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

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

Chiral epichlorohydrin (ECH) is an attractive intermediate for chiral pharmaceuticals and chemicals preparation. The asymmetric synthesis of chiral ECH using 1,3-dicholoro-2-propanol (1,3-DCP) catalyzed by a haloalcohol dehalogenase (HHDH) was considered as a feasible approach. However, the reverse ring opening reaction caused low optical purity of chiral ECH, thus severely restricts the industrial application of HHDHs. In the present study, a novel selective conformation adjustment strategy was developed with an engineered HheCPS to regulate the kinetic parameters of the forward and reverse reactions, based on site saturation mutation and molecular simulation analysis. The HheCPS mutant E85P was constructed with a markable change in the conformation of (S)-ECH in the substrate pocket and a slight impact on the interaction between 1,3-DCP and the enzyme, which resulted in the kinetic deceleration of the reverse reactions. Compared with HheCPS, the catalytic efficiency (kcat(S)-ECH/Km(S)-ECH) of the reversed reaction dropped to 0.23-fold (from 0.13 to 0.03 mM−1 s−1), while the catalytic efficiency (kcat(1,3-DCP)/Km(1,3-DCP)) of the forward reaction only reduced from 0.83 to 0.71 mM−1 s−1. With 40 mM 1,3-DCP as substrate, HheCPS E85P catalyzed the synthesis of (S)-ECH with the yield up to 55.35% and the e.e. increased from 92.54 to >99%. Our work provided an effective approach for understanding the stereoselective catalytic mechanism as well as the green manufacturing of chiral epoxides. [ABSTRACT FROM AUTHOR]

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]

D-Allulose 3-epimerase (DAE) is a vital biocatalyst for the industrial synthesis of D-allulose, an ultra-low calorie rare sugar. However, limited thermostability of DAEs hinders their use at high-temperature production. In this research, hyperthermophilic TI-DAE (Tm = 98.4 ± 0.7 ℃) from Thermotoga sp. was identified via in silico screening. A comparative study of the structure and function of site-directed saturation mutagenesis mutants pinpointed the residue I100 as pivotal in maintaining the high-temperature activity and thermostability of TI-DAE. Employing TI-DAE as a biocatalyst, D-allulose was produced from D-fructose with a conversion rate of 32.5%. Moreover, TI-DAE demonstrated excellent catalytic synergy with glucose isomerase CAGI, enabling the one-step conversion of D-glucose to D-allulose with a conversion rate of 21.6%. This study offers a promising resource for the enzyme engineering of DAEs and a high-performance biocatalyst for industrial D-allulose production. [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]

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

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]

S-adenosyl-l-methionine (SAM), a vital physiologically active substance in living organisms, is produced by fermentation over Saccharomyces cerevisiae. The main limitation in SAM production was the low biosynthesis ability of SAM in S. cerevisiae. The aim of this work is to breed an SAM-overproducing mutant through UV mutagenesis coupled with high-throughput selection. Firstly, a high-throughput screening method by rapid identification of positive colonies was conducted. White colonies on YND medium were selected as positive strains. Then, nystatin/sinefungin was chosen as a resistant agent in directed mutagenesis. After several cycles of mutagenesis, a stable mutant 616–19-5 was successfully obtained and exhibited higher SAM production (0.41 g/L vs 1.39 g/L). Furthermore, the transcript levels of the genes SAM2, ADO1, and CHO2 involved in SAM biosynthesis increased, while ergosterol biosynthesis genes in mutant 616–19-5 significantly decreased. Finally, building on the above work, S. cerevisiae 616–19-5 could produce 10.92 ± 0.2 g/L SAM in a 5-L fermenter after 96 h of fermentation, showing a 2.02-fold increase in the product yield compared with the parent strain. Paving the way of breeding SAM-overproducing strain has improved the good basis for SAM industrial production. [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]

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

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

With potent herbicidal activity, biocatalysis synthesis of l-glufosinate has drawn attention. In present research, NAP-Das2.3, a deacetylase capable of stereoselectively resolving N-acetyl-l-glufosinate to l-glufosinate mined from Arenimonas malthae, was heterologously expressed and characterized. In Escherichia coli, NAP-Das2.3 activity only reached 0.25 U/L due to the formation of inclusive bodies. Efficient soluble expression of NAP-Das2.3 was achieved in Pichia pastoris. In shake flask and 5 L bioreactor fermentation, NAP-Das2.3 activity by recombinant P. pastoris reached 107.39 U/L and 1287.52 U/L, respectively. The optimum temperature and pH for N-acetyl-glufosinate hydrolysis by NAP-Das2.3 were 45 °C and pH 8.0, respectively. The Km and Vmax of NAP-Das2.3 towards N-acetyl-glufosinate were 25.32 mM and 19.23 μmol mg−1 min−1, respectively. Within 90 min, 92.71% of L-enantiomer in 100 mM racemic N-acetyl-glufosinate was converted by NAP-Das2.3. l-glufosinate with high optical purity (e.e.P above 99.9%) was obtained. Therefore, the recombinant NAP-Das2.3 might be an alternative for l-glufosinate biosynthesis. [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]

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]

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

Cellobiose 2-epimerase (CE) is ideally suited to synthesize lactulose from lactose, but the poor thermostability and catalytic efficiency restrict enzymatic application. Herein, a non-characterized CE originating from Caldicellulosiruptor morganii (CmCE) was discovered in the NCBI database. Then, a smart mutation library was constructed based on FoldX ΔΔG calculation and modeling structure analysis, from which a positive mutant D226G located within the α8/α9 loop exhibited longer half-lives at 65–75 °C as well as lower Km and higher kcat/Km values compared with CmCE. Molecular modeling demonstrated that the improvement of D226G was largely attributed to the rigidification of the flexible loop, the compactness of the catalysis pocket and the increment of substrate-binding capability. Finally, the yield of synthesizing lactulose catalyzed by D226G reached 45.5%, higher than the 35.9% achieved with CmCE. The disclosed effect of the flexible loop on enzymatic stability and catalysis provides insight to redesign efficient CEs to biosynthesize lactulose. [ABSTRACT FROM AUTHOR]

Erythritol is a novelty 4-carbon sugar polyol and has great potential to be used as the precursor of some platform chemicals. The increasing cost of glucose poses researchers shifting insights to the cheaper biodiesel raw materials. Herein, we engineered a non-degradation, non-byproducts Yarrowia lipolytica for the erythritol production with high-titer from glycerol. Initially, the degradation and competition modules were blocked by URA3 counter-selection marker. Subsequently, a shortened biosynthetic pathway was explored to elevate its synthetic flux by multi-modules combination expression of functional genes. Furthermore, a screened glycerol transporter ScFPS1 was integrated into ERY6 genome to promote the glycerol uptake. The constructed strain ERY8 produced 176.66 g/L erythritol in the 5-L bioreactor with a yield and productivity of 0.631 g/g and 1.23 g/L/h, respectively, which achieved the highest fermentation production efficiency till date. This study proposed a novel multi-modules combination strategy for effectively engineering Y. lipolytica to produce erythritol using glycerol. [ABSTRACT FROM AUTHOR]

Fructose, which is produced by the isomerization of glucose isomerase, is a crucial precursor for the biosynthesis of rare sugars. In this study, thermophilic glucose isomerases (GI) from Caldicellulosiruptor acetigenus (CAGI), Thermoanaerobacter thermocopriae (TTGI), and Thermotoga petrophila (TPGI) were screened from GenBank database by a virtual probe and were successfully expressed in Escherichia coli BL21(DE3). The results of characterization demonstrated that the optimal pH for CAGI and TTGI were 8.0 and were maintained at 80% in a slightly acidic environment. The relative residual activities of CAGI and TTGI were found to be 40.6% and 52.6%, respectively, following an incubation period of 24 h at 90 ℃. Furthermore, CAGI and TTGI exhibited superior catalytic performance that their reaction equilibrium both reached only after an hour at 85 ℃ with 200 g/L glucose, and the highest conversion rates were 54.2% and 54.1%, respectively. This study identifies competitive enzyme candidates for fructose production in the industry with appreciable cost reduction. [ABSTRACT FROM AUTHOR]

Echinocandin B (ECB) is the key precursor compound of the antifungal drug Anidulafungin. The effects of the five precursor amino acids on ECB biosynthesis were firstly investigated. It showed that although L-threonine was a main compound of the hexapeptide scaffold of ECB, exogenous addition of L-threonine had no significant effect on the increase of ECB fermentation titer. Meanwhile, the ECB fermentation titer with methyl oleate showed two times higher than that of the other carbon sources. Transcription level analysis of the key genes for ECB biosynthesis indicated that the gene an655543 related to L-threonine biosynthesis showed higher value during the fermentation process, therefore, the exogenous addition of L-threonine had no obvious affection. Furthermore, it indicated that the transcription level of gene ecdA might be the main restriction factor for the ECB biosynthesis. The study provided the research foundation for the modification of the ECB producing strains in the following work. [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]

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

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]

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]

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]

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

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

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]

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

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

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

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]

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