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Transaminase (TA) is a crucial biocatalyst for enantioselective production of the herbicide L-phosphinothricin (L-PPT). The use of enzymatic cascades has been shown to effectively overcome the unfavorable thermodynamic equilibrium of TA-catalyzed transamination reaction, also increasing demand for TA stability. In this work, a novel thermostable transaminase (PtTA) from Pseudomonas thermotolerans was mined and characterized. The PtTA showed a high specific activity (28.63 U/mg) towards 2‐oxo‐4‐[(hydroxy)(methyl)phosphinoyl]butyric acid (PPO), with excellent thermostability and substrate tolerance. Two cascade systems driven by PtTA were developed for L-PPT biosynthesis, including asymmetric synthesis of L-PPT from PPO and deracemization of D, L-PPT. For the asymmetric synthesis of L-PPT from PPO, a three-enzyme cascade was constructed as a recombinant Escherichia coli (E. coli G), by co-expressing PtTA, glutamate dehydrogenase (GluDH) and D-glucose dehydrogenase (GDH). Complete conversion of 400 mM PPO was achieved using only 40 mM amino donor L-glutamate. Furthermore, by coupling D-amino acid aminotransferase (Ym DAAT) from Bacillus sp. YM‐1 and PtTA, a two-transaminase cascade was developed for the one-pot deracemization of D, L-PPT. Under the highest reported substrate concentration (800 mM D, L-PPT), a 90.43% L-PPT yield was realized. The superior catalytic performance of the PtTA-driven cascade demonstrated that the thermodynamic limitation was overcome, highlighting its application prospect for L-PPT biosynthesis. Key points: • A novel thermostable transaminase was mined for L-phosphinothricin biosynthesis. • The asymmetric synthesis of L-phosphinothricin was achieved via a three-enzyme cascade. • Development of a two-transaminase cascade for D, L-phosphinothricin deracemization. [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]

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]

Driven by the massive demand in recent years, the production of β-alanine has significantly progressed in chemical and biological ways. Although the chemical method is relatively mature compared to biological synthesis, its high cost of waste disposal and environmental pollution does not meet the environmental protection standard. Hence, the biological method has become more prevalent as a potential alternative to the chemical synthesis of β-alanine in recent years. As a result, the aspartate pathway from l-aspartate to β-alanine (the most significant rate-limiting step in the β-alanine synthesis) catalyzed by l-aspartate-α-decarboxylase (ADC) has become a research hotspot in recent years. Therefore, it is vital to comprehensively understand the different enzymes that possess a similar catalytic ability to ADC. This review will investigate the exploratory process of unique synthesis features and catalytic properties of ADC/ADC-like enzymes in particular creatures with similar catalytic capacity or high sequence homology. At the same time, we will discuss the different β-alanine production methods which can apply to future industrialization. [ABSTRACT FROM AUTHOR]

The plant growth hormone gibberellic acid (GA3), as one of the representative secondary metabolites, is widely used in agriculture, horticulture and brewing industry. GA3 is detected in both plants and several fungi with the ability to stimulate plant growth. Currently, the main mode of industrial production of GA3 is depended on the microbial fermentation via long-period submerged fermentation using Fusarium fujikuroi as the only producing strain, qualified for its natural productivity. However, the demand of large-sale industrialization of GA3 was still restricted by the low productivity. The biosynthetic route of GA3 in F. fujikuroi is now well-defined. Furthermore, the multi-level regulation mechanisms involved in the whole network of GA3 production have also been gradually unveiled by the past two decades based on the identification and characterization of several global regulators and their mutual functions. Combined with the quick development of genetic manipulation techniques, the rational modification of producing strain F. fujikuroi development become practical for higher productivity achievement. Herein, we review the latest advances in the molecular regulation of GA3 biosynthesis in F. fujikuroi and conclude a comprehensive network involving nitrogen depression, global regulator, histone modification and G protein signaling pathway. Correspondingly, the bioengineering strategies covering conventional random mutation, genetic manipulating platform development, metabolic edition and fermentation optimization were also systematically proposed. [ABSTRACT FROM AUTHOR]

With the ban of highly toxic herbicides, such as paraquat and glyphosate, phosphinothricin (PPT) is becoming the most popular broad-spectrum and highly effective herbicide. The current PPT products in the market are usually a racemic mixture with two configurations, the D-type and L-type, of which only the L-PPT has the herbicidal activity. The racemic product is not atom economic, more toxic and may cause soil damage. Asymmetric synthesis of L-PPT has become a research focus in recent years, while biological synthesis methods are preferred for its character of environmental friendly and requiring less reaction steps when being compared to the chemical methods. We have developed a biological synthesis route to produce optically pure L-PPT from D,L-PPT in two steps using 2-carbonyl-4- (hydroxymethyl phosphonyl) butyric acid as the intermediate. In this study, we expressed the glutamate dehydrogenase and glucose dehydrogenase using Pichia pastoris as the first time. After a series of optimization, the total L-PPT yield reached 84%. The developed synthesis system showed a high potential for future industrial application. Compare to the previous plasmid-carrying-E. coli expression system, the established method may avoid antibiotic usage and provided an alternative way for industrial synthesis of optically pure L-PPT. [ABSTRACT FROM AUTHOR]

2-chloronicotinic acid (2-CA) is a key precursor for the synthesis of a series of pesticides and pharmaceuticals. Nitrilase-catalyzed bioprocess is a promising method for 2-CA production from 2-chloronicotinonitrile (2-CN). In this study, a mutant of nitrilase from Rhodococcus zopfii (RzNIT/W167G) was constitutively overexpressed with Escherichia coli as host, which exhibited a onefold increase in enzymatic activity compared with inducible expression. Biosynthesis of 2-CA using whole cells harboring nitrilase as biocatalysts were investigated and 318.5 mM 2-CA was produced, which was the highest level for 2-CA production catalyzed by nitrilase to date. 2-CA was recovered from the reaction mixture through a simple acidification step with a recovery yield of 90%. This study developed an efficient bioprocess for 2-CA with great potential for industrial application. [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]

Nicotinamide mononucleotide (NMN) or Nicotinamide-1-ium-1-β-D-ribofuranoside 5′-phosphate is a nucleotide that can be converted into nicotinamide adenine dinucleotide (NAD) in human cells. NMN has recently attracted great attention because of its potential as an anti-aging drug, leading to great efforts for its effective manufacture. The chemical synthesis of NMN is a challenging task since it is an isomeric compound with a complicated structure. The majority of biological synthetic routes for NMN is through the intermediate phosphoribosyl diphosphate (PRPP), which is further converted to NMN by nicotinamide phosphoribosyltransferase (Nampt). There are various routes for the synthesis of PRPP from simple starting materials such as ribose, adenosine, and xylose, but all of these require the expensive phosphate donor adenosine triphosphate (ATP). Thus, an ATP regeneration system can be included, leading to diminished ATP consumption during the catalytic process. The regulations of enzymes that are not directly involved in the synthesis of NMN are also critical for the production of NMN. The aim of this review is to present an overview of the biological production of NMN with respect to the critical enzymes, reaction conditions, and productivity. [ABSTRACT FROM AUTHOR]

Phosphinothricin (PPT) is one of the most prevalently using herbicides. The commercial phosphinothricin products are generally in the form of a racemic mixture, of which only the l-phosphinothricin (L-PPT) gives herbicidal function. Synthesis of optically pure L-PPT by deracemization of D/L-PPT is a promising way to cut down the environmental burden and manufacturing cost. To convert D/L-PPT to L-PPT, we expressed the catalytic enzymes by genomic integration in E. coli. The whole production was implemented in two steps in one pot using four catalytic enzymes, namely d-amino acid oxidase, catalase, glutamate dehydrogenase, and glucose dehydrogenase. Finally, after a series of process optimization, the results showed that with our system the overall L-PPT yield reached 86%. Our study demonstrated a new strategy for L-PPT synthesis, based on enzymes from chromosomal integrated expression, which does not depend on antibiotic selection, and shows a high potential for future industrial application. [ABSTRACT FROM AUTHOR]

R-2-(4-hydroxyphenoxy)propionic acid (R-HPPA) is a key chiral intermediate for phenoxypropionic acid herbicide synthesis. In this study, to improve the production of R-HPPA with B. bassiana ZJB16007, the cultivation conditions in solid-state fermentation (SSF) were investigated. The effects of various substrates on R-HPPA production were evaluated and the process parameters were also optimized. The results showed that rice bran was the optimal substrate for R-HPPA production. The optimal medium components and cultivation conditions were: rice bran: silkworm chrysalis powder = 5.25: 2.25 (g: g), nutrient salts solution 12 mL which contained 50 g/L R-PPA, pH 5.0, and cultivated at 28 °C for 11 days. Under the optimized conditions, the transformation of R-HPPA was significantly improved and the yield of R-HPPA reached 77.78%, which was 15.14% higher than that of the control (67.55%). Therefore, SSF may serve as an alternative for R-HPPA production by B. bassiana ZJB16007. [ABSTRACT FROM AUTHOR]

Background: Amphotericin B (AmB) is widely used against fungal infection and produced mainly by Streptomyces nodosus. Various intracellular metabolites of S. nodosus were identified during AmB fermentation, and the key compounds that related to the cell growth and biosynthesis of AmB were analyzed by principal component analysis (PCA) and partial least squares (PLS). Results: Rational design that based on the results of metabolomics was employed to improve the AmB productivity of Streptomyces nodosus, including the overexpression of genes involved in oxygen-taking, precursor-acquiring and product-exporting. The AmB yield of modified strain S. nodosus VMR4A was 6.58 g/L, which was increased significantly in comparison with that of strain S. nodosus ZJB2016050 (5.16 g/L). This was the highest yield of AmB reported so far, and meanwhile, the amount of by-product amphotericin A (AmA) was decreased by 45%. Moreover, the fermentation time of strain S. nodosus VMR4A was shortened by 24 h compared with that of strain. The results indicated that strain S. nodosus VMR4A was an excellent candidate for the industrial production of AmB because of its high production yield, low by-product content and the fast cell growth. Conclusions: This study would lay the foundation for improving the AmB productivity through metabolomics analysis and overexpression of key enzymes. [ABSTRACT FROM AUTHOR]

Enantioselective hydrolysis of epoxides by epoxide hydrolase (EH) is one of the most attractive approaches for the synthesis of chiral epoxides. So far, attempts to develop an efficient epoxide hydrolase -mediated biotransformation have been limited by either the low activity or insufficient enantioselectivity of epoxide hydrolase. In this study, iterative saturation mutagenesis (ISM) of epoxide hydrolase from Agrobacterium radiobacter AD1 (ArEH) was performed for efficient production of ( R)-epichlorohydrin. Six amino acid residues, I108, A110, D131, I133, T247, and G245, were selected for site saturation mutagenesis, and a sequential combination of positive mutants using ISM was constructed. Targeted mutagenesis generated five mutants (T247K, I108L, D131S, T247K/I108L, and T247K/I108L/D131S) with improved activity and enantioselectivity. Kinetics analysis showed that the best mutant, T247K/I108L/D131S, exhibited a 4.5-fold higher catalytic efficiency ( k / K ) value and a 2.1-fold higher enantioselectivity ( E value) towards epichlorohydrin than the wild-type (WT) enzyme. Molecular docking computations support the source of notably improved enantioselectivity. In addition, the triple mutant also displayed a significantly enhanced thermostability, with > 8-fold longer half-life at 50 °C than WT. A gram-scale kinetic resolution of ( R, S)-epichlorohydrin was performed using T247K/I108L/D131S mutant as biocatalyst, affording a ( R)-epichlorohydrin yield of 40.2% (> 99.9% enantiomeric excess) and an average productivity of 1410 g L d. The engineered T247K/I108L/D131S variant is a promising biocatalyst for the enzymatic synthesis of ( R)-epichlorohydrin. [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]

Objective: To improve the operational stability and reusability of an epoxide hydrolase (EH) for the biosynthesis of optically active epoxides. Results: A covalently immobilization strategy was employed to improve the stability of Agrobacterium radiobacter EH by using ethylenediamine (EDA)-functionalised epoxy resin LX-1000EP as carrier. Under the optimal conditions, the activity recovery of immobilized enzyme was 72 % and the specific activity was 634 U/g. Immobilized EH exhibited significantly enhanced thermal stability with a half-life of more than 6.8-fold at 50 °C than that of the free enzyme. A gram-scale kinetic resolution of ( R, S)-epichlorohydrin using immobilized preparation as biocatalyst was performed and ( R)-epichlorohydrin was obtained with 35 % yield and 99 % enantiomeric excess. The immobilized EH showed good operational stability and even after six reactions, it retained >85 % of the initial activity. Conclusion: The operational stability and recyclability of immobilized EH on an EDA-functionalized epoxy supports demonstrated its potential for producing ( R)-epichlorohydrin. [ABSTRACT FROM AUTHOR]

A carbonyl reductase (SCR2) gene was synthesized and expressed in Escherichia coli after codon optimization to investigate its biochemical properties and application in biosynthesis of ethyl ( S)-4-chloro-3-hydroxybutanoate (( S)-CHBE), which is an important chiral synthon for the side chain of cholesterol-lowering drug. The recombinant SCR2 was purified and characterized using ethyl 4-chloro-3-oxobutanoate (COBE) as substrate. The specific activity of purified enzyme was 11.9 U mg. The optimum temperature and pH for enzyme activity were 45 °C and pH 6.0, respectively. The half-lives of recombinant SCR2 were 16.5, 7.7, 2.2, 0.41, and 0.05 h at 30 °C, 35 °C, 40 °C, 45 °C, and 50 °C, respectively, and it was highly stable in acidic environment. This SCR2 displayed a relatively narrow substrate specificity. The apparent K and V values of purified enzyme for COBE are 6.4 mM and 63.3 μmol min mg, respectively. The biocatalytic process for the synthesis of ( S)-CHBE was constructed by this SCR2 in an aqueous-organic solvent system with a substrate fed-batch strategy. At the final COBE concentration of 1 M, ( S)-CHBE with yield of 95.3 % and e.e. of 99 % was obtained after 6-h reaction. In this process, the space-time yield per gram of biomass (dry cell weight, DCW) and turnover number of NADP to ( S)-CHBE were 26.5 mmol L h g DCW and 40,000 mol/mol, respectively, which were the highest values as compared with other works. [ABSTRACT FROM AUTHOR]

The substrate concentration and yield were shown to be very low in the production of ( R)-epichlorohydrin by hydrolysis of racemic epichlorohydrin using epoxide hydrolases in previous studies. In this work, we synthesized an epoxide hydrolase gene from Agrobacterium radiobacter and expressed it in Escherichia coli by the PCR assembly method. The recombinant A. radiobacter epoxide hydrolase (ArEH) was applied in the preparation of ( R)-epichlorohydrin and, a yield of 42.7% with ≥99% enantiomeric excess ( ee) from 25.6 mM racemic epichlorohydrin was obtained. However, the ee of ( R)-epichlorohydrin was not able to reach 99% due to substrate and product inhibition when the substrate concentration was over 320 mM. Inhibition studies revealed that ( S)-3-chloro-1,2-propanediol displayed non-competitive inhibition in the conversion of ( S)-epichlorohydrin but non-significant inhibition for ( R)-epichlorohydrin. Moreover, ArEH was successfully applied in the preparation of ( R)-epichlorohydrin at high substrate concentration by eliminating the substrate inhibition. The substrate concentration increased to 448 mM by intermittent feeding of the substrate and to 512 mM by using a two-phase reaction system, with a high yield (>27%) and ee (>98%) of ( R)-epichlorohydrin. This is the first report of high-yield production of ( R)-epichlorohydrin at high substrate concentration, laying the foundations for its application on the industrial scale. [ABSTRACT FROM AUTHOR]

Biotransformation of 1,3-dichloro-2-propanol (DCP) to epichlorohydrin (ECH) by the whole cells of recombinant Escherichia coli expressing halohydrin dehalogenase was limited by product inhibition. To solve this problem and improve the ECH yield, a biotransformation strategy using resin-based in situ product removal (ISPR) was investigated. Seven macroporous resins were examined to adsorb ECH: resin HZD-9 was the best. When 10 % (w/v) HZD-9 was added to batch biotransformation, 53.3 mM ECH was obtained with a molar yield of 88.3 %. The supplement of the HZD-9 increased the ECH volumetric productivity from 0.5 to 2.8 mmol/l min compared to without addition of resin. In fed-batch biotransformation, this approach increased ECH from 31 to 87 mM. These results provide a promising basis for the biosynthesis of ECH. [ABSTRACT FROM AUTHOR]

In this study, nitriles were used as sole sources of nitrogen in the enrichments to isolate nitrile-converting microorganisms. A novel fungus named ZJB-09150 possessing nitrile-converting enzymes was obtained with 3-cyanopyridine as sole source of nitrogen, which was identified by morphology, biology and 18S rDNA gene sequence as Fusarium proliferatum. It was found that F. proliferatum had ability to convert nitriles to corresponding acids or amides and showed wide substrate specificity to aliphatic nitriles, aromatic nitriles and ortho-substituted heterocyclic nitriles. The nitrile converting enzymes including nitrilase and nitrile hydratase in ZJB-09150 were induced by ε-caprolactam. Nitrilase obtained in this study showed high activity toward 3-cyanopyridine. It was active within pH 3.0-12.0 and temperature ranging from 25 to 65 °C with optimal at pH 9.0 and temperature 50-55 °C. The enzyme was thermostable and its half-life was 12.5 and 6 h at 45 and 55 °C, respectively. Under optimized reaction conditions, 60 mM 3-cyanopyridine was converted to nicotinic acid in 15 min, which indicated ZJB-09150 has potentials of application in large scale production of nicotinic acid. [ABSTRACT FROM AUTHOR]

Iminodiacetic acid (IDA) is widely used as an intermediate in the manufacture of chelating agents, glyphosate herbicides and surfactants. In the current work, the fragment with the length of 1,110 bp encoding the Acidovorax facilis nitrilase was obtained. The recombinant nitrilase expressed in Escherichia coli BL21 (DE3) was successfully used in the production of IDA from iminodiacetonitrile. To improve the stability of operation, the recombinant cells were entrapped in polyvinyl alcohol (PVA) and sodium alginate (SA) copolymer. The maximum relative nitrilase activity with 98.1% was further observed at 1.0% SA, 8.0% PVA, 1.0% CaCl2, and 5.0% wet cells, under conditions of 1.0% iminodiacetonitrile in distilled water and a temperature of 40°C, respectively. The entrapped cells facilitated easy separation and good recycling compared with free cells. Moreover, the immobilized cells showed good operation and storage stability. This report is the first to describe IDA preparation using immobilized recombinant E. coli harboring nitrilase. Copyright © 2012 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Abstract: Bioconversion of p-methoxyphenylacetonitrile (MOPAN) to p-methoxyphenylacetic acid (MOPAA) by immobilized cells of Bacillus subtilis ZJB-063, a nitrilase producing strain, was investigated in batch and repeated batch. Four entrapment matrixes were screened to search for a suitable support in terms of enzyme activity and reusability of the bead, and alginate exhibited significant process advantages over its other counterparts. The effects of sodium alginate concentration, bead diameter, CaCl2 concentration were examined. Maximum activity was achieved when the above parameters were set as 2.5% sodium alginate, 2mm of bead diameter, 4.0% CaCl2. An initial MOPAN concentration of 10mM was completely converted to MOPAA by entrapped cells after 8h reaction under the optimized conditions. Efficient reusability of the biocatalyst up to nine batches was achieved by immobilization, and treatment with polyethyleneimine (PEI) and glutaraldehyde (GA) further extended the longevity to 18 batches. [Copyright &y& Elsevier]

Different carbon sources lead to differential acarbose production in Actinoplanes. To uncover the underlying differentiation in the context of genes and pathways, we performed transcriptome sequencing of Actinoplanes utahensis ZJB-03852 grown on different saccharides, such as glucose, maltose, or the saccharide complex consisting of glucose plus maltose. The differentially expressed genes were classified into GO (gene ontology) terms and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways for functional annotations. Key enriched modules were uncovered. Our data revealed that both maltose and its complex with glucose gave improved acarbose titer. Sugar transportation, cytochrome oxidase, protein synthesis and amino acid metabolism modules were enriched under the saccharide complex condition, while ferritin metabolism gene expressions were enriched in the glucose medium. Our results provided the foundation for uncovering the mechanism of carbon source on acarbose production in A. utahensis. [ABSTRACT FROM AUTHOR]

Background: l-2-aminobutyric acid (l-ABA) is an unnatural amino acid that is a key intermediate for the synthesis of several important pharmaceuticals. To make the biosynthesis of l-ABA environmental friendly and more suitable for the industrial-scale production. We expand the nature metabolic network of Escherichia coli using metabolic engineering approach for the production of l-ABA. Results: In this study, Escherichia coli THR strain with a modified pathway for threonine-hyperproduction was engineered via deletion of the rhtA gene from the chromosome. To redirect carbon flux from 2-ketobutyrate (2-KB) to l-ABA, the ilvIH gene was deleted to block the l-isoleucine pathway. Furthermore, the ilvA gene from Escherichia coli W3110 and the leuDH gene from Thermoactinomyces intermedius were amplified and co-overexpressed. The promoter was altered to regulate the expression strength of ilvA* and leuDH. The final engineered strain E. coli THR ΔrhtAΔilvIH/Gap-ilvA*-Pbs-leuDH was able to produce 9.33 g/L of l-ABA with a yield of 0.19 g/L/h by fed-batch fermentation in a 5 L bioreactor. Conclusions: This novel metabolically tailored strain offers a promising approach to fulfill industrial requirements for production of l-ABA. [ABSTRACT FROM AUTHOR]

Microbial fermentation for L-methionine (L-Met) production based on natural renewable resources is attractive and challenging. In this work, the effects of medium composition and fermentation conditions were investigated to improve L-Met production by genetically engineered Escherichia coli MET-3. Statistical optimization techniques including Plackett-Burman (PB) design and Box-Behnken design (BBD) were adopted first to optimize the culture medium. Results of PB-designed experiments indicated that the culture medium components including glucose, yeast extract, KH2PO4, and MgSO4.7H2O had significant effects on L-Met biosynthesis. With their best-predicted concentration established by BBD (glucose 37.43 g/L, yeast extract 0.95 g/L, KH2PO4 1.82 g/L, and MgSO4.7H2O 4.51 g/L), L-Met titer was increased to 3.04 g/L from less than 2.0 g/L. For further enhancement of L-Met biosynthesis, the fermentation conditions of batch cultivation carried out in a 5-L fermentor were optimized, and the optimum results were obtained at an agitation rate of 300 rpm, medium pH of 7.0, and induction temperature of 28 °C. Based on the optimization parameters, fed-batch fermentation with the modified medium was conducted. As a result, great improvement of L-Met titer (12.80 g/L) and yield (0.13 mol/mol) were achieved, with an increase of 38.53% and 30.0% compared with those of the basal medium, respectively. Furthermore, higher L-Met productivity of 0.261 g/L/h was obtained, representing 2.13-fold higher in comparison to the original medium. The results may provide a helpful reference for further study on strain improvement and fermentation control. [ABSTRACT FROM AUTHOR]

O-succinyl-L-homoserine (OSH) is a promising platform chemical for the production of C4 chemicals with huge market potential which can be produced by fermentation from glucose. To construct a strain capable of producing OSH with high yield, the metJ (encodes transcriptional repressor) and metI (encodes a subunit of DL-methionine transporter) were deleted in Escherichia coli W3110 to obtain a strain E. coli ∆JI. Then, overexpression of metL (encodes bifunctional aspartate kinase/homoserine dehydrogenase II) and inactivation of metB (encodes cystathionine γ-synthase) were implemented in one step, and the OSH titer of the resulting strain E. coli ∆JIB* TrcmetL was dramatically increased to 7.30 g/L. The feedback regulation was further relieved by progressively overexpressing metAfbr (encodes homoserine O-succinyltransferase), yjeH (encodes L-methionine exporter), and thrAfbr (encodes bifunctional aspartate kinase/homoserine dehydrogenase I) to increase the metabolic flux from aspartate to OSH. The 100% rationally designed strain E. coli ∆JIB* TrcmetL/pTrc-metAfbr-Trc-thrAfbr-yjeH produced 9.31 g/L OSH from 20 g/L glucose (0.466 g/g glucose) in batch fermentation, which represents the highest OSH yield from glucose reported to date. The culture profiles of the newly constructed strains were recorded to investigate their productive properties. The effects of L-methionine addition on the fermentation process of the optimal strain were also studied. Our results demonstrate that tuning the expression level of metL, inactivation of metB, and attenuation of feedback resistance of the crucial enzymes in the biosynthetic pathway are the key factors that impact the OSH production in E. coli. [ABSTRACT FROM AUTHOR]

Adaptable exploitation of the catalytic potential of membrane-bound d-sorbitol dehydrogenase (mSLDH) from Gluconobacter oxydans is desperately needed in the industrial-scale production of miglitol. In the present study, a carbonyl group-dependent colorimetric quantification method was developed for the assay of miglitol key intermediate 6-(N-hydroxyethyl)-amino-6-deoxy-α-l-sorbofuranose (6NSL), and a high-throughput screening process of positive mutants was processed. Combined with several rounds of ultraviolet irradiation mutagenesis and screening procedure, a positive mutant strain G. oxydans ZJB16009 was obtained with significant increase in mSLDH catalytic activity by 1.5-fold, which exhibited an extremely accelerated uptake rate of d-sorbitol, and the fermentation time was significantly shortened from 22 to 11 h. In a 5-L biotransformation system, 60 g/L substrate N-2-hydroxyethyl glucamine (NHEG) was catalyzed by the resting cells of the mutant strain within 36 h and accumulated 53.6 g/L 6NSL, showing a 33.6% increase in the product yield. Therefore, it was indicated that the established high-throughput screening method could provide a highly efficient platform for the breading of G. oxydans strain for the industrial biosynthesis of miglitol intermediate 6NSL. [ABSTRACT FROM AUTHOR]

d -(−)-Pantolactone (DPL) is a key intermediate for the production of d -(+)-pantothenate (vitamin B5). Deracemization of d , l -pantolactone (D, L -PL) through oxidizing l -(+)-pantolactone (LPL) to ketopantoyl lactone (KPL) and subsequently reducing KPL to DPL is a promising route for synthesizing DPL. Herein, a newly mined l -pantolactone dehydrogenase from Rhodococcus hoagie (Rho LPLDH) was used for the oxidative dehydrogenation of LPL. To alleviate inclusion bodies formed by membrane-bound Rho LPLDH intracellular expression in E. coli , strategies involving chaperone assistance and decreasing induction temperature were used to achieve Rho LPLDH soluble expression. To enhance its activity, directed evolution and hydrophilicity-based engineering yielded increased catalytic activity and thermostability. 1 M LPL was efficiently converted to KPL by engineering strain CM5 co-expressing Rho LPLDHL254I/V241I/I156L/F224Q/N164K and chaperone. A "two stages in one-pot" method was employed in deracemization of 1 M D, L -PL with 91.2% yield. These results demonstrated that CM5 catalyst exhibits great potential in enzyme cascade deracemization for the production of DPL. • First heterologous soluble expression of l -pantolactone dehydrogenase (LPLDH). • Hydrophilicity-based engineering enhanced the activity and thermostability of LPLDH. • Catalyst CM5 was applied in deracemization of 1 M D, L -PL obtaining 91.2% DPL yield. [ABSTRACT FROM AUTHOR]

l -Methionine biosynthesis is through multilevel regulated and multibranched biosynthetic pathway (MRMBP). Because of the complex regulatory mechanism and the imbalanced metabolic flux between branched pathways, microbial production of l -methionine has not been commercialized. In this study, local metabolic response in MRMBP of l -methionine was investigated and various crucial genes in branched pathways were determined. In l -serine pathway, the crucial gene was serABC. In O -succinyl homoserine (OSH) pathway, which was the C4 backbone of l -methionine, metB and metL controlled the metabolic flux jointly. In l -cysteine pathway, the crucial gene cysE fbr could disturb the flux distribution of local network in l -methionine biosynthesis. However, no crucial gene for l -methionine production in 5-methyl tetrahydrofolate (CH 3 -THF) pathway was found. The relation between these pathways was also researched. l -Serine pathway, as the upstream pathway of l -cysteine and CH 3 -THF, played a crucial role in l -methionine biosynthesis. l -Cysteine pathway showed the strongest controlling force of the metabolic flux, and OSH pathway was second to l -cysteine pathway. In contrast, CH 3 -THF pathway was the weakest, which was probably the mainly limited steps at present and had great potential in further research. In addition, constructed W3110 IJAHFEBC/pA∗HAmL was able to produce 2.62 g/L l -methionine in flask. This study is instructive for l -methionine biosynthesis and provides a new research method of biosynthesizing other metabolic products in MRMBPs. [Display omitted] • Multilevel regulated and multibranched biosynthetic pathways (MRMBPs) for l -methionine biosynthesis were analyzed. • Various crucial genes in MRMBPs for l -methionine biosynthesis were determined. • The balance between MRMBPs for l -methionine biosynthesis was clarified. [ABSTRACT FROM AUTHOR]

The miglitol intermediate, 6-(N -hydroxyethyl)-amino-6-deoxy-α- l -sorbofuranose (6NSL), is catalyzed from N -2-hydroxyethyl glucamine (NHEG) by resting cells of Gluconobacter oxydans. One of the key factors limiting 6NSL production was the availability of oxygen during both cell cultivation and biotransformation of NHEG to 6NSL. Based on G. oxydans /pBBR1 -sldAB-pqqABCDE-tldD (G. oxydans /AB-PQQ), the Vitreoscilla hemoglobin (VHb) was heterologously expressed in G. oxydans to enhance oxygen transfer efficiency and improve 6NSL production. The recombinant G. oxydans /AB-PQQ-VHb displayed higher biomass and NHEG oxidation activity than the control stain. The transcription levels of respiratory chain-related enzyme genes in G. oxydans /AB-PQQ-VHb exhibited up-regulation, indicating that the presence of VHb promoted the respiration. The dissolved oxygen (DO) concentration for cell cultivation was optimized in a 5-L stirred bioreactor. At a DO concentration of 20%, the maximum volumetric oxidation activity of NHEG of G. oxydans /AB-PQQ-VHb in the stirred bioreactor reached 168.3 ± 3.2 U/L. Furthermore, the biotransformation of NHEG to 6NSL using G. oxydans /AB-PQQ-VHb was carried out under different oxygen tensions to investigate the effect of oxygen on 6NSL production. Finally, up to 87.5 ± 5.9 g/L 6NSL was accumulated in the reaction mixture within 16 h when the DO was controlled at 30%. • Vitreoscilla hemoglobin (VHb) was engineered to enhance oxygen transfer efficiency. • The transcription levels of respiratory chain-related enzyme genes were up-regulated. • The DO level of 20% was the optimal condition for cell proliferation. • The biotransformation of NHEG to 6NSL by resting cells was performed at 30% DO. [ABSTRACT FROM AUTHOR]

• The key amino acid residues of MetA were modified through site-saturation mutation. • Single and double mutants of MetA were obtained for improved l -methionine production. • Expression of L63F mutant in ΔIJA-HFEBC-P-SerA* strain increased product to 3.5 g/L. l -Methionine biosynthesis in Eschericha coli consists of multiple unit modules with various enzymes involved and the imbalance between different modules always restricted its productivity. In this study, the key enzymes participating in the pathway were investigated for their effect on l -methionine production and the pivotal enzyme homoserine O -succinyltransferase (MetA) was designed to be regulated. The surface amino acid residues of MetA were effectively modified through site-saturation mutagenesis and single mutants L63F, A28V, P298L and double mutant L63F/A28V were obtained with improved l -methionine productivity. The structure analysis revealed that the involved residues were on the surface loop regions, which was proposed to be conducive to the refolding of MetA and thus reduce the inhibition effect caused by l -methionine. After expression of the selected single mutant L63F in engineered E. coli ΔIJA-HFEBC strain with l -methionine efflux pump and mutated 3-phosphoglycerate dehydrogenase, the l -methionine production was significantly improved, with a final yield of 3528 mg/L. The results demonstrated the efficiency of MetA regulation for enhanced production of l -methionine and meanwhile provided important guidance for further engineering of MetA with increased l -methionine productivity. [ABSTRACT FROM AUTHOR]

[Display omitted] • A novel carbonyl reductase was isolated and modified based on based on molecular modification of hydrogen bonding network regulation. • An organic-aqueous biphasic system was first constructed for Vibegron intermediate synthesis. • The substrate concentration of 200 g/L and the spatiotemporal yield of 1.55 g/(L·h·g DCW) were higher than ever reported. Vibegron is a novel, potent, highly selective β 3 -adrenergic receptor agonist for the treatment of overactive bladder with higher therapeutic capacity and lower side effects. Methyl(2 S ,3 R)-2-((tert -butoxycarbonyl)amino)-3-hydroxy-3-phenylpropanoate ((2 S ,3 R)-aminohydroxy ester) is a key chiral intermediate for the synthesis of Vibegron. A novel carbonyl reductase from Exiguobacterium sp. s126 (Ea SDR6) was isolated using data mining technology from GenBank database with preferable catalytic activity. Hydrogen bond network regulation was performed using site-directed saturation mutagenesis and combination mutagenesis. The mutant Ea SDR6A138L/S193A was obtained with the activity improvement by 4.58 folds compared with the wild type Ea SDR6. The K m of Ea SDR6A138L/S193A was decreased from 1.57 mM to 0.67 mM, k cat was increased by 2.17 folds, and the overall catalytic efficiency k cat / K m was increased by 5.07 folds. The organic-aqueous biphasic bioreaction system for the asymmetric synthesis of (2 S ,3 R)-aminohydroxy ester was constructed for the first time. Under the substrate concentration of 150 g/L, the yield of (2 S ,3 R)-aminohydroxy ester was > 99.99%, the e.e. was > 99.99%, and the spatiotemporal yield was 1.55 g/(L·h·g DCW) after 12 h reaction. While the substrate concentration was increased to 200 g/L and the reaction lasted for 36 h, the yield of (2 S ,3 R)-aminohydroxy ester was > 99.99%, the e.e. was > 99.99% and the spatiotemporal yield was 1.05 g/(L·h·g DCW). The substrate concentration and spatiotemporal yield were higher than ever reported. [ABSTRACT FROM AUTHOR]

• A transaminase from P. fluorescens (Pf -TA) has been identified and expressed. • Pf -TA shows good temperature and pH stability. • Pf -TA catalyzes the synthesis of L-phosphinotricin from PPO using E. coli whole cells. • Up to 500 mM PPO can be used with L-alanine as amine donor. • 75% isolated yield of L-PPT with 99.9% ee was achieved. L-Phosphinotricin (L-PPT) is an active compound of a broad-spectrum herbicide used worldwide. Asymmetric synthesis of L-PPT from 4-(hydroxy(methyl)phosphoryl)-2-oxobutanoic acid (PPO) using transaminase is of great significance due to the 100% theoretical yield and high enantioselectivity. In this study, a new transaminase from Pseudomonas fluorescens ZJB09-108 (named as Pf -TA) was cloned and expressed in E. coli BL21 (DE3). The amino acid sequence analysis showed that the maximum sequence identity of Pf -TA with other transaminases is 74%. This recombinant Pf -TA exhibited a good temperature stability and pH stability. The K m and k cat of Pf -TA toward PPO were 34.02 mmol L−1 and 11.3 s−1, respectively. After optimization of the L-PPT biosynthesis catalyzed by whole-cells of E. coli harboring transaminase, >99% yield of L-PPT with 99.9% of enantiomeric excess was attained at a PPO concentration of 100 mM. High yield of L-PPT (79%) with 99.9% enantiomeric excess was achieved at high PPO concentration of 500 mM as well. These results suggested the recombinant transaminase is a potential candidate for the biosynthesis of L-PPT from PPO. [ABSTRACT FROM AUTHOR]

• Enzyme activity of mSLDH towards NHEG was improved by 2.15-fold after recombination of pBBR1-gHp0169- sldAB in G. oxydans ZJB-605. • PQQ concentration became limited for achieving higher mSLDH activity in recombinants. • Enhancement in PQQ production was achieved by a combined supplementation of Glu, Ile, Ser, Arg in culture medium, which further improved mSLDH activity by 1.33-fold. • Efficient bioconversion process from NHEG to 6NSL was performed by three repeated-batch and 184.28 g·L−1 of 6NSL was obtained with a yield of 81.9%. 6-(N-hydroxyethyl) amino-6-deoxy- l -sorbofuranose (6NSL) is the direct precursor of miglitol for diabetes therapy. The regio- and stereo-selective dehydrogenation offered by the membrane-bound d -sorbitol dehydrogenase (mSLDH) from Gluconobacter oxydans provides an elegant enzymatic method for 6NSL production. In this study, two subunits sldA and sldB of mSLDH were introduced into G. oxydans ZJB-605, and the specific enzyme activity of mSLDH towards NHEG was enhanced by 2.15-fold. However, the endogenous PQQ level was dramatically reduced in the recombinant strain and became a bottleneck to support the holo-enzyme activity. A combined supplementation of four amino acids (Glu, Ile, Ser, Arg) involved in biosynthesis of PQQ in conventional media effectively increased extracellular accumulation of PQQ by 1.49-fold, which further enhanced mSLDH activity by 1.33-fold. The synergic improvement of mSLDH activity provided in this study supports the superior high dehydrogenate activity towards substrate N-2-hydroxyethyl-glucamine, 184.28 g·L−1 of 6NSL was produced after a repeated bioconversion process catalyzed by the resting cells of G. oxydans/pBB-sldAB , all of which presenting a great potential of their industrial application in 6NSL biosynthesis. [ABSTRACT FROM AUTHOR]

Highlights • A novel (R)-selective As -TA was screened for biosynthesis of (R)-3-amino-1-butanol. • The catalytic process was optimized for enhanced production of (R)-3-amino-1-butanol. • High concentration of substrate could be effectively converted by a fed-batch method. • The product with a maximum yield of 29.6 g/L and e.e. value of 99.9% was obtained. Abstract (R)-3-amino-1-butanol is a key intermediate of Dolutegravir for the treatment of HIV/AIDS and its green and efficient biosynthesis has attracted a great deal of attention. Transaminases are currently used as promising biocatalyst for the synthesis of chiral amines. However, many transaminases have (S)-specificity and (R)-selective transaminases were less exploited and studied, making the production of (R)-amines remain challenging. In this study, a novel transaminase from Actinobacteria sp. (As -TA) was obtained and applied for the biosynthesis of (R)-3-amino-1-butanol by transferring the amino group from isopropylamine to 4-hydroxy-2-butanone. After optimization of the reaction condition and using a substrate fed-batch strategy, the conversion of 100, 200, 300, 400 and 500 mM 4-hydroxy-2-butanone reached 100%, 94.9%, 86.1%, 76.1% and 70.9%, respectively. (R)-3-amino-1-butanol with a maximum yield of 29.6 g/L and 99.9% e.e. value was obtained. This was the first time demonstrating the successful biosynthesis of (R)-3-amino-1-butanol with transaminase as biocatalyst and the obtained As -TA enriched the enzyme pool of transaminase with (R)-specificity. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Method was developed for screening enzymes or strains producing L-Phosphinothricin. • Method has good detection range, precision and accuracy. • Strain K. cryocrescens ZJB-17005 was newly isolated by the screening method. • 45% of conversion and >99.9% of e.e. were achieved at 100 g L−1 substrates. Abstract A novel and simple methodology for the detection of phosphinothricin produced by a biosynthesis approach in 96-well microtiter plates was developed based on the fluorometric determination of an isoindole derivative. The assay method to determine the generation of L-phosphinothricin was conducted with the help of a derivatization reaction. The linear detection range of the method was demonstrated to be from 0.74 to 100 μg ml−1 for the catalysis by resting cells and from 1.6 to 100 μg ml-1 for the catalysis by secretory enzymes. Meanwhile, the value of the relative standard deviation was less than 2.2% and the recovery was 99–101% of true value. Based on the method constructed in this study, one bacterial strain, Kluyvera cryocrescens ZJB-17005, with high stereoselectivity (>99%) and excellent yield (45%) was isolated from 13,284 strains in the production of L-phosphinothricin by a biosynthesis approach. [ABSTRACT FROM AUTHOR]

Asymmetric synthesis of chiral epichlorohydrin (ECH) from 1,3-dichloro-2-propanol (1,3-DCP) using halohydrin dehalogenase (HHDH) is of great value due to the 100% theoretical yield and high enantioselectivity. In this study, HheC (P175S/W249P) was immobilized on an A502Ps resin and used for the preparation of ( S )-ECH. In aqueous system, the immobilized HheC catalyzed the biosynthesis of ( S )-ECH with 83.78% yield and 92.53% enantiomeric excess (ee) at 1,3-DCP concentration of 20 mM. The non-aqueous system was further developed using water saturated ethyl acetate as solvent and reaction phase. The non-aqueous bioconversion system showed higher enantioselectivity (>98% ee) toward ( S )-ECH production with modest conversion (52.34%) compared with ever reported aqueous reactions. Batch reactions were performed in a packed-bed bioreactor for 45 batches in aqueous phase and 24 batches in non-aqueous phase. The present work demonstrated the potential of immobilized HheC (P175S/W249P) in aqueous and non-aqueous phase biosynthesis of chiral ECH. [ABSTRACT FROM AUTHOR]

tert -Butyl (3 R ,5 S )-6-chloro-3,5-dihydroxyhexanoate ((3 R ,5 S )-CDHH) is the key intermediate for synthesis of atorvastatin and rosuvastatin. Carbonyl reductase exhibits excellent activity toward tert -butyl ( S )-6-chloro-5-hydroxy-3-oxohexanoate (( S )-CHOH) to synthesize (3 R ,5 S )-CDHH. In this study, a whole cell biosynthesis reaction system to produce (3 R ,5 S )-CDHH was constructed in organic solvents. A solution of 10% ( v/v ) Tween-80 was introduced to the reaction system as a co-solvent, which greatly enhanced biotransformation process, giving 98.9% yield, >99% ee and 1.8-fold higher space time yield in 5 h bioconversion of 1 M ( S )-CHOH, compared with 98.7% yield and >99% ee in 9 h bioconversion of a purely aqueous reaction system. Moreover, a water-octanol biphasic reaction system was built and 20% of octanol was added as reservoir of substrate resulting in 98% yield, >99% ee and 4.08 mmol L −1  h −1  g −1 (wet cell weight) space time yield. This study paved a way for the whole cell biosynthesis of (3 R ,5 S )-CDHH in mono and biphasic media. [ABSTRACT FROM AUTHOR]

The addition of various sulfates for enhanced cordyceps polysaccharide (CP) production in submerged cultivation of H. sinensis was investigated, and manganese sulfate was found the most effective. 2 mM of manganese sulfate on 0 day (d) was investigated as the optimal adding condition, and the CP production reached optimum with 5.33%, increasing by 93.3% compared with the control. Furthermore, the consumption of three main precursors of CP was studied over cultivation under two conditions. Intracellular mannose content decreased by 43.1% throughout 6 days cultivation, which corresponded to CP accumulation rate sharply increased from 0 d to 6 d, and mannose was considered as the most preferred precursor for generating CP. Subsequently, mannose biosynthetic pathway was constructed and verified for the first time in H. sinensis , which constituted the important part of CP biosynthesis, and transcriptional levels of the biosynthetic genes were studied. Transcriptional level of gene cpsA was significantly up-regulated 5.35-fold and it was a key gene involved both in mannose and CP biosynthesis. This study demonstrated that manganese sulfate addition is an efficient and simple way to improve CP production. Transcriptional analysis based on biosynthetic pathway was helpful to find key genes and better understand CP biosynthesis. [ABSTRACT FROM AUTHOR]

β-Alanine is the only natural β-amino acid and an important precursor for pantothenic acid (vitamin B 5) synthesis. In this work, to improve the β-alanine fermentative production, collaborative modifications mainly focused on aspartate ammonia-lyase (aspA), phosphoenolpyruvate (PEP) – pyruvate (PYR) – oxaloacetate (OAA) – L -aspartate nodes, glucose uptake system, and β-alanine uptake system were performed, including knock out of aspA , reducing the consumption of central metabolic nodes PEP and PYR, enforcement of the non-PEP-dependent transferase system (non-PTS), inactivation of gluconeogenesis through deleting pck gene, and disruption of β-alanine uptake system. As a result, a significantly enhanced β-alanine accumulation was achieved. The β-alanine titer was increased to 6.67 g/L (final strain B15) from 2.46 g/L (original strain B1) in shake flask fermentation, and a titer of 41.12 g/L was achieved in 5-L fermentor fermentation. With the presence of succinic acid (0.25 g/L), the β-alanine titer eventually reached 52.61 g/L at 72 h, and a high productivity (0.73 g/L/h) and yield (0.268 g/g glucose) were also obtained. The yield was relatively higher compared with the reported levels and the B15 exhibited a remarkable fermentation stability over a long period. This result laid foundation for industrialization of β-alanine fermentative production. [Display omitted] • Deletion of aspA gene improved the β-alanine biosynthesis (34.15 %). • Knocking out gene dadx resulted in a nearly 10 % increased β-alanine titer. • Knocking out gene pck resulted in 45.89 % increased β-alanine titer. • Addition of succinic acid (0.25 g/L) could improve β-alanine production. [ABSTRACT FROM AUTHOR]

[Display omitted] • Rate-limiting steps of GA 3 biosynthesis were characterized in F. fujikuroi. • Multivariate modular metabolic engineering was achieved in GA 3 biosynthesis. • Precursor pool and pathway-specific channel modules were analyzed and integrated. • CPR, P450-1 and bacterial VHB contributed to P450-mediated oxidation module. • 2.89 g/L of gibberellic acid was harvested via submerged fermentation. Gibberellic acid (GA 3) is one of natural phytohormones, widely used in agriculture and downstream fields. Qualified for the nature productivity, Fusarium fujikuroi was currently employed for the industrial biotransformation from agriculture residues into GA 3. Herein, Multivariate modular metabolic engineering (MMME) was assigned to reconstitute the metabolic balance in F. fujikuroi for enhancing GA 3 production. Three modules including precursor pool, cluster-specific channel and P450-mediated oxidation in GA 3 biosynthetic pathway were defined and optimized separately. The enhancement of both precursor pool and cluster-specific channel pushed metabolic flux transfer into the GA 3 -specific pathway. Moreover, both introduction of Vitreoscilla hemoglobin and reinforcement of NADPH-dependent cytochrome P450 reductase facilitated oxidation cofactor transfer and subsequently boosted mycelium growth and GA 3 biosynthesis. Integration of three modules in the engineered strain accumulated 2.89 g/L GA 3 in shake flask via submerged fermentation, presenting a promising modular metabolic engineering model for efficient microbial transformation in agro-industrial application. [ABSTRACT FROM AUTHOR]

A novel halohydrin dehalogenase (HHDH Tm ) has been identified in Tistrella mobilis ZJB1405 and subsequently cloned and over-expressed in Escherichia coli . HHDH Tm shares low similarity (less than 38%), and has different biochemical catalytic characteristics from other HHDHs. The purified HHDH Tm displays maximum enzymatic activity toward 1,3-dichloro-2-propanol (DCP) at the more alkaline pH, 45 °C. HHDH Tm was stable up to 55 °C and approximately 60% of residual activity retained at 60 °C for 1 h. HHDH Tm shows higher activities toward halohydrins with modest enantioselectivities compared with other reported HHDHs. Interesting, ( S )-epichlorohydrin (ECH) was enantioselectively biotransformed from the prochiral 1,3-DCP with about 75% yield. Moreover, 100 g/L ethyl ( S )-4-chloro-3-hydroxybutyrate (CHBE) can be converted to ethyl ( R )-4-cyano-3-hydroxybutyrate (HN) with >99% ee and 89% isolated recovery by the recombinant HHDH Tm . Therefore, HHDH Tm is a promising biocatalyst for ( S )-ECH and ( R )-HN production. [ABSTRACT FROM AUTHOR]

[Display omitted] • Reinforcement of the precursor supply module enhanced the D-pantothenate production. • Enhancing the supply of one carbon unit module increased the yield of D-pantothenate. • Reinforcement of cofactor and energy provision enhanced D-pantothenate biosynthesis. • Omics analysis characterized metabolic variation and offered targets of modification. • 45.35 g/L D-pantothenate with a yield of 0.31 g/g glucose was produced. Biosynthesis of D-pantothenate has been widely studied as D-pantothenate is one kind of important vitamins used in food and pharmaceuticals. However, the engineered strain for D-pantothenate production was focused solely on the main biosynthetic pathway, while other important factors such as one carbon unit were ignored. Here the systematic modular engineering on different factors coupled with omics analysis were studied in Escherichia coli for efficient D-pantothenate production. Through reinforcing the precursor pool, refactoring the one carbon unit generation pathway, optimization of reducing power and energy supply, the D-pantothenate titer reached 34.12 g/L with the yield at 0.28 g/g glucose under fed-batch fermentation in 5-L bioreactor. With a further comparative transcriptome and metabolomics studies, the addition of citrate was implemented and 45.35 g/L D-pantothenate was accumulated with a yield of 0.31 g/g glucose. The systematic modular engineering coupled with omics studies provide useful strategies for the industrial production of D-pantothenate. [ABSTRACT FROM AUTHOR]

Abstract: For efficient production of 2-chloronicotinic acid from 2-chloronicotinamide, an amidase gene was synthesized by codon-optimized and expressed. The optimum pH and temperature for the biocatalytic process were 7.5 and 45–50°C, respectively. The recombinant E. coli whole cells exhibited tolerance against a high substrate concentration of up to 180mM. It could also be repeatedly used in the biotransformation and the residual activity after 10 batches was still over 50% of the initial activity. These results indicated this recombinant amidase is of great potential for the practical production of 2-chloronicotinic acid. [Copyright &y& Elsevier]

[Display omitted] • A heterologous panD was inserted into genome to obtain initial β-alanine producer. • Deletion of β-alanine uptake system significantly improved β-alanine yield. • Modification the glucose uptake system increased the β-alanine production. • Overexpression of heterologous panD and aspB further increased β-alanine titer. • A 43.94 g/L β-alanine was obtained in fed-batch fermentation. β-Alanine is the only naturally occurring β-amino acid, widely used in the fine chemical and pharmaceutical fields. In this study, metabolic design strategies were attempted in Escherichia coli W3110 for enhancing β-alanine biosynthesis. Specifically, heterologous L-aspartate-α-decarboxylase was used, the aspartate kinase I and III involved in competitive pathways were down-regulated, the β-alanine uptake system was disrupted, the phosphoenolpyruvate carboxylase was overexpressed, and the isocitrate lyase repressor repressing glyoxylate cycle shunt was delete, the glucose uptake system was modified, and the regeneration of amino donor was up-regulated. On this basis, a plasmid harboring the heterologous panD and aspB was constructed. The resultant strain ALA17/pTrc99a- panD BS - aspB CG could yield 4.20 g/L β-alanine in shake flask and 43.94 g/L β-alanine (a yield of 0.20 g/g glucose) in 5-L bioreactor via fed-batch cultivation. These modification strategies were proved effective and the constructed β-alanine producer was a promising microbial cell factory for industrial production of β-alanine. [ABSTRACT FROM AUTHOR]

Nitrilases are of significant interest both due to their potential for industrial production of valuable products as well as degradation of hazardous nitrile-containing wastes. All known functional members of the nitrilase superfamily have an underlying dimer structure. The true nitrilases expand upon this basic dimer and form large spiral or helical homo-oligomers. The formation of this larger structure is linked to both the activity and substrate specificity of these nitrilases. The sequences of the spiral nitrilases differ from the non-spiral forming homologs by the presence of two insertion regions. Homology modeling suggests that these regions are responsible for associating the nitrilase dimers into the oligomer. Here we used cysteine scanning across these two regions, in the spiral forming nitrilase cyanide dihydratase from Bacillus pumilus (CynD), to identify residues altering the oligomeric state or activity of the nitrilase. Several mutations were found to cause changes to the size of the oligomer as well as reduction in activity. Additionally one mutation, R67C, caused a partial defect in oligomerization with the accumulation of smaller oligomer variants. These results support the hypothesis that these insertion regions contribute to the unique quaternary structure of the spiral microbial nitrilases. [Display omitted] • Solubility-enhancing peptide tags and linkers were designed and screened. • Nitrilase with GKGKG tag showed improved soluble expression, thermostability and activity. • A high space-time yield of 2006.5 g/L/d was reached in 1-CA production. [ABSTRACT FROM AUTHOR]

• The genetic engineering method was employed to improve the production of 6NSL. • mSLDH and PQQ synthases were co-expressed in G. oxydans to enhance mSLDH activity. • Regulation of PQQ biosynthesis through the expression of pqqABCDE and tldD. • Repeated batch biotransformation was performed by G. oxydans /pGA004. 6-(N -hydroxyethyl)-amino-6-deoxy- l -sorbofuranose (6NSL), a key precursor in the synthesis of miglitol, is produced from N -2-hydroxyethyl-glucamine (NHEG) by the regioselective oxidation of Gluconobacter oxydans. The limitation of PQQ biosynthesis became a bottleneck for improvement of PQQ-dependent D -sorbitol dehydrogenase (mSLDH) activity. Five expression plasmids were constructed for the co-expression of the pqqABCDE gene cluster and the tldD gene on the basis of pBBR1- gHp0169 - sldAB in G. oxydans to increase the biosynthesis of PQQ. The G. oxydans /pGA004, in which pqqABCDE and tldD were expressed as a cluster under the control of gHp0169 promoter, showed the optimal performance. The intracellular PQQ concentration and specific activity of mSLDH in cells increased by 79.3 % and 53.7 %, respectively, compared to that in G. oxydans /pBBR- sldAB. Then, the repeated batch biotransformation of NHEG to 6NSL by G. oxydans /pGA004 was carried out. Up to 75.0 ± 3.0 g/L of 6NSL production with 94.5 ± 3.6 % of average conversion rate of NHEG to 6NSL was achieved after four cycles of run. These results indicated that G. oxydans /pGA004 with high productivity had great potential for 6NSL production in industrial bioprocess. [ABSTRACT FROM AUTHOR]

• Aqueous-organic two-phase system was applied for (S)-epichlorohydrin production. • Structure-based engineering was performed on halohydrin dehalogenases (HheC ps). • A zigzag tunnel and depressed cave region were key to the properties of HheC ps. • Positive mutants were obtained with increased activity and enantioselectivity. Halohydrin dehalogenases (HHDHs) represent as important biocatalysts for the production of valuable chiral epoxides and β-substituted alcohols. The HHDH from A. radiobacter (HheC) showed great potential for biosynthesis of (S)-epichlorohydrin ((S)-ECH). However, two bottlenecks including self-degradation and enzyme-mediated racemization of product in aqueous solution restricted its wide-spread applications. To solve the problems, the biocatalytic process was performed in aqueous-organic two-phase system and structure-based engineered of HheC was performed. A newly found zigzag tunnel connecting the active site region and outside space, as well as a depressed cave structure in formation of an extra three-pot herringbone channel were demonstrated to play a pivotal role affecting the activity and enantioselectivity of the enzyme. By mutation of the involved amino acid residues, two variants E255 T and E255 G showed significantly improved activity, with specific activity increased from 59.9 U/mg to 101.6 U/mg and 77.5 U/mg, respectively, while one variant Y93 L exhibited high enantioselectivity, with ee value of (S)-ECH maintained at >99 %. In addition, the synergic variant Y93 L/E255 T had both improved catalytic efficiency and enantioselectivity. The results not only facilitate the applicability of HheC for efficient production of (S)-ECH, but also give new insights into the structural-functional relationship of the enzyme. [ABSTRACT FROM AUTHOR]

• A novel covalent co-immobilization method of transaminase and PLP was constructed. • Addition of exogenous PLP was eliminated when harboring the biocatalyst. • The biocatalyst showed good operational stability in a 700-batch reaction. Transaminases (TAs, EC 2.6.1.X) are a class of promising catalysts for the synthesis of chiral amines. The addition of exogenous expensive cofactor, pyridoxal 5′-phosphate (PLP) leads to the high cost and complicated reaction composition and thus limits the industrial implementation of TAs. In this study, a novel self-sufficient biocatalyst was developed based on transaminase and PLP covalent co-immobilization with the activity recovery of 83.6% and the specific activity of 343.0 U/g catalyst. The self-sufficient BgTA biocatalyst was employed in the continuous biosynthesis of (R)-sitagliptin in a recirculating packed bed reactor (RPBR) for 700 batches reaction without extra addition of PLP. The yield and e.e. of (R)-sitagliptin for each batch were above 90% and 99% respectively. The space-time yield was 40.0 g/(L·h) which was higher than ever reported. This is the first report about the covalent co-immobilization of TA and PLP with high activity and stability, and our work also contributed to the economic flexibility of chiral amine pharmaceutical intermediates biosynthesis. [ABSTRACT FROM AUTHOR]