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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]

Alcohol dehydrogenases (ADHs), which belong to the oxidoreductase superfamily, catalyze the interconversion between alcohols and aldehydes or ketones with high stereoselectivity under mild conditions. ADHs are widely employed as biocatalysts for the dynamic kinetic resolution of racemic substrates and for the preparation of enantiomerically pure chemicals. This review provides an overview of biotechnological applications for ADHs in the production of chiral pharmaceuticals and fine chemicals. [ABSTRACT FROM AUTHOR]

This paper investigates the coordinated motion control problem of autonomous underwater vehicles (AUVs), and an adaptive cooperative formation control method based on event-triggered mechanism is designed. In the estimation of external disturbance and model uncertainty, adaptive parameters are adopted to represent the upper bound of the learning weight matrix, which reduces the computational pressure of the system. It is proved theoretically that the signals in the system are semi-global uniformly ultimately bounded and independent of the initial state of the system. An event-triggered control mechanism is introduced, and the system updates the control law only when the event is triggered. Simulation results show that the controller can achieve the expected formation shape and save the communication resources of the system. [ABSTRACT FROM AUTHOR]

A new Keggin-based 3D framework with 1D U-shaped channels, [Ag 3 (H 2 O)(L) 2 ](H 2 PMo 12 O 40)·H 2 O (1) (HL = 4-(1H-1,2,4-triazole-1-methyl) benzoic acid), was synthesized under hydrothermal conditions, which displays good properties of photocatalytic degradation of MB and electrocatalytic reduction of BrO 3 - and NO 2 – ions. Moreover, it also has the antimicrobial activities against Escherichia coli , Monilia albican and Xanthomonas axonopodis pv. Citri. [Display omitted] • A new 3D framework constructed from Keggin clusters and 4-(1H-1,2,4-triazole-1-methyl) benzoic acid was synthesized by hydrothermal reaction. • 1D U-shaped channels exist in compound 1. • Compound 1 displays good properties of photocatalytic degradation of MB and electrocatalytic reduction of BrO 3 - and NO 2 – ions. • The antimicrobial activities against Escherichia coli , Monilia albican and Xanthomonas axonopodis pv. Citri have also been investigated. A novel polyoxometalate-based metal–organic framework (POMOF), [Ag 3 (H 2 O)(L) 2 ](H 2 PMo 12 O 40)·H 2 O (1) (HL = 4-(1H-1,2,4-triazole-1-methyl) benzoic acid), was synthesized under hydrothermal conditions. The structure was characterized by single crystal X-ray diffraction, elemental analysis, IR and TGA. In compound 1 , Ag ions are connected by organic ligands into a 1D U-shaped channel, and the 1D U-shaped channels are further linked into a 3D framework by {PMo 12 }. Compound 1 displays good properties of photocatalytic degradation of MB and electrocatalytic reduction of BrO 3 - and NO 2 – ions. Moreover, the antimicrobial activities against Escherichia coli , Monilia albican and Xanthomonas axonopodis pv. Citri have also been investigated. [ABSTRACT FROM AUTHOR]

The enantioselective resolution of ( R , S )-2,3-dichloro-1-propanol (( R , S )-DCP) to ( S )-DCP by whole cells of a recombinant Escherichia coli expressing halohydrin dehalogenase (HHDH) activity was limited by product inhibition. To solve this problem to improve the productivity of ( S )-DCP, an n -heptane–aqueous biphasic system was adopted in this work. The influential operational parameters including phase volumetric ratio, buffer pH and reaction temperature were optimized. Under the optimal reaction conditions, significant improvements of substrate concentration and biocatalyst productivity (375 mM and 7.64 mmol ( S )-DCP g −1 cell) were achieved in this n -heptane–aqueous biphasic system compared with aqueous single-phase system (150 mM and 2.97 mmol g −1 cell). The scale-up biosynthesis of ( S )-DCP was successfully performed in a 2-L stirred reactor, resulting in a 128.8 mM ( S )-DCP with enantiomeric excess of 99.1% and average productivity of 2.07 g ( S )-DCP L −1 h −1 , respectively. [ABSTRACT FROM AUTHOR]

Highlights: [•] A novel bioprocess for (S)-3-cyano-5-methyl hexanoic acid by esterase was reported. [•] Activity and selectivity of the esterase were affected by the alcohol moiety of esters. [•] E-value of the hydrolysis reaction was notably enhanced by addition of 50% DMSO. [•] The whole-cell catalysts showed strong tolerance against high DMSO concentration. [ABSTRACT FROM AUTHOR]

Abstract: 1,3-Dihydroxyacetone can be produced by biotransformation of glycerol with glycerol dehydrogenase from Gluconobacter oxydans cells. Firstly, improvement the activity of glycerol dehydrogenase was carried out by medium optimization. The optimal medium for cell cultivation was composed of 5.6g/l yeast extract, 4.7g/l glycerol, 42.1g/l mannitol, 0.5g/l K2HPO4, 0.5g/l KH2PO4, 0.1g/l MgSO4·7H2O, and 2.0g/l CaCO3 with the initial pH of 4.9. Secondly, an internal loop airlift bioreactor was applied for DHA production from glycerol by resting cells of G. oxydans ZJB09113. Furthermore, the effects of pH, aeration rate and cell content on DHA production and glycerol feeding strategy were investigated. 156.3±7.8g/l of maximal DHA concentration with 89.8±2.4% of conversion rate of glycerol to DHA was achieved after 72h of biotransformation using 10g/l resting cells at 30°C, pH 5.0 and 1.5vvm of aeration rate. [Copyright &y& Elsevier]

Abstract: Trehalase, with the target to control insects, nematodes and fungi, is of increasing interest and has been investigated extensively in recent years. Validamycin compounds, as competitive trehalase inhibitors and lead compounds with broad applications have attracted substantial attention as well. In this study, the characterizations of termites trehalase were investigated and the inhibitory effects of validamycin compounds on the termites trehalase were studied as well. Results showed that the termites trehalase is presumably belonging to the acid trehalase with optimal pH of 3.3 and optimal temperature of 37°C. It was investigated that the concentrations of validoxylamine A (VAA), validoxylamine B (VBB), validamycin A (VA) and validamycin B (VB) required for 50% inhibition IC50 of termites trehalase were calculated to be 14.73mgl−1, 20.80mgl−1, 3.17×103 mgl−1and 2.24×103 mgl−1, respectively. The inhibition kinetic constant Ki values for the above validamycin compounds were 3.2×10−6 moll−1, 1.03×10−5 moll−1 , 4.02×10−4 moll−1and 2.69×10−4 moll−1, respectively. Validoxylamine A appeared to be the most potential termites trehalase inhibitor among the four compounds. [Copyright &y& Elsevier]

Abstract: A nitrilase-producing strain ZJUTB06-99, capable of biotransforming acrylonitrile into acrylic acid, was newly isolated from soil samples. Based on the morphology, physiological tests, ATB system and its 16S rDNA sequence, strain ZJUTB06-99 was identified as Arthrobacter nitroguajacolicus. Optimal reaction conditions were investigated by the manipulation and measurement of various parameters including pH, temperature and certain cationic metals. The highest nitrilase activity was obtained when reaction was carried out at a pH of 6.5 phosphate buffer and in temperature of 40°C water bath. The nitrilase of A. nitroguajacolicus ZJUTB06-99 exhibited excellent thermostability. Nitrilase activity was strongly inhibited by Hg2+, Ag+ and Cu2+, but Ni2+ and Ca2+ increased enzyme activity to 163% and 158%, respectively. The investigation of substrates spectrum showed that A. nitroguajacolicus ZJUTB06-99 exhibited the highest nitrilase activity towards aromatic nitriles such as phenylacetonitrile. However, no detectable activity was recorded when any of the tested amides were used as substrates. [Copyright &y& Elsevier]

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]

Abstract: Resting cells of Rhodococcus sp. G20 were used for the transformation of β-aminopropionitrile to β-alanine, an important beta amino acid. A 23 central composite experimental design was performed with the purpose of optimizing the β-alanine production in a bubble column reactor with 200mL working volume using response surface methodology (RSM). The individual and interactive effects of three independent variables (cells loading, substrate concentration, airflow rate) on β-alanine production were investigated. A quadratic polynomial predictive model was obtained after statistical analysis to predict the optimum biotransformation conditions. The optimum bioconversion conditions of β-aminopropionitrile in a batch operation for β-alanine production were as follows: cells loading of 16.50gww/200mL, substrate concentration of 1.29% (v/v), and airflow rate of 86.56L/h, under which an overall 40.6% increase in productivity of β-alanine was obtained. The influences of the temperature and pH on the conversion were also studied, and the optimums were 30°C and pH 7.5. The measured activation energy (E a) was found to be 22,199J/mol, thus indicating the presence of diffusional resistance. [Copyright &y& Elsevier]

Abstract: Valienamine is an important medicinal intermediate with broad use in the synthesis of some stronger α-glucosidase inhibitors. In order to improve valienamine concentration in the fermentation broth and make the downstream treatment easy, a fed-batch process for the enhanced production of valienamine by Stenotrophomonas maltrophilia in a stirred tank bioreactor was developed. Results showed that supplementation of validamycin A in the process of cultivation could increase the valienamine concentration. One-pulse feeding was observed to be the best strategy. The maximum valienamine concentration of 2.35gL−1 was obtained at 156h when 86.4g of validamycin A was added to a 15-L bioreactor containing 8L fermentation medium with one-pulse feeding. The maximum valienamine concentration had a great improvement and was increased above 100% compared to batch fermentation in the stirred tank bioreactor. The pH-controlled experiments showed that controlling the pH in the process of one-pulse feeding fermentation had not obvious effect on the production of valienamine. [Copyright &y& Elsevier]

Abstract: Valienamine, an aminocyclitol with similar configuration to α-glucose, has a strong inhibitory effect on α-glucosidase. α-Glucosidase plays an important role in insect carbohydrate metabolism. The inhibitory effect of valienamine on the enzymatic activity of honeybee (Apis cerana Fabr.) α-glucosidase was investigated. Our results show that valienamine inhibition of honeybee α-glucosidase was pH- and dose-dependent, but temperature-independent. Valienamine is shown to be a potent and competitive reversible inhibitor of honeybee α-glucosidase in vitro with an IC50 value of 5.22×10−5 M and K i value of 3.54×10−4 M at pH 6.5, 45°C. Valienamine has the potential to be developed into novel insecticides. [Copyright &y& Elsevier]

A method for the separation and preparation of validamycin A and validamycin B by anion-exchange resin is described. The two compounds, validamycin A and validamycin B, could be separated effectively in the 10 × 400 mm column with anion-exchange resin A600 under the optimized conditions: elution flow rate of 0.25 mL/min, added quantity of 0.7 g, bed height of 330 mm, and deionized water as eluent. The yields of validamycin A and validamycin B were 58.9% and 89.5%, respectively. [ABSTRACT FROM AUTHOR]

Abstract: Valienamine, an aminocyclitol, is an important medicinal intermediate with broad use in the synthesis of some stronger α-glucosidase inhibitors. The bacterial strain Stenotrophomonas maltrophilia CCTCC M 204024 was recently isolated in our laboratory for production of valienamine from validamycin A. In this study, conditions were varied to investigate valienamine production using this strain. We found the optimized conditions included (1) use of validamycin A as the sole carbon source (initial concentration of 15gL−1), (2) use of (NH4)2SO4 as the sole nitrogen source, (3) pH 7.0, (4) temperature range between 28 and 37°C, and (5) k L a of 133h−1. Under these optimal conditions, an average of 1.15gL−1 valienamine was obtained after fermentation for 6d. Fermentation in a 15-L mechanically stirred tank bioreactor containing 10L of culture medium was also done to observe valienamine production. The concentration in culture broth reached 1.09gL−1 after 6d of fermentation. It is concluded that degradation of validamycin A by S. maltrophilia is a promising method to achieve an effective way to production of valienamine. [Copyright &y& Elsevier]

Abstract: The effects of initial culture pH ranging from 3.5 to 6.5 on biomass content and astaxanthin concentration by Xanthophyllomyces dendrorhous were investigated in shake flask fermentation. An optimal initial pH of 6.0 for cell growth was obtained with cell density at 17.21g/l, while an optimum pH of 5.0 for astaxanthin accumulation was obtained with volumetric concentration of astaxanthin at 20.48mg/l. Batch bioprocess of astaxanthin fermentation by X. dendrorhous at various pH values ranging from 4.0 to 6.0 was examined. In batch fermentation under a controlled pH, the optimum pH for cell growth was 6.0 with specific cell growth 0.049h−1, and the optimum pH for astaxanthin formation in cell was 4.0 with specific product yield (Y p/x) at 1.52mg/g. A pH-shift strategy was developed to gain higher volumetric concentration of astaxanthin in batch fermentation. The maximum volumetric concentration of astaxanthin reached 27.05mg/l, increased by 24.1% as comparison with that of constant pH fermentation. [Copyright &y& Elsevier]

Abstract: The ability of Nocardia sp. 108 to hydrate indole-3-acetonitrile to indole-3-acetamide was identified. Cell culture showed that Nocardia sp. 108 acrylonitrile-hydrating activity reached its maximum, 1209.8U/g DCW, after cultivated at 28°C for 45h. Under the biotransformation conditions of 28°C and pH 7.5, the maximal indole-3-acetonitrile conversion ratio and indole-3-acetamide yield were 34.34 and 32.68% (w/w), respectively. Moreover, the K m and V max values for the indole-3-acetonitrile biotransformation catalyzed by Nocardia sp. 108 were 1.18×10−2 mol/l and 1.16×10−3 mol/lmin. Ethanol supplemented to the reaction system dramatically enhanced indole-3-acetonitrile aqueous solubility. Furthermore, 4% (v/v) ethyl acetate raised the indole-3-acetonitrile bioconversion capacity 78%. [Copyright &y& Elsevier]

Abstract: Rice straw is a by-product of rice production, and a great bioresource as raw biomass material for manufacturing value-adding protein for animal feedstock, which has been paid more and more attention. In the present work, utilizing rice straw hydrolysate as a substrate for microbial biomass production in 11.5L external-loop airlift bioreactors was investigated. Rice straw hydrolysate obtained through acid-hydrolyzing rice straw was used for the culture of yeast Candida arborea AS1.257. The influences of gas flow rate, initial liquid volume, hole diameter of gas sparger and numbers of sieve plates on microbial biomass production were examined. The best results in the external-loop airlift bioreactor were obtained under 9.0L initial liquid volume, 1.1(v/v)/min gas flow rate during culture time of 0–24h and 1.4(v/v)/min gas flow rate of 24–48h at 29±1°C. The addition of the sieve plates in the riser of the external-loop airlift bioreactor increased productivity. After 48h, under optimized operation conditions, crude protein productivity with one sieve and two sieves were 13.6mg/mL and 13.7mg/mL, respectively, comparing 12.7mg/mL without sieves in the airlift bioreactor and 11.7mg/mL in the in the 10-L mechanically stirred tank bioreactor. It is feasible to operate the external-loop airlift bioreactors and possible to reduce the production cost for microbial biomass production from the rice straw hydrolysate. [Copyright &y& Elsevier]

Immobilized Pseudomonas sp. HZ519 cells have been used for transformation of validamycin A to valienamine and the degradation pathway of validamycin A by Pseudomonas sp. HZ519 has also been studied. Substrate inhibition in immobilized cell system was avoided. An average of 8.6 g L -1 valienamine concentration was obtained when concentration of validamycin A was increased up to 120 g L -1 . Through a treatment of the immobilized cells with 0.3 mol L -1 substrate, the activity of the immobilized cells was increased distinctly. Compared with free cells, the productivity of valienamine by CA-immobilized cells was improved about three times. The reusability of the immobilized cells was evaluated with repeated–batch degradation experiments. The Tiele modulus was obtained from the experimental effectiveness factor. The result showed that the degradation process in the immobilized system was governed by intraparticle diffusion and chemical reaction. [ABSTRACT FROM AUTHOR]

Different cation resins, IR-120 and HZ-001, were used as catalysts to prepare validoxylamine A through validamycin A degradation. Herein, influences of reaction time, reaction temperature, resin usage, initial validamycin A concentration, as well as external and internal diffusion limitation, on the degradation ratio of validamycin A were studied. Results demonstrated that validamycin A could almost completely be degraded into validoxylamine A under optimized operation conditions of 5.0% (w/v) resin usage, 1.0% (w/v) initial validamycin A concentration, agitation speed of 200 rpm, and heating at 100 °C for 5 h. [Copyright &y& Elsevier]

Hydrogenation plays an important role in the production of bulk and fine chemicals. In the present work, the hydrogenation process of 1-cyanocyclohexaneacetic acid (1-CA), a key intermediate in the chemo-enzymatic synthesis of the antiepileptic drug gabapentin, was investigated. The catalysts were screened, the operating parameters for the 1-CA hydrogenation were determined by response surface methodology, and the effects of biological impurities on the reaction and catalysts were investigated. The reaction temperature, reaction solution pH and reaction time were identified as the main factors for hydrogenation by single-factor experiments. Under the optimal reaction conditions: reaction temperature 110 °C, solution pH 12.33, reaction time 3.74 h, hydrogen pressure 2 MPa and stirring speed 500 rpm, the substrate conversion reached 100%, and the total product yield reached 96.40%, which was very close to the prediction (96.88%). It was demonstrated that biological impurities greatly affect the reaction rate of hydrogenation, as well as the activity and reusability of the catalyst, in which protein impurities lead to catalyst deactivation through cumulative deposition on the catalyst surface and within the pores. [ABSTRACT FROM AUTHOR]

The unspecific peroxygenase (UPO) from Cyclocybe aegerita (AaeUPO) can selectively oxidize C–H bonds using hydrogen peroxide as an oxygen donor without cofactors, which has drawn significant industrial attention. Many studies have made efforts to enhance the overall activity of AaeUPO expressed in Komagataella phaffii by employing strategies such as enzyme-directed evolution, utilizing appropriate promoters, and screening secretion peptides. Building upon these previous studies, the objective of this study was to further enhance the expression of a mutant of AaeUPO with improved activity (PaDa-I) by increasing the gene copy number, co-expressing chaperones, and optimizing culture conditions. Our results demonstrated that a strain carrying approximately three copies of expression cassettes and co-expressing the protein disulfide isomerase showed an approximately 10.7-fold increase in volumetric enzyme activity, using the 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) as the substrate. After optimizing the culture conditions, the volumetric enzyme activity of this strain further increased by approximately 48.7%, reaching 117.3 U/mL. Additionally, the purified catalytic domain of PaDa-I displayed regioselective hydroxylation of R-2-phenoxypropionic acid. The results of this study may facilitate the industrial application of UPOs. Key points: • The secretion of the catalytic domain of PaDa-I can be significantly enhanced through increasing gene copy numbers and co-expressing of protein disulfide isomerase. • After optimizing the culture conditions, the volumetric enzyme activity can reach 117.3 U/mL, using the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) as the substrate. • The R-2-phenoxypropionic acid can undergo the specific hydroxylation reaction catalyzed by catalytic domain of PaDa-I, resulting in the formation of R-2-(4-hydroxyphenoxy)propionic acid. [ABSTRACT FROM AUTHOR]

A facile and efficient one-pot, single-step method for deracemizing a broad range of 2-hydroxyacids to (R)-2-hydroxyacids was established by combination of resting cells of an (S)-hydroxyacid dehydrogenase-producing microorganism and an (R)-ketoacid reductase-producing microorganism. [ABSTRACT FROM AUTHOR]

Nitrilases represent a distinct class of enzymes that play a pivotal role in catalyzing the hydrolysis of nitrile compounds, leading to the formation of corresponding carboxylic acids. These enzymatic entities have garnered significant attention across a spectrum of industries, encompassing pharmaceuticals, agrochemicals, and fine chemicals. Moreover, their significance has been accentuated by mounting environmental pressures, propelling them into the forefront of biodegradation and bioremediation endeavors. Nevertheless, the natural nitrilases exhibit intrinsic limitations such as low thermal stability, narrow substrate selectivity, and inadaptability to varying environmental conditions. In the past decade, substantial efforts have been made in elucidating the structural underpinnings and catalytic mechanisms of nitrilase, providing basis for engineering of nitrilases. Significant breakthroughs have been made in the regulation of nitrilases with ideal catalytic properties and application of the enzymes for industrial productions. This review endeavors to provide a comprehensive discourse and summary of recent research advancements related to nitrilases, with a particular emphasis on the elucidation of the structural attributes, catalytic mechanisms, catalytic characteristics, and strategies for improving catalytic performance of nitrilases. Moreover, the exploration extends to the domain of process engineering and the multifarious applications of nitrilases. Furthermore, the future development trend of nitrilases is prospected, providing important guidance for research and application in the related fields. • The substrate promiscuity of nitrilases enhanced their versatility. • The catalytic characteristics and improvement strategies of nitrilase are overviewed. • The process engineering expanded the application scenarios of nitrilase. • The industrial and environmental applications of nitrilase are summarized. • Challenges and future research directions of nitrilase engineering are discussed. [ABSTRACT FROM AUTHOR]

D-Pantothenic acid, as a momentous vitamin, is extensively applied to feed, medicine, cosmetics and other fields. However, there are still limitations to produce D-pantothenic acid by microbial fermentation at present. In this paper, we constructed a recombinant strain for D-pantothenic acid production by blocking the organic acid pathway, boosting pyruvate biosynthesis, relieving feedback inhibition of acetolactate synthase, improving glucose intake capacity, and modifying essential genes in the metabolic pathway. In addition, a new acetolactate isomeroreductase mutant V412A origin from Escherichia coli (EcAHAIR) encoded by ilvC was obtained to explore its substrate promiscuity. Compared with the wild type, the variant EcAHAIR-V412A has reduced steric hindrance and enhanced intermolecular forces, resulting in a high affinity for 2-acetolactate. Eventually, the fermentation production of the final strain DPAN19/trc-ilvCV412A reached 4.65 g/L, increased by 192.5% compared with strain DPA8 in shake flask cultivation and produced 62.82 g/L D-pantothenic acid in a 5 L bioreactor. The metabolic engineering strategies and enzyme modification approaches described in this paper provide a particular perspective for the bio-manufacturing of D-pantothenic acid, branched-chain amino acids and its derivates. [ABSTRACT FROM AUTHOR]

Steroid-based drugs are now mainly produced by the microbial transformation of phytosterol, and a two-step bioprocess is adopted to reach high space–time yields, but byproducts are frequently observed during the bioprocessing. In this study, the catabolic switch between the C19- and C22-steroidal subpathways was investigated in resting cells of Mycobacterium neoaurum NRRL B-3805, and a dose-dependent transcriptional response toward the induction of phytosterol with increased concentrations was found in the putative node enzymes including ChoM2, KstD1, OpccR, Sal, and Hsd4A. Aldolase Sal presented a dominant role in the C22 steroidal side-chain cleavage, and the byproduct was eliminated after sequential deletion of opccR and sal. Meanwhile, the molar yield of androst-1,4-diene-3,17-dione (ADD) was increased from 59.4 to 71.3%. With the regard of insufficient activity of rate-limiting enzymes may also cause byproduct accumulation, a chromosomal integration platform for target gene overexpression was established supported by a strong promoter L2 combined with site-specific recombination in the engineered cell. Rate-limiting steps of ADD bioconversion were further characterized and overcome. Overexpression of the kstD1 gene further strengthened the bioconversion from AD to ADD. After subsequential optimization of the bioconversion system, the directed biotransformation route was developed and allowed up to 82.0% molar yield with a space–time yield of 4.22 g·L−1·day−1. The catabolic diversion elements and the genetic overexpression tools as confirmed and developed in present study offer new ideas of M. neoaurum cell factory development for directed biotransformation for C19- and C22-steroidal drug intermediates from phytosterol. Key points: • Resting cells exhibited a catabolic switch between the C19- and C22-steroidal subpathways. • The C22-steroidal byproduct was eliminated after sequential deletion of opccR and sal. • Rate-limiting steps were overcome by promoter engineering and chromosomal integration. [ABSTRACT FROM AUTHOR]

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]

Tacrolimus (FK506) is a widely used and clinically important immunosuppressant drug that can be produced by fermentation of Streptomyces tsukubaensis. The industrial strains are typically obtained through multiple rounds of mutagenesis and screening, a labor-intensive process. We have established an efficient yeast cell based screening method for the evolutionary process of high-FK506-yielding strain. The S. tsukubaensis strains of different FK506 yields were tested for zone of growth inhibition of the wild type and calcineurin mutant (cnb1∆) yeast strains. We found that different FK506 yields correspond well to altered yeast growth inhibitions. Based on the combinational inhibition effects of FK506 with different antifungals that have been frequently reported, we also tested the zone of inhibition by addition of fluconazole, amphotericin B and caspofungin to the medium. In the end, for the best screening performance, we systemically evaluated the strategy when different yeast strains and different antifungals were used according to the clarity, size, and divergence of the inhibition circles. Using different yeast strains and antifungals, we successfully broadened the screening spectrum. An efficient high-FK506-yield S. tsukubaensis screening method has been established and optimized. [ABSTRACT FROM AUTHOR]

Steroid-based drugs are now mainly produced by the microbial transformation of phytosterol, and a two-step bioprocess is adopted to reach high space–time yields, but byproducts are frequently observed during the bioprocessing. In this study, the catabolic switch between the C19- and C22-steroidal subpathways was investigated in resting cells of Mycobacterium neoaurum NRRL B-3805, and a dose-dependent transcriptional response toward the induction of phytosterol with increased concentrations was found in the putative node enzymes including ChoM2, KstD1, OpccR, Sal, and Hsd4A. Aldolase Sal presented a dominant role in the C22 steroidal side-chain cleavage, and the byproduct was eliminated after sequential deletion of opccR and sal. Meanwhile, the molar yield of androst-1,4-diene-3,17-dione (ADD) was increased from 59.4 to 71.3%. With the regard of insufficient activity of rate-limiting enzymes may also cause byproduct accumulation, a chromosomal integration platform for target gene overexpression was established supported by a strong promoter L2 combined with site-specific recombination in the engineered cell. Rate-limiting steps of ADD bioconversion were further characterized and overcome. Overexpression of the kstD1 gene further strengthened the bioconversion from AD to ADD. After subsequential optimization of the bioconversion system, the directed biotransformation route was developed and allowed up to 82.0% molar yield with a space–time yield of 4.22 g·L−1·day−1. The catabolic diversion elements and the genetic overexpression tools as confirmed and developed in present study offer new ideas of M. neoaurum cell factory development for directed biotransformation for C19- and C22-steroidal drug intermediates from phytosterol. Key points: • Resting cells exhibited a catabolic switch between the C19- and C22-steroidal subpathways. • The C22-steroidal byproduct was eliminated after sequential deletion of opccR and sal. • Rate-limiting steps were overcome by promoter engineering and chromosomal integration. [ABSTRACT FROM AUTHOR]

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]

The structural integrity and esthetic appeal of concrete can be compromised by concrete cracks. Promise has been shown by microbe-induced calcium carbonate precipitation (MICP) as a solution for concrete cracking, with a focus on urease-producing microorganisms in research. Bacillus cereus was isolated from soil and employed for this purpose in this study due to its high urease activity. The strain exhibited strong tolerance for alkaline media and high salt levels, which grew at a pH of 13 and 4% salt concentration. The repair of concrete cracks with this strain was evaluated by assessing the effects of four different thickeners at varying concentrations. The most effective results were achieved with 10 g/L of sodium carboxymethyl cellulose (CMC-Na). The data showed that over 90% repair of cracks was achieved by this system with an initial water penetration time of 30 s. The study also assessed the quantity and sizes of crystals generated during the bacterial mineralization process over time to improve our understanding of the process. Key points: • MICP using Bacillus cereus shows potential for repairing concrete cracks. • Strain tolerates alkaline media and high salt levels, growing at pH 13 and 4% salt concentration. • Sodium carboxymethyl cellulose (CMC-Na) at 10 g/L achieved over 90% repair of cracks. [ABSTRACT FROM AUTHOR]

Surface-crosslinked hydrogel particles encapsulating E. coli cells harboring nitrilase were constructed and used as a durable biocatalyst in the synthesis of gabapentin intermediate. E. coli cells (50 g/L) expressing nitrilase were encapsulated in 2% (w/v) alginate hydrogel, 0.5%(w/v) glutaraldehyde and 1%(w/v) polyethyleneimine were sequentially used as crosslinker. The constructed alginate particles with GA&PEI composite crosslinking had a similar catalytic capacity compared to equivalent free cells, which could completely hydrolyze 1.0 M substrate 1-cyanocyclohexaneacetonitrile within 4 h at pH 7.5 and 35 °C, and have a specific activity of 134.6 U/g, an optimum temperature at 55 °C and a corresponding half-life up to 19.1 ± 1.9 h. The particles showed excellent storage stability, reusability and mechanical strength, which retained 90.3% activity after 2-month storage at 4 °C, and 83.2% activity after 14-day storage at 35 °C. In batch reactions, 50 g/L particles could be reused for maximum 32 cycles, maintained 63.5% residual activity and 50.7% particle integrity, providing an average product yield of 94.6% in the first 30 batches and a productivity of 1467.03 g/L/day. Besides, the stability and catalytic activity of the particles were almost unaffected in several organic co-solvents and in purified water, showing their good industrial potential. [Display omitted] • Surface-crosslinked hydrogel particles encapsulating E. coli cells were prepared as biocatalysts. • The particles have similar catalytic capacity compared to equivalent free cells. • High activity and strength enable it to completely hydrolyze 1 M substrate within 4 h. • The particles retained 90.3% activity after 2 months of storage and could be reused up to 32 times. [ABSTRACT FROM AUTHOR]

Methionine is the only one of the essential amino acids that contain sulfur, widely used as a feed additive in agriculture. In this study, the availability of 5-methyl-tetrahydrofolate was confirmed as the main limitation in the complex multibranched biosynthetic pathway of L-methionine. The cycle of one-carbon units was thoroughly investigated and modified to supply 5-methyl-tetrahydrofolate for L-methionine production, such as enhancing the supply of precursor, expediting the conversion rate of the cycle, introducing exogenous serine hydroxymethyltransferase and increasing pool size of one-carbon units carrier. The final strain MYA/pAmFA-4 was able to produce 20.89 g/L L-methionine by fed-batch fermentation, which was the highest titer reported in the literatures. This study is instructive for other metabolites biosynthesized needing one-carbon units or having a complex multibranched biosynthetic pathway. [ABSTRACT FROM AUTHOR]

O-Acetyl-L-homoserine (OAH) is a potentially important platform metabolic intermediate for the production of homoserine lactone, methionine, 1,4-butanediol and 1,3-propanediol which have giant market value. Currently, multiple strategies have been adopted to explore sustainable production of OAH. However, the production of OAH by consuming cheap bio-based feedstocks with Escherichia coli as the chassis is still in its infancy. Construction of high yield OAH-producing strains is of great significance in industry. In this study, we introduced an exogenous metA from Bacillus cereus (metXbc) and engineered an OAH-producing strain by combinatorial metabolic engineering. Initially, exogenous metXs/metA were screened and used to reconstruct an initial biosynthesis pathway of OAH in E. coli. Subsequently, the disruption of degradation and competitive pathways combined with optimal expression of metXbc were carried out, accumulating 5.47 g/L OAH. Meanwhile, the homoserine pool was enriched by overexpressing metL with producing 7.42 g/L OAH. Lastly, the carbon flux of central carbon metabolism was redistributed to balance the metabolic flux of homoserine and acetyl coenzyme A (acetyl-CoA) in OAH biosynthesis with accumulating 8.29 g/L OAH. The engineered strain produced 24.33 g/L OAH with a yield of 0.23 g/g glucose in fed-batch fermentation. By these strategies, the key nodes for OAH synthesis were clarified and corresponding strategies were proposed. This study would lay a foundation for OAH bioproduction. [ABSTRACT FROM AUTHOR]

Methionine is the only one of the essential amino acids that contain sulfur, widely used as a feed additive in agriculture. In this study, the availability of 5-methyl-tetrahydrofolate was confirmed as the main limitation in the complex multibranched biosynthetic pathway of L-methionine. The cycle of one-carbon units was thoroughly investigated and modified to supply 5-methyl-tetrahydrofolate for L-methionine production, such as enhancing the supply of precursor, expediting the conversion rate of the cycle, introducing exogenous serine hydroxymethyltransferase and increasing pool size of one-carbon units carrier. The final strain MYA/pAmFA-4 was able to produce 20.89 g/L L-methionine by fed-batch fermentation, which was the highest titer reported in the literatures. This study is instructive for other metabolites biosynthesized needing one-carbon units or having a complex multibranched biosynthetic pathway. [ABSTRACT FROM AUTHOR]

O-Acetyl-L-homoserine (OAH) is a potentially important platform metabolic intermediate for the production of homoserine lactone, methionine, 1,4-butanediol and 1,3-propanediol which have giant market value. Currently, multiple strategies have been adopted to explore sustainable production of OAH. However, the production of OAH by consuming cheap bio-based feedstocks with Escherichia coli as the chassis is still in its infancy. Construction of high yield OAH-producing strains is of great significance in industry. In this study, we introduced an exogenous metA from Bacillus cereus (metXbc) and engineered an OAH-producing strain by combinatorial metabolic engineering. Initially, exogenous metXs/metA were screened and used to reconstruct an initial biosynthesis pathway of OAH in E. coli. Subsequently, the disruption of degradation and competitive pathways combined with optimal expression of metXbc were carried out, accumulating 5.47 g/L OAH. Meanwhile, the homoserine pool was enriched by overexpressing metL with producing 7.42 g/L OAH. Lastly, the carbon flux of central carbon metabolism was redistributed to balance the metabolic flux of homoserine and acetyl coenzyme A (acetyl-CoA) in OAH biosynthesis with accumulating 8.29 g/L OAH. The engineered strain produced 24.33 g/L OAH with a yield of 0.23 g/g glucose in fed-batch fermentation. By these strategies, the key nodes for OAH synthesis were clarified and corresponding strategies were proposed. This study would lay a foundation for OAH bioproduction. [ABSTRACT FROM AUTHOR]

Chiral amino acids are extensively applied in the pharmaceutical, food, cosmetic, agricultural, and feedstuff industries. The development of synthetic methodologies for optically pure amino acids has been driven by their significant applications. Among the various synthesis methods for the production of chiral amino acids, enzymatic asymmetric synthesis is a unique preparation strategy that shows great potential. This review provides an overview of the reported methods for enzymatic asymmetric synthesis of chiral amino acids, including asymmetric reductive amination of keto acids, asymmetric transfer of an amino group to keto acids, enantioselective addition of ammonia to α,β-unsaturated acids, and aldol condensation of an amino acid to aldehydes. [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]

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]

D -Pantothenic acid (D -PA) is an essential vitamin with wide applications. However, the biotechnological production of D -PA is still not competitive with the chemical synthesis in terms of production cost. Ketopantoate hydroxymethyltransferase is a crucial enzyme in the D -PA synthetic pathway in Escherichia coli encoded by the panB gene. Here a hot spots study was applied to a ketopantoate hydroxymethyltransferase from Corynebacterium glutamicum (Cg KPHMT) to relieve the product inhibitory effect and thus improve the D -PA production. Compared with the wild type, the double-site variant Cg KPHMT-K25A/E189S showed 1.8 times higher enzyme activity and 2.1 times higher catalytic efficiency, 1.88 and 3.32 times higher inhibitory constant of α-ketoisovalerate and D -PA, respectively. The D -PA yield using E. coli W3110 adopted the double-site variant was 41.17 g·L−1 within 48 h, a 9.80 g·L−1 increase. Structural analysis of K25A/E189S revealed the expansion of the entry channel and the change of the electric charge from negative to uncharged due to the substitution from glutamic acid to serine at site 189. Our study emphasized the positive roles of ketopantoate hydroxymethyltransferase in D -PA production and paved the way by analyzing critical enzymes in the synthetic pathway of E. coli to increase the D -PA yield. [Display omitted] • A double-site variant of KPHMT with improved properties was obtained by hot spot based-engineering. • The double-site variant K25A/E189S was increased by 2.8-fold and 2.1-fold to the wild-type in EA and Kcat. • The inhibitory effect of ketoisovalerate and D -PA on variant K25A/E189S was partially relieved. • The D -PA yield was increased by 9.80 g/L by mutagenesis of KPHMT in E. coli in a 5-L scale fermentation. [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]

Glycoside hydrolases (GHs) exhibit high activity and stability under harsh conditions, such as high temperatures and extreme pHs, given their wide use in industrial biotechnology. However, strategies for improving the acidophilic and alkalophilic adaptations of GHs are poorly summarized due to the complexity of the mechanisms of these adaptations. This review not only highlights the adaptation mechanisms of acidophilic and alkalophilic GHs under extreme pH conditions, but also summarizes the recent advances in engineering the pH performances of GHs with a focus on four strategies of protein engineering, enzyme immobilization, chemical modification, and medium engineering (additives). The examples described here summarize the methods used in modulating the pH performances of GHs and indicate that methods integrated in different protein engineering techniques or methods are efficient to generate industrial biocatalysts with the desired pH performance and other adapted enzyme properties. [ABSTRACT FROM AUTHOR]

Glucose isomerase (GI) is used in vitro to convert d -glucose to d -fructose, which is capable of commercial producing high fructose corn syrup (HFCS). To manufacture HFCS at elevated temperature and reduce the cost of enriching syrups, novel refractory GIs from Thermoanaerobacterium xylanolyticum (TxGI), Thermus oshimai (ToGI), Geobacillus thermocatenulatus (GtGI) and Thermoanaerobacter siderophilus (TsGI) were screened via genome mining approach. The enzymatic characteristics research showed that ToGI had higher catalytic efficiency and superior thermostability toward d -glucose among the screened GIs. Its optimum temperature reached 95 °C and could retain more than 80% of initial activity in the presence of 20 mM Mn 2+ at 85 °C for 48 h. The K m and k cat / K m values for ToGI were 81.46 mM and 21.77 min −1 mM −1 , respectively. Furthermore, the maximum conversion yield of 400 g/L d -glucose to d -fructose at 85 °C was 52.16%. Considering its excellent high thermostability and ameliorable application performance, ToGI might be promising for realization of future industrial production of HFCS at elevated temperature. [ABSTRACT FROM AUTHOR]

Amidase signature (AS) family amidases are known to exhibit broad substrate specificity. According to the available genome sequence data, a novel AS family amidase, Pl -Ami, was identified and cloned from the genome of Parvibaculum lavamentivorans ZJB14001. The recombinant amidase was overexpressed in Escherichia coli BL21, purified and functionally characterized. The optimal pH and temperature for Pl -Ami were 9.5 and 45 °C, respectively. Pl -Ami preferred long chain aliphatic amides as substrates, while no activity was detected towards aromatic, heterocyclic and other amides. The highest enzyme activity of 128 U/mg was obtained when hexanoamide was used as substrate. Kinetic analysis indicated that the extension of chain length of aliphatic amides considerably decreased the K m values, and the turnover number ( k cat ) was higher with long chain aliphatic amides as substrates. The obtained results provided a distinct understanding of substrate specificity of AS family amidases. [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]

(S)-2-chlorophenylglycine ((S)-CPG) is a key chiral intermediate for the synthesis of clopidogrel. Herein, a novel, efficient and environmentally friendly chemo-enzymatic route for the preparation of optically pure (S)-CPG was developed. A straightforward chemical synthesis of the corresponding prochiral keto acid substrate (2-chlorophenyl)glyoxylic acid (CPGA) was developed with 91.7% yield, which was enantioselectively aminated by leucine dehydrogenase (LeuDH) to (S)-CPG. Moreover, protein engineering of LeuDH was performed via directed evolution and semi-rational design. A beneficial variant Es LeuDH-F362L with enlarged substrate-binding pocket and increased hydrogen bond between K77 and substrate CPGA was constructed, which exhibited 2.1-fold enhanced specific activity but decreased thermal stability. Coupled with a glucose dehydrogenase from Bacillus megaterium (Bm GDH) for NADH regeneration, Es LeuDH-F362L completely converted up to 0.5 M CPGA to (S)-CPG in 8 h at 40 °C. • A new chemo-enzymatic catalytic route for (S)− 2-chlorophenylglycine was developed. • Es LeuDH-F362L exhibited enhanced catalytic activity but decreased thermal stability. • Es LeuDH-F362L completely converted up to 0.5 M (2-chlorophenyl)glyoxylic acid in 8 h. [ABSTRACT FROM AUTHOR]

Steroidal resource occupies a vital proportion in the pharmaceutical industry attributing to their important therapeutic effects on fertility, anti-inflammatory and antiviral activities. Currently, microbial transformation from phytosterol has become the dominant strategy of steroidal drug intermediate synthesis that bypasses the traditional chemical route. Mycobacterium sp. serve as the main industrial microbial strains that are capable of introducing selective functional modifications of steroidal intermediate, which has become an indispensable platform for steroid biomanufacturing. By reviewing the progress in past two decades, the present paper concentrates mainly on the microbial rational modification aspects that include metabolic pathway editing, key enzymes engineering, material transport pathway reinforcement, toxic metabolic intermediates removal and byproduct reconciliation. In addition, progress on omics analysis and direct genetic manipulation are summarized and classified that may help reform the industrial hosts with more efficiency. The paper provides an insightful present for steroid biomanufacturing especially on the current trends and prospects of mycobacteria. [ABSTRACT FROM AUTHOR]

The article focuses on a research related to one-pot, single-step deracemization of 2-hydroxyacids by tandem biocatalytic oxidation and reduction.

Microbial production of D-pantothenic acid (D-PA) has become an important research area, as D-PA is an essential vitamin widely applied in food, feed, chemical and pharmaceutical industries. Here both dynamic regulations of redox metabolism and fermentation strategies were applied to enhancing D-PA biosynthesis in Escherichia coli W3110. Firstly, a quorum-sensing (QS) system for redox regulation developed, in which zwf and nadK overexpression were shown to be effective for enhancing D-PA production. Based on this, a strain DPA11/p LBCWK was constructed with D-PA titer at 4.19 g/L in flask. Moreover, the fermentation strategies including pH-feedback feeding and gradient β-alanine feeding were introduced, under optimal conditions, the strain DPA11/p LBCWK could produce 48.21 g/L D-PA in 5 L fermenter, with the specific productivity of 0.72 g/L·h and the yield of 0.33 g/g glucose. These modification strategies were proved effective and would provide valuable reference for biosynthesis of other chemicals. [Display omitted] • The redox regulation improved the yield of D-pantothenic acid. • A quorum-sensing system was used for D-pantothenic acid synthesis for the first time. • A 48.21 g/L D-pantothenic acid was obtained in feeding fermentation. [ABSTRACT FROM AUTHOR]