no successful attempt to use carbonyl reductases in non‐aqueous phase. RESULTS: In this study, a non‐aqueous catalytic system of carbonyl reductase was developed for the first time with a carbonyl reductase–cofactor co‐immobilized biocatalyst. Using n‐hexane as the reaction medium, the key chiral diol intermediate of the top‐selling hypolipidemic drug rosuvastatin, tert‐butyl (3R,5S)‐6‐chloro‐3,5‐dihydroxyhexanoate ((3R,5S)‐CDHH) was prepared in a continuous packed‐bed bioreactor, with a total yield of 95.51%, enantiomeric excess (e.e.) > 99.5% and diastereomeric excess (d.e.) > 99.5% in a 44‐batch reaction. The yield of per unit biocatalyst was 34.42 mg U−1, and the space–time yield was 91.01 g h−1 L−1. CONCLUSION: Our work corroborated the valuable potential of carbonyl reductases in non‐aqueous biosynthesis of chiral pharmaceuticals and fine chemicals. © 2021 Society of Chemical Industry (SCI). [ABSTRACT FROM AUTHOR]

BACKGROUND: Miglitol is a potent alpha‐glucosidase inhibitor that is used as a hypoglycemic agent. The industrial synthesis route of miglitol involves the biotransformation from N‐2‐hydroxyethyl glucamine to 6‐(N‐hydroxyethyl)‐amino‐6‐deoxy‐alpha‐l‐sorbofuranose and the subsequent hydrogenation from 6‐(N‐hydroxyethyl)‐amino‐6‐deoxy‐alpha‐l‐sorbofuranose to miglitol. This study comprehensively investigated the hydrogenation process and catalyst, and the effect of operating parameters on the product yield. RESULTS: A 55.6% miglitol yield was obtained under the following optimal reaction conditions: 5 g L−1 palladium/carbon (Pd/C) (5% Pd); ethanol addition 1:1 ethanol:water (v/v); 80 g L−16‐(N‐hydroxyethyl)‐amino‐6‐deoxy‐alpha‐l‐sorbofuranose; reaction temperature 25 °C; solution pH 6; stirring speed 500 rpm; hydrogen pressure 1 MPa. The yield obtained was much higher than the average 36.8% in current industrial production. Catalyst deactivation during reuse was found to be caused by biological impurities, especially the macromolecular impurities introduced in previous biotransformation. The deposition of impurities on the porous support of the Pd/C catalyst may be the direct cause of catalyst deactivation. CONCLUSION: Optimal conditions for the hydrogenation in the synthesis of miglitol were determined. It also was confirmed that biological impurities need to be completely removed to avoid catalyst deactivation and to improve catalyst reusability. [ABSTRACT FROM AUTHOR]

Enzyme engineering usually generates trade‐offs between activity, stability, and selectivity. Herein, we report semirational engineering of an aldo–keto reductase (AKR) KmAKR for simultaneously enhancing its thermostability and catalytic activity. Previously, we constructed KmAKRM9 (W297H/Y296W/K29H/Y28A/T63M/A30P/T302S/N109K/S196C), which showed outstanding activity towards t‐butyl 6‐chloro‐(3R,5S)‐dihydroxyhexanoate ((3R,5S)‐CDHH), and t‐butyl 6‐cyano‐(3R,5R)‐dihydroxyhexanoate, the key chiral building blocks of rosuvastatin and atorvastatin. Under the guidance of computer‐aided design including consensus residues analysis and molecular dynamics (MD) simulations, K164, S182, S232, and Q266 were dug out for their thermostability conferring roles, generating the "best" mutant KmAKRM13(W297H/Y296W/K29H/Y28A/T63M/A30P/T302S/N109K/S196C/K164E/S232A/S182H/Q266D). The Tm and T5015 values of KmAKRM13 were 10.4 and 6.1°C higher than that of KmAKRM9, respectively. Moreover, it displayed a significantly elevated organic solvent tolerance over KmAKRM9. Structural analysis indicated that stabilization of the α‐helixes mainly contributed to thermostability enhancement. Under the optimized conditions, KmAKRM13 completely asymmetrically reduced 400 g/l t‐butyl 6‐chloro‐(5S)‐hydroxy‐3‐oxohexanoate ((5S)‐CHOH) in 8.0 h at a high substrate to catalyst ratio (S/C) of 106.7 g/g, giving diastereomerically pure (3R,5S)‐CDHH (>99.5% d.e.P) with a space‐time yield (STY) of 449.2 g/l·d. [ABSTRACT FROM AUTHOR]

Ezetimibe is a top‐selling hypolipidemic drug for the treatment of cardiovascular diseases. Biosynthesis of (4S)‐3‐[(5S)‐5‐(4‐fluorophenyl)‐5‐hydroxypentanoyl]‐4‐phenyl‐1,3‐oxazolidin‐2‐one ((S)‐ET‐5) using carbonyl reductase has shown advantages including high catalytic efficiency, excellent stereoselectivity, mild reaction conditions, and environmental friendness, and was considered as the key step for ezetimibe production. The regeneration efficiency of the cofactor, nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) is one of the main restricted factor. Recombinant Escherichia coli strain (smCR125) coexpressing carbonyl reductase (CR125) and glucose dehydrogenase were successfully constructed and applied for the production of (S)‐ET‐5 for the first time. Without extra addition of the coenzyme NADPH, the yield of 99.8% and the enantiomeric excess (e.e.) of 99.9% were achieved under ET‐4 concentration of 200 g/L. Using a substrate fed‐batch strategy, under the optimal conditions, the substrate ET‐4 concentration was increased to 250 g/L with the yield of 98.9% and the e.e. of 99.9% after 12 hr reaction. The space–time yield of 494.5 g L−1 d−1 and the space–time yield per gram biocatalyst of 24.7 g L−1 d−1 g−1 DCW were achieved, which were higher than ever reported for the biosynthesis of the ezetimibe intermediate. [ABSTRACT FROM AUTHOR]

R‐2‐(4‐hydroxyphenoxy)propionicacid (HPOPA) is a valuable intermediate for the synthesis of enantiomerically pure aryloxyphenoxypropionic acid herbicides. In this work, to improve the HPOPA biosynthesis by Beauveria bassiana ZJB16002 from the substrate R‐2‐phenoxypropionic acid (POPA), the original HPOPA producer B. bassiana ZJB16002 was subjected to physical mutagenesis with 137Cs‐γ irradiation and chemical mutagen N‐methyl‐N'‐nitro‐N‐nitrasoguanidine (NTG) induced mutagenesis. The effects of different treatment doses of the mutagens on the lethal rate and positive mutation rate were investigated, and the results showed that the optimal 137Cs‐γirradiation dose and NTG concentration was 850 Gy and 500 µg/mL, respectively. Under these conditions, a mutant strain CCN‐7 with the highest HPOPA production capacity was obtained through two rounds of 137Cs‐γ irradiation treatment followed by one round of NTG mutagenesis. At the substrate (POPA) concentration of 50 g/L, HPOPA titer of CCN‐7 reached 36.88 g/L, which was 9.73‐fold higher than the parental strain. The morphology of the wild‐type and mutant strain was compared and the results might provide helpful information in exploration of the correlation of morphology and biochemical features of B. bassiana. [ABSTRACT FROM AUTHOR]

Protein engineering is a powerful tool for improving the properties of enzymes. However, large changes in enzyme properties are still challenging for traditional evolution strategies because they usually require multiple amino acid substitutions. In this study, a feasible evolution approach by a combination of fragment swapping and semi‐rational design was developed for the engineering of nitrilase. A chimera BaNIT harboring 12 amino acid substitutions was obtained using nitrilase from Arabis alpine (AaNIT) and Brassica rapa (BrNIT) as parent enzymes, which exhibited higher enantioselectivity and activity toward isobutylsuccinonitrile for the biosynthesis of pregabalin precursor. The semi‐rational design was executed on BaNIT to further generate variant BaNIT/L223Q/H263D/Q279E with the concurrent improvement of activity, enantioselectivity, and solubility. The robust nitrilase displayed a 5.4‐fold increase in whole‐cell activity and the enantiomeric ratio (E) increased from 180 to higher than 300. Molecular dynamics simulation and molecular docking demonstrated that the substitution of residues on the A and C surface contributed to the conformation alteration of nitrilase, leading to the simultaneous enhancement of enzyme properties. The results obtained not only successfully engineered the nitrilase with great industrial potential for the production of pregabalin precursor, but also provided a new perspective for the development of novel industrially important enzymes. [ABSTRACT FROM AUTHOR]

Enzyme membrane reactors (EMRs) are becoming increasingly interesting for application in bioconversion processes in food processing, pharmaceutics, biorefinery and wastewater treatment. Integrating the highly efficient enzymatic reaction with selectable membrane separation technology, the EMRs are able to reduce product inhibition, improve the stability of the enzyme, increase the number of reaction cycles and sustainably separate products from biotransformation solutions. Generally, in EMRs, enzymes can be either free in the solution, immobilized on an additional carrier or immobilized directly in/on a porous structure of the membrane, of which the system with the enzyme immobilized directly in/on the membrane (EIM) is a relatively simple and most widely applied method. The EIM system not only facilitates recycling of the enzymes but also in many cases additionally enhances enzyme properties such as the stability and viability. A membrane with a porous structure is usually considered as a potential carrier for enzyme immobilization. A series of immobilization methods has been developed to fix the enzymes on the membranes. The purpose of this review is to systemically summarize the immobilization methods of enzymes in/on membranes and the applications of the EIM system for biocatalysis. [ABSTRACT FROM AUTHOR]

Most amino acids contain chiral centres and exist as both D‐enantiomer and L‐enantiomer. The optically pure enantiomer is often more valuable than the racemate. Enzymatic resolution provides an effective strategy to obtain optically pure amino acids but often results in large amounts of unwanted isomer. In this study, optically pure L‐glufosinate (L‐PPT) was obtained by coupling amidase‐mediated hydrolysis of N‐phenylacetyl‐D,L‐glufosinate with racemization of N‐phenylacetyl‐D‐glufosinate (NPDG), which exclusively exhibits effective herbicidal properties compared with its D‐enantiomer. To improve the yield of L‐PPT, the racemization reaction conditions were optimized, and through single‐factor experiments, the optimal reaction temperature, reaction time, and mole ratio of phenylacetic acid to NPDG were determined to be 150°C, 30 minutes, and 1.5, respectively. The response surface methodology was applied to further optimize the racemization conditions, and the final yield of L‐PPT reached 96.13% with optimum reaction temperature of 154°C, reaction time of 23 minutes, and phenylacetic acid/NPDG mole ratio of 1.7, respectively. Moreover, adding a small amount of acetic anhydride further raised the yield of L‐PPT to 97.02%. [ABSTRACT FROM AUTHOR]

Abstract: Biotic resistance may influence invasion success; however, the relative roles of species richness, functional or phylogenetic distance in predicting invasion success are not fully understood. We used biomass fraction of Chromolaena odorata, an invasive species in tropical and subtropical areas, as a measure of ‘invasion success’ in a series of artificial communities varying in species richness. Communities were constructed using species from Mexico (native range) or China (non‐native range). We found strong evidence of biotic resistance: species richness and community biomass were negatively related with invasion success; invader biomass was greater in plant communities from China than from Mexico. Harvesting time had a greater effect on invasion success in plant communities from China than on those from Mexico. Functional and phylogenetic distances both correlated with invasion success and more functionally distant communities were more easily invaded. The effects of plant‐soil fungi and plant allelochemical interactions on invasion success were species‐specific. [ABSTRACT FROM AUTHOR]

Halohydrin dehalogenase (HHDH)‐mediated dehalogenation of 1,3‐dichloro‐2‐propanol (1,3‐DCP) is a key step in the chemoenzymatic synthesis of epichlorohydrin (ECH) from glycerol. In this study, a covalent immobilization strategy was employed to enhance the stability of Agrobacterium tumefaciens HHDH using epoxy resin ES‐103B as a carrier. Under optimal conditions, the activity recovery of ES‐103B‐immobilized HHDH (HHDH@ES‐103B) was 62.4% and the specific activity was 1604 U/g. The HHDH@ES‐103B exhibited excellent thermostability, with a half‐life of 68.6 days at 40°C, which is 8.0‐times higher than that of the free HHDH. A semicontinuous biotransformation of 1,3‐DCP to ECH was performed using HHDH@ES‐103B as biocatalyst in a recirculating packed bed reactor (RPBR), resulting in an ECH yield of 94.2%, with an average productivity of 5.2 g/L/h. The RPBR system exhibited a high operational stability and even after 50 cycles of reaction, it retained > 90% of the initial conversion. Furthermore, an integrated bioprocess based on in situ product recovery (ISPR) was developed in RPBR to overcome product inhibition. The integrated bioreactor equipped with an external macroporous adsorption resin HZD‐9 column led to another 1.6‐fold increase in ECH productivity to 8.46 g/L/h. This improved stability and reusability of HHDH@ES‐103B demonstrated its potential for the biotransformation of 1,3‐DCP to ECH. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:784–792, 2018 [ABSTRACT FROM AUTHOR]

Abstract: Amphotericin B is an effective antifungal antibiotic. However, its production in the wild‐type strain of Streptomyces nodosus is relatively low. In this study, a strain of ZJB 20130827, capable of producing amphotericin B, was isolated and identified as S. nodosus based on its physiological and biochemical characteristics as well as its 16S rRNA gene sequence analysis. With N‐methyl‐N‐nitroso‐N′‐nitroguanidine (NTG) and ultraviolet (UV) treatment, this wild‐type strain was mutated for improving the yield of amphotericin B. After NTG and UV treatment, the best mutant N3 was obtained for optimization of fermentation conditions. The production of amphotericin B with N3 was 5,260 mg/L, while the wild‐type strain ZJB 20130827 was about 580 mg/L, an increase of 906.9%. A genetically stable mutant strain with high yield of amphotericin B was obtained using NTG and UV. The mutant obtained in this work could potentially be utilized in industrial production of amphotericin B. [ABSTRACT FROM AUTHOR]

t-Butyl 6-cyano-(3 R,5 R)-dihydroxyhexanoate ((3 R,5 R)- 2) is a key chiral diol precursor of atorvastatin calcium (Lipitor®). We have constructed a Kluyveromyces lactis aldo-keto reductase mutant KlAKR-Y295W/W296L ( KlAKRm) by rational design in previous research, which displayed high activity and excellent diastereoselectivity ( dep > 99.5%) toward t-butyl 6-cyano-(5 R)-hydroxy-3-oxohexanoate ((5 R)- 1). To realize in situ cofactor regeneration, a robust KlAKRm and Exiguobacterium sibiricum glucose dehydrogenase ( EsGDH) co-producer E. coli BL 21(DE3) pETDuet- esgdh (MCS1)/pET-28b (+)- klakrm was constructed in this work. Under the optimized conditions, AKR and GDH activities of E. coli BL 21(DE3) pETDuet- esgdh (MCS1)/pET-28b (+)- klakrm peaked at 249.9 U/g DCW (dry cellular weight) and 29100 U/g DCW, respectively. It completely converted (5 R)- 1 at substrate loading size of up to 60.0 g/L (5 R)- 1 in the absence of exogenous NADH, which was one-fifth higher than that of the separately prepared KlAKRm and EsGDH under the same conditions. In this manner, a biocatalytic process for (3 R,5 R)- 2 with productivity of 243.2 kg/m3 d was developed. Compared with the combination of separate expressed KlAKRm with EsGDH, co-expression of KlAKRm and EsGDH has the advantages of alleviating cell cultivation burden and elevating substrate load. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1235-1242, 2017 [ABSTRACT FROM AUTHOR]

3-Hydroxypropionic acid (3-HP) is an important platform synthesis block for sets of chemicals, but the relatively low production of 3-HP from biological sources presented major barriers for its industrial applications. In this study, a dual-substrate fermentative strategy by glycerol and glucose was proposed, and the aim was to evaluate the effect of different substrate addition strategies on the fermentation process. The results indicated that the optimal cosubstrate was glucose (20 g/L), and the enzymatic activity of aldehyde dehydrogenase (AldH) could be improved 3.5-fold as compared with no glucose addition. Continuous fed-batch fermentation at a constant speed displayed better 3-HP production of 17.20 g/L and highest specific 3-HP productivity of 1.79 mmol/(g cell·H) than the other fed-batch mode. The addition of glucose could greatly reduce the imbalance of the activity between glycerol dehydratase and AldH and provide a feasible method for improving 3-HP production. These results would be helpful in developing the 3-HP fermentation process. [ABSTRACT FROM AUTHOR]

To biosynthesize the (3R,5S)-CDHH in an industrial scale, a newly synthesized stereoselective short chain carbonyl reductase (SCR) was successfully cloned and expressed in Escherichia coli. The fermentation of recombinant E. coli harboring SCR was carried out in 500 L and 5000 L fermenters, with biomass and specific activity of 9.7 g DCW/L, 15749.95 U/g DCW, and 10.97 g DCW/L, 19210.12 U/g DCW, respectively. The recombinant SCR was successfully applied for efficient production of (3R,5S)-CDHH. The scale-up synthesis of (3R,5S)-CDHH was performed in 5000 L bioreactor with 400 g/L of (S)-CHOH at 30°C, resulting in a space-time yield of 13.7 mM/h/g DCW, which was the highest ever reported. After isolation and purification, the yield and d.e. of (3R,5S)-CDHH reached 97.5% and 99.5%, respectively. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:612-620, 2017 [ABSTRACT FROM AUTHOR]

ABSTRACT L-methionine has attracted a great deal of attention for its nutritional, pharmaceutical, and clinical applications. In this study, Escherichia coli W3110 was engineered via deletion of a negative transcriptional regulator MetJ and over-expression of homoserine O-succinyltransferase MetA together with efflux transporter YjeH, resulting in L-methionine overproduction which is up to 413.16 mg/L. The partial inactivation of the L-methionine import system MetD via disruption of metI made the engineered E. coli Δ metJ Δ metI/pTrcA*H more tolerant to high L-ethionine concentration and accumulated L-methionine to a level 43.65% higher than that of E. coli W3110 Δ metJ/pTrcA*H. Furthermore, deletion of lysA, which blocks the lysine biosynthesis pathway, led to a further 8.5-fold increase in L-methionine titer of E. coli Δ metJ Δ metI Δ lysA/pTrcA*H. Finally, addition of Na2S2O3 to the media led to an increase of fermentation titer of 11.45%. After optimization, constructed E. coli Δ metJ Δ metI Δ lysA/pTrcA*H was able to produce 9.75 g/L L-methionine with productivity of 0.20 g/L/h in a 5 L bioreactor. This novel metabolically tailored strain of E. coli provides an efficient platform for microbial production of L-methionine. Biotechnol. Bioeng. 2017;114: 843-851. © 2016 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Optically pure t-butyl 6-cyano-( 3R, 5R)-dihydroxyhexanoate (( R)- 1b) is the key precursor for atorvastatin calcium, the most widely used cholesterol-lowering drug. In this work, a strain ZJB-09224 capable of asymmetrically reducing t-butyl 6-cyano-( 5R)-hydroxy-3-oxohexanoate ( 1a) to corresponding optically pure ( R)- 1b was successfully isolated from soil sample, identified belonging to Rhodotorula glutinis based on the morphology, physiological tests, and the 18S rDNA sequence analysis. It was found that heat treatment of cell suspension at 45 °C for 25 Min significantly improved R. glutinis ZJB-09224 stereoselectivity. The asymmetric bioreduction of 1a was most efficient at pH 7.5, 35 °C, 50 mM (15.0 g L−1) substrate concentration, 40.0 g DCW L−1 cell loading size, 0.54 M (60.0 g L−1) sodium lactate acting as co-substrate. Under these optimal conditions, 0.046 M ( R)- 1b was produced with de (diastereomeric excess) value of 99.2% after 40 H conversion. Moreover, R. glutinis ZJB-09224 has a broad substrate spectrum, making it a potential tool for some valuable chiral alcohol pharmaceutical intermediates synthesis. [ABSTRACT FROM AUTHOR]

( R)-(−)-Mandelic acid ( R-MA) is widely used both as a versatile intermediate for pharmaceuticals and a resolving agent in chiral resolution processes. In the current study, to improve the stability of operation, recombinant Escherichia coli cells expressing nitrilase from Alcaligenes faecalis were immobilized for the enantioselective hydrolysis of ( R, S)-mandelonitrile to R-MA. Different immobilization methods including entrapment matrices, entrapment matrices cross-linked by cross-linking and polymerization agents, and direct cross-linking cells using glutaraldehyde (GA) or bionic silicon were investigated. To facilitate industrial solid-liquid separation, the direct cross-linking recombinant E. coli cells using diatomite/GA/polyethyleneimine with 135.95% relative activity compared with free cells was chosen using water as the reaction medium. The operational stability of the immobilized cells was obviously superior to that of free cells, without significant activity loss after 28 cycles of batch reaction and the successive production of R-MA could reach 1.88 M. Moreover, the immobilized cells showed good storage stability with about 52% relative activity after storing for 30 days at 4 °C. Therefore, the immobilized biocatalyst is very promising for upscale production of optically pure R-MA with high performance and low cost. [ABSTRACT FROM AUTHOR]

BACKGROUND The regioselective hydration of alicyclic α,ω-dinitrile 1-cyanocyclohexaneacetonitrile (1- CCHAN), followed by hydrogenation of ω-cyano group is a green and elegant route to gabapentin. As the selective chemical hydration of dinitrile is virtually impossible, a bioprocess for regioselective hydration of 1- CCHAN was developed using microbial nitrile hydratase. RESULTS A newly isolated NHase producing strain, Rhodococcus aetherivorans ZJB1208, was successfully used for hydration of 1- CCHAN. Some key parameters of the biocatalytic process, including reaction temperature, pH, catalyst loading and substrate loading, were optimized. The fed-batch biotransformation was performed in non-buffered water system with the continuous precipitation of 1-cyanocyclohexaneacetamide. The substrate loading was increased up to 864 g L−1 (6.0 mol L−1), giving a product concentration of 966.7 g L−1 and biocatalyst yield (gproduct/gcat) of 204.2. CONCLUSION This study is the first example of microbial nitrile hydratase with both excellent regioselectivity and strong substrate tolerance for alicyclic dinitrile and affords a potentially industrial route to gabapentin. © 2015 Society of Chemical Industry [ABSTRACT FROM AUTHOR]