Search

Vibegron functions as a potent and selective β 3 -adrenergic receptor agonist, with its chiral precursor (2S,3R)-aminohydroxy ester (1b) being crucial to its synthesis. In this study, loop engineering was applied to the carbonyl reductase (EaSDR6) from Exiguobacterium algae to achieve an asymmetric reduction of the (rac)-aminoketone ester 1a. The variant M5 (A138L/A190V/S193A/Y201F/N204A) was obtained and demonstrated an 868-fold increase in catalytic efficiency (k cat /K m  = 260.3 s -1  mM -1 ) and a desirable stereoselectivity (>99% enantiomeric excess, e.e.; >99% diastereomeric excess, d.e.) for the target product 1b in contrast to the wild-type EaSDR6 (WT). Structural alignment with WT indicated that loops 137-154 and 182-210 potentially play vital roles in facilitating catalysis and substrate binding. Moreover, molecular dynamics (MD) simulations of WT-1a and M5-1a complex illustrated that M5-1a exhibits a more effective nucleophilic attack distance and more readily adopts a pre-reaction state. The interaction analysis unveiled that M5 enhanced hydrophobic interactions with substrate 1a on cavities A and B while diminishing unfavorable hydrophilic interactions on cavity C. Computational analysis of binding free energies indicated that M5 displayed heightened affinity towards substrate 1a compared to the WT, aligning with its decreased K m value. Under organic-aqueous biphasic conditions, the M5 mutant showed >99% conversion within 12 h with 300 g/L substrate 1a (highest substrate loading as reported). This study enhanced the catalytic performance of carbonyl reductase through functional loops engineering and established a robust framework for the large-scale biosynthesis of the vibegron intermediate., (© 2024 Wiley Periodicals LLC.)

l-homoserine is an important platform compound of many valuable products. Construction of microbial cell factory for l-homoserine production from glucose has attracted a great deal of attention. In this study, l-homoserine biosynthesis pathway was divided into three modules, the glucose uptake and upstream pathway, the downstream pathway, and the energy supply module. Metabolomics of the chassis strain HS indicated that the supply of ATP was inadequate, therefore, the energy supply module was firstly modified. By balancing the ATP supply module, the l-homoserine production increased by 66% to 12.55 g/L. Further, the results indicated that the upstream pathway was blocked, and increasing the culture temperature to 37°C could solve this problem and the l-homoserine production reached 21.38 g/L. Then, the downstream synthesis pathways were further strengthened to balance the fluxes, and the l-homoserine production reached the highest reported level of 32.55 g/L in shake flasks. Finally, fed-batch fermentation in a 5-L bioreactor was conducted, and l-homoserine production could reach to 119.96 g/L after 92 h cultivation, with the yield of 0.41 g/g glucose and productivity of 1.31 g/L/h. The study provides a well research foundation for l-homoserine production by microbial fermentation with the capacity for industrial application., (© 2024 Wiley Periodicals LLC.)

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 panD BS and aspB CG for an optimal flux match between two key pathways. Subsequently, additional copies of key genes (panD BS K104S 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., (© 2024 Wiley Periodicals LLC.)

Hydroxysteroid dehydrogenases (HSDHs) catalyze the oxidation/reduction of hydroxyl/keto groups of steroids with high regio- or stereoselectivity, playing an essential role in producing optically pure chemicals. In this work, a novel approach was developed to simultaneously improve the stability and activity of 7β-hydroxysteroid dehydrogenase (7β-HSDH) by combining B-factor analysis and computer-aided prediction. Several advantageous mutants were identified, and the most promising variant, S51Y/P202Y, exhibited 2.3-fold improvements in catalytic activity, 3.3-fold in half-life at 40°C, and 4.7-fold in catalytic efficiency ( k cat /K m ), respectively. Structural modeling analysis showed that the shortened reversible oxidation reaction catalytic distance and the strengthened residue interactions compared to the wild type were attributed to the improved stability and activity of the obtained mutants. To synthesize ursodeoxycholic acid cost-effectively by mutant S51Y/P202Y, a NAD-kinase was employed to facilitate the substitution of nicotinamide adenine dinucleotide phosphate (NADP + ) with nicotinamide adenine dinucleotide (NAD + ) in the whole-cell catalysis system. The substrate 7-ketolithocholic acid (100 mM) was converted completely in 0.5 h, achieving a space-time yield of 1,887.3 g L -1 d -1 . This work provided a general target-oriented strategy for obtaining stable and highly active dehydrogenase for efficient biosynthesis., Importance: Hydroxysteroid dehydrogenases have emerged as indispensable tools in the synthesis of steroids, bile acids, and other steroid derivatives for the pharmaceutical and chemical industries. In this study, a novel approach was developed to simultaneously improve the stability and activity of a hydroxysteroid dehydrogenase by combining B-factor analysis and computer-aided prediction. This semi-rational method was demonstrated to be highly effective for enzyme engineering. In addition, NAD kinase was introduced to convert NAD + to NADP + for effective coenzyme regeneration in the whole-cell multienzyme-catalyzed system. This strategy reduces the significant economic costs associated with externally supplemented cofactors in NADP-dependent biosynthetic pathways., Competing Interests: The authors declare no conflict of interest.

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., (© 2024 Wiley Periodicals LLC.)

Through iterative rounds of mutation and selection, proteins can be engineered to enhance their desired biological functions. Nevertheless, identifying optimal mutation sites for directed evolution remains challenging due to the vastness of the protein sequence landscape and the epistatic mutational effects across residues. To address this challenge, we introduce MLSmut, a deep learning-based approach that leverages multi-level structural features of proteins. MLSmut extracts salient information from protein co-evolution, sequence semantics, and geometric features to predict the mutational effect. Extensive benchmark evaluations on 10 single-site and two multi-site deep mutation scanning datasets demonstrate that MLSmut surpasses existing methods in predicting mutational outcomes. To overcome the limited training data availability, we employ a two-stage training strategy: initial coarse-tuning on a large corpus of unlabeled protein data followed by fine-tuning on a curated dataset of 40-100 experimental measurements. This approach enables our model to achieve satisfactory performance on downstream protein prediction tasks. Importantly, our model holds the potential to predict the mutational effects of any protein sequence. Collectively, these findings suggest that our approach can substantially reduce the reliance on laborious wet lab experiments and deepen our understanding of the intricate relationships between mutations and protein function., (© 2024 Wiley‐VCH GmbH.)

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 HheC PS to regulate the kinetic parameters of the forward and reverse reactions, based on site saturation mutation and molecular simulation analysis. The HheC PS 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 HheC PS , the catalytic efficiency (k cat(S)-ECH /K m(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 (k cat(1,3-DCP) /K m(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, HheC PS 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., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Substrate access tunnel engineering is a useful strategy for enzyme modification. In this study, we improved the catalytic performance of Fe-type Nitrile hydratase (Fe-type NHase) from Pseudomonas fluorescens ZJUT001 (PfNHase) by mutating residue Q86 at the entrance of the substrate access tunnel. The catalytic activity of the mutant PfNHase-αQ86W towards benzonitrile, 2-cyanopyridine, 3-cyanopyridine, and 4-hydroxybenzonitrile was enhanced by 9.35-, 3.30-, 6.55-, and 2.71-fold, respectively, compared to that of the wild-type PfNHase (PfNHase-WT). In addition, the mutant PfNHase-αQ86W showed a catalytic efficiency (k cat /K m ) towards benzonitrile 17.32-fold higher than the PfNHase-WT. Interestingly, the substrate preference of PfNHase-αQ86W shifted from aliphatic nitriles to aromatic nitrile substrates. Our analysis delved into the structural changes that led to this altered substrate preference, highlighting an expanded entrance tunnel region, theenlarged substrate-binding pocket, and the increased hydrophobic interactions between the substrate and enzyme. Molecular dynamic simulations and dynamic cross-correlation Matrix (DCCM) further supported these findings, providing a comprehensive explanation for the enhanced catalytic activity towards aromatic nitrile substrates., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The d-amino acid oxidase (DAAO) is pivotal in obtaining optically pure l-glufosinate (l-PPT) by converting d-glufosinate (d-PPT) to its deamination product. We screened and designed a Rasamsonia emersonii DAAO (ReDAAO), making it more suitable for oxidizing d-PPT. Using Caver 3.0, we delineated three substrate binding pockets and, via alanine scanning, identified nearby key residues. Pinpointing key residues influencing activity, we applied virtual saturation mutagenesis (VSM), and experimentally validated mutants which reduced substrate binding energy. Analysis of positive mutants revealed elongated side-chain prevalence in substrate binding pocket periphery. Although computer-aided approaches can rapidly identify advantageous mutants and guide further design, the mutations obtained in the first round may not be suitable for combination with other advantageous mutations. Therefore, each round of combination requires reasonable iteration. Employing VSM-assisted screening multiple times and after four rounds of combining mutations, we ultimately obtained a mutant, N53V/F57Q/V94R/V242R, resulting in a mutant with a 5097% increase in enzyme activity compared to the wild type. It provides valuable insights into the structural determinants of enzyme activity and introduces a novel rational design procedure., (© 2024 Wiley‐VCH GmbH.)

Ketopantoate hydroxymethyltransferase (KPHMT) plays a pivotal role in d-pantothenic acid biosynthesis. Most KPHMTs are homodecamers with low thermal stability, posing challenges for protein engineering and limiting output enhancement. Previously, a high-enzyme activity KPHMT mutant (K25A/E189S) from Corynebacterium glutamicum was screened as mother strain (M0). Building upon this strain, our study focused on interface engineering modifications, employing a multifaceted approach including integrating folding-free energy calculation, B -factor analysis, and conserved site analysis. Preliminary screening led to the selection of five mutants in the interface─E106S, E98T, E98N, S247I, and S247D─showing improved thermal stability, culminating in the double-site mutant M8 (M0-E98N/S247D). M8 exhibited a T 1/2 value of 288.79 min at 50 °C, showing a 3.29-fold increase compared to M0. Meanwhile, the T m value of M8 was elevated from 53.2 to 59.6 °C. Investigations of structural and molecular dynamics simulations revealed alterations in surface electrostatic charge distribution and the formation of increased hydrogen bonds between subunits, contributing to enhanced thermal stability. This investigation corroborates the efficacy of interface engineering modifications in bolstering KPHMT stability while showing its potential for positively impacting industrial d-pantothenic acid synthesis.

The nicotinamide phosphoribosyltransferase (NAMPT)-catalyzed substitution reaction plays a pivotal role in the biosynthesis of nucleotide compounds. However, industrial applications are hindered by the low activity of NAMPTs. In this study, a novel dual-channel protein engineering strategy was developed to increase NAMPT activity by enhancing substrate accessibility. The best mutant ( Cp NAMPT Y13G+Y15S+F76P ) with a remarkable 5-fold increase in enzyme activity was obtained. By utilizing Cp NAMPT Y13G+Y15S+F76P as a biocatalyst, the accumulation of β-nicotinamide mononucleotide reached as high as 19.94 g L -1 within 3 h with an impressive substrate conversion rate of 99.8%. Further analysis revealed that the newly generated substrate channel, formed through crack propagation, facilitated substrate binding and enhanced byproduct tolerance. In addition, three NAMPTs from different sources were designed based on the dual-channel protein engineering strategy, and the corresponding dual-channel mutants with improved enzyme activity were obtained, which proved the effectiveness and practicability of the approach.

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., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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., (© 2024. The Author(s).)

Actinoplanes utahensis deacylase (AAC)-catalyzed deacylation of echinocandin B (ECB) is a promising method for the synthesis of anidulafungin, the newest of the echinocandin antifungal agents. However, the low activity of AAC significantly limits its practical application. In this work, we have devised a multi-dimensional rational design strategy for AAC, conducting separate analyses on the substrate-binding pocket's volume, curvature, and length. Furthermore, we quantitatively analyzed substrate properties, particularly on hydrophilic and hydrophobic. Accordingly, we tailored the linoleic acid-binding pocket of AAC to accommodate the extended long lipid chain of ECB. By fine-tuning the key residues, the resulting AAC mutants can accommodate the ECB lipid chain with a lower curvature binding pocket. The D53A/I55F/G57M/F154L/Q661L mutant (MT) displayed 331 % higher catalytic efficiency than the wild-type (WT) enzyme. The MT product conversion was 94.6 %, reaching the highest reported level. Utilizing a multi-dimensional rational design for a customized mutation strategy of the substrate-binding pocket is an effective approach to enhance the catalytic efficiency of enzymes in handling complicated substrates., Competing Interests: Declaration of competing interest The authors declare on conflicts of interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

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, LsCR M4 (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 LsCR M4 -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 LsCR M4 . Our computer-assisted design and combinatorial strategy dramatically increased the efficiency of thermostable enzyme production., (© 2024 Wiley Periodicals LLC.)

Catalytic activity is undoubtedly a key focus in enzyme engineering. The complicated reaction conditions hinder some enzymes from industrialization even though they have relatively promising activity. This has occurred to some dehydrogenases. Hydroxysteroid dehydrogenases (HSDHs) specifically catalyze the conversion between hydroxyl and keto groups, and hold immense potential in the synthesis of steroid medicines. We underscored the importance of 7α-HSDH activity, and analyzed the overall robustness and underlying mechanisms. Employing a high-throughput screening approach, we comprehensively assessed a mutation library, and obtained a mutant with enhanced enzymatic activity and overall stability/tolerance. The superior mutant (I201M) was identified to harbor improved thermal stability, substrate susceptibility, cofactor affinity, as well as the yield. This mutant displayed a 1.88-fold increase in enzymatic activity, a 1.37-fold improvement in substrate tolerance, and a 1.45-fold increase in thermal stability when compared with the wild type (WT) enzyme. The I201M mutant showed a 2.25-fold increase in the k cat / K M ratio (indicative of a stronger binding affinity for the cofactor). This mutant did not exhibit the highest enzyme activity compared with all the tested mutants, but these improved characteristics contributed synergistically to the highest yield. When a substrate at 100 mM was present, the 24 h yield by I201M reached 89.7%, significantly higher than the 61.2% yield elicited by the WT enzyme. This is the first report revealing enhancement of the catalytic efficiency, cofactor affinity, substrate tolerance, and thermal stability of NAD(H)-dependent 7α-HSDH through a single-point mutation. The mutated enzyme reached the highest enzymatic activity of 7α-HSDH ever reported. High enzymatic activity is undoubtedly crucial for enabling the industrialization of an enzyme. Our findings demonstrated that, when compared with other mutants boasting even higher enzymatic activity, mutants with excellent overall robustness were superior for industrial applications. This principle was exemplified by highly active enzymes such as 7α-HSDH.

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 ( Ec AHAIR) encoded by ilvC was obtained to explore its substrate promiscuity. Compared with the wild type, the variant Ec AHAIR-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- ilvC V412A 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., Competing Interests: Conflict of interestAll authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© King Abdulaziz City for Science and Technology 2024. Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.)

4-cyanobenzoic acid serves as a crucial intermediate for the synthesis of various high-value organic compounds. The enzymatic hydrolysis of terephthalonitrile to produce 4-cyanobenzoic acid using nitrilase offers the advantages of a simple reaction pathway, environmental friendliness, and easy product separation. In order to efficiently develop nitrilases that meet industrial production requirements, the virtual screening method used in the study is established and mature. From a total of 371 amino acids in the nitrilase AfNIT, which exhibits activity in terephthalonitrile hydrolysis, three candidate sites (F168, S192, and T201) were identified, and a "small and accurate" mutant library was constructed. The triple mutant F168V/T201N/S192F was screened from this small mutant library with a specific activity of 227.3 U mg -1 , which was 3.8 times higher than that of the wild-type AfNIT. Using the whole-cell biocatalyst containing the mutant F168V/T201N/S192F, terephthalonitrile was successfully hydrolyzed at a concentration of 150 g L -1 to produce 4-cyanobenzoic acid with a final yield of 170.3 g L -1 and a conversion rate of 98.7%. The obtained nitrilase mutant F168V/T201N/S192F in this study can be effectively applied in the biomanufacturing of 4-cyanobenzoic acid using terephthalonitrile as a substrate. Furthermore, the results also demonstrate the significant improvement in predictive accuracy achieved through the latest AI-assisted computer simulation methods. This approach represents a promising and feasible new technological pathway for assisting enzyme engineering research, laying a theoretical foundation for other related studies., (© 2024 Wiley-VCH GmbH.)

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., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

In response to the persistent expansion of global resource demands, considerable attention has been directed toward the synthetic microbial consortia (SMC) within the domain of microbial engineering, aiming to address the sustainable management and valorization of biomass wastes. This comprehensive review systematically encapsulates the most recent advancements in research and technological applications concerning the utilization of SMC for biomass waste treatment. The construction strategies of SMC are briefly outlined, and the diverse applications of SMC in biomass wastes treatment are explored, with particular emphasis on its potential advantages in waste degradation, hazardous substances control, and high value-added products conversion. Finally, recommendations for the future development of SMC technology are proposed, and prospects for its sustainable application are discussed., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The aldo-keto reductase (AKR) KdAKR from Kluyvermyces dobzhanskii can reduce t-butyl 6-chloro-(5S)-hydroxy-3-oxohexanoate ((5S)-CHOH) to t-butyl 6-chloro-(3R,5S)-dihydroxyhexanoate ((3R,5S)-CDHH), which is the key chiral intermediate of rosuvastatin. Herein, a computer-aided design that combined the use of PROSS platform and consensus design was employed to improve the stability of a previously constructed mutant KdAKR M6 . Experimental verification revealed that S196C, T232A, V264I and V45L produced improved thermostability and activity. The "best" mutant KdAKR M10 (KdAKR M6 -S196C/T232A/V264I/V45L) was constructed by combining the four beneficial mutations, which displayed enhanced thermostability. Its T 50 15 and T m values were increased by 10.2 and 10.0°C, respectively, and half-life (t 1/2 ) at 40°C was increased by 17.6 h. Additionally, KdAKR M10 demonstrated improved resistance to organic solvents compared to that of KdAKR M6 . Structural analysis revealed that the increased number of hydrogen bonds and stabilized hydrophobic core contributed to the rigidity of KdAKR M10 , thus improving its stability. The results validated the feasibility of the computer-aided design strategy in improving the stability of AKRs., (© 2024 Wiley-VCH GmbH.)

Nicotinamide mononucleotide (NMN) has garnered substantial interest as a functional food product. Industrial NMN production relies on chemical methods, facing challenges in separation, purification, and regulatory complexities, leading to elevated prices. In contrast, NMN biosynthesis through fermentation or enzyme catalysis offers notable benefits like eco-friendliness, recyclability, and efficiency, positioning it as a primary avenue for future NMN synthesis. Enzymatic NMN synthesis encompasses the nicotinamide-initial route and nicotinamide ribose-initial routes. Key among these is nicotinamide riboside kinase (NRK), pivotal in the latter route. The NRK-mediated biosynthesis is emerging as a prominent trend due to its streamlined route, simplicity, and precise specificity. The essential aspect is to obtain an engineered NRK that exhibits elevated activity and robust stability. This review comprehensively assesses diverse NMN synthesis methods, offering valuable insights into efficient, sustainable, and economical production routes. It spotlights the emerging NRK-mediated biosynthesis pathway and its significance. The establishment of an adenosine triphosphate (ATP) regeneration system plays a pivotal role in enhancing NMN synthesis efficiency through NRK-catalyzed routes. The review aims to be a reference for researchers developing green and sustainable NMN synthesis, as well as those optimizing NMN production.

Pichia pastoris ( P. pastoris ) is one of the most popular cell factories for expressing exogenous proteins and producing useful chemicals. The alcohol oxidase 1 promoter (P AOX1 ) is the most commonly used strong promoter in P. pastoris and has the characteristic of biphasic expression. However, the inducer for P AOX1 , methanol, has toxicity and poses risks in industrial settings. In the present study, analyzing transcriptomic data of cells collected at different stages of growth found that the formate dehydrogenase (FDH) gene ranked 4960th in relative expression among 5032 genes during the early logarithmic growth phase but rose to the 10th and 1st during the middle and late logarithmic growth phases, respectively, displaying a strict biphasic expression characteristic. The unique transcriptional regulatory profile of the FDH gene prompted us to investigate the properties of its promoter (P FDH800 ). Under single-copy conditions, when a green fluorescent protein variant was used as the expression target, the P FDH800 achieved 119% and 69% of the activity of the glyceraldehyde-3-phosphate dehydrogenase promoter and P AOX1 , respectively. After increasing the copy number of the expression cassette in the strain to approximately four copies, the expression level of GFPuv driven by P FDH800 increased to approximately 2.5 times that of the strain containing GFPuv driven by a single copy of P AOX1 . Our P FDH800 -based expression system exhibited precise biphasic expression, ease of construction, minimal impact on normal cellular metabolism, and high strength. Therefore, it has the potential to serve as a new expression system to replace the P AOX1 promoter.IMPORTANCEThe alcohol oxidase 1 promoter (PAOX1) expression system has the characteristics of biphasic expression and high expression levels, making it the most widely used promoter in the yeast Pichia pastoris . However, PAOX1 requires methanol induction, which can be toxic and poses a fire hazard in large quantities. Our research has found that the activity of PFDH800 is closely related to the growth state of cells and can achieve biphasic expression without the need for an inducer. Compared to other reported non-methanol-induced biphasic expression systems, the system based on the PFDH800 offers several advantages, including high expression levels, simple construction, minimal impact on cellular metabolism, no need for an inducer, and the ability to fine-tune expression., Competing Interests: The authors declare no conflict of interest.

Enzymatic synthesis of β-nicotinamide mononucleotide (NMN) from D-ribose has garnered widespread attention due to its cheap material, the use of mild reaction conditions, and the ability to produce highly pure products with the desired optical properties. However, the overall NMN yield of this method is impeded by the low activity of rate-limiting enzymes. The ribose-phosphate diphosphokinase (PRS) and nicotinamide phosphoribosyltransferase (NAMPT), that control the rate of the reaction, were engineered to improve the reaction efficacy. The actives of mutants PRS-H150Q and NAMPT-Y15S were 334% and 57% higher than that of their corresponding wild-type enzymes, respectively. Furthermore, by adding pyrophosphatase, the byproduct pyrophosphate which can inhibit the activity of NAMPT was degraded, leading to a 6.72% increase in NMN yield. Following with reaction-process reinforcement, a high yield of 8.10 g L -1 NMN was obtained after 3 h of reaction, which was 56.86-fold higher than that of the stepwise reaction synthesis (0.14 g L -1 ), indicating that the in vitro enzymatic synthesis of NMN from D-ribose and niacinamide is an economical and feasible route., (© 2024 Wiley-VCH GmbH.)

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., (© 2024. The Author(s).)

Pseudomonas putida KT2440, a GRAS strain, has been used for synthesizing bulk and fine chemicals. However, the gene editing tool to metabolically engineer KT2440 showed low efficiency. In this study, a novel sacB-based system pK51mobsacB was established to improve the efficiency for marker-free gene disruption. Then the rhamnolipid synthetic pathway was introduced in KT2440 and genes of the competitive pathways were deleted to lower the metabolic burden based on pK51mobsacB. A series of endogenous and synthetic promoters were used for fine tuning rhlAB expression. The limited supply of dTDP-L-rhamnose was enhanced by heterologous rmlBDAC expression. Cell growth and rhamnolipid production were well balanced by using glucose/glycerol as mixed carbon sources. The final strain produced 3.64 g/L at shake-flask and 19.77 g/L rhamnolipid in a 5 L fermenter, the highest obtained among metabolically engineered KT2440, which implied the potential of KT2440 as a promising microbial cell factory for industrial rhamnolipid production., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

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., (© 2024. The Author(s).)

Atorvastatin, a lipid-lowering drug that is widely used in the treatment of cardiovascular diseases, has significant clinical significance. This article focuses on the synthetic procedures of atorvastatin, including Paal-Knorr synthesis and several new synthetic strategies. It also outlines chemical and chemo-enzymatic methods for synthesizing optically active side chain of atorvastatin. In addition, a comprehensive overview of the analytical monitoring techniques for atorvastatin and its metabolites and impurities is reported, alongside a discussion of their strengths and limitations., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

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., Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03870-y., Competing Interests: Conflict of interestThe authors declare that they have no competing interests and the manuscript has been approved by all authors for publication., (© King Abdulaziz City for Science and Technology 2023. Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.)

The application of (R)-ω-transaminases as biocatalysts for chiral amine synthesis has been hampered by inadequate stereoselectivity and narrow substrate spectrum. Herein, an effective evolution strategy for (R)-ω-transaminase designing for the asymmetric synthesis of sitagliptin intermediate is presented. Since natural transaminases lack activity toward bulky prositagliptin ketone, transaminase scaffolds with catalytic machinery and activity toward the truncated prositagliptin ketone were firstly screened based on substrate walking principle. A transaminase chimera was established synchronously conferring catalytic activity and (R)-selectivity toward prositagliptin ketone through motif swapping, followed by stepwise evolution. The process resulted in a "best" engineered variant MwTA M8 , which exhibited 79.2-fold higher activity than the chimeric scaffold MwTA Mc . Structural analysis revealed that the heightened activity is mainly due to the enlarged and adaptive substrate pocket and tunnel. The novel (R)-transaminase exhibited unsatisfied industrial operation stability, which is expected to further modify the protein to enhance its tolerance to temperature, pH, and organic solvents to meet sustainable industrial demands. This study underscores a useful evolution strategy of engineering biocatalysts to confer new properties and functions on enzymes for synthesizing high-value drug intermediates., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this work., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The d-amino acid oxidase (DAAO) from Rhodotorula taiwanensis has proven to have great potential for applications due to its excellent catalytic kinetic parameters. However, its poor thermal stability has limited its performance in biocatalysis. Herein, starting from the variant SHVG of Rtw DAAO, this study employed a comprehensive computational design approach for protein stability engineering, resulting in positive substitutions at specific sites (A43S, T45M, C234L, E195Y). The generated variant combination, SHVG/SMLY, exhibited a significant synergistic effect, leading to an extension of the half-life and T m app . The ancestral sequence reconstruction revealed the conservation of the variant sites. The association of the variant sites with the highly stable ancestral enzyme was further explored. After determining the contribution of the variant sites to thermal stability, it was applied to other homologous sequences and validated. Molecular dynamics simulations indicated that the increased hydrophobicity of the variant SHVG/SMLY was a key factor for the increased stability, with strengthened intersubunit interactions playing an important role. In addition, the physical properties of the amino acids themselves were identified as crucial factors for thermal stability generality in homologous enzymes, which is important for the rapid acquisition of a series of stable enzymes.

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., (© 2023 Wiley Periodicals LLC.)

Background: 7-Dehydrocholesterol (7-DHC) can be directly converted to vitamin D 3 by UV irradiation and de novo synthesis of 7-DHC in engineered Saccharomyces cerevisiae has been recognized as an attractive substitution to traditional chemical synthesis. Introduction of sterol extracellular transport pathway for the secretory production of 7-DHC is a promising approach to achieve higher titer and simplify the downstream purification processing., Methods and Results: A series of genes involved in ergosterol pathway were combined reinforced and reengineered in S. cerevisiae. A biphasic fermentation system was introduced and 7-DHC was found to be enriched in oil-phase with an increased titer by 1.5-folds. Quantitative PCR revealed that say1, atf2, pdr5, pry1-3 involved in sterol storage and transport were all significantly induced in sterol overproduced strain. To enhance the secretion capacity, lipid transporters of pathogen-related yeast proteins (Pry), Niemann-Pick disease type C2 (NPC2), ATP-binding cassette (ABC)-family, and their homologues were screened. Both individual and synergetic overexpression of Plant pathogenesis Related protein-1 (Pr-1) and Sterol transport1 (St1) largely increased the de novo biosynthesis and secretory productivity of 7-DHC, and the final titer reached 28.2 mg g -1 with a secretion ratio of 41.4%, which was 26.5-folds higher than the original strain. In addition, the cooperation between Pr-1 and St1 in sterol transport was further confirmed by confocal microscopy, molecular docking, and directed site-mutation., Conclusion: Selective secretion of different sterol intermediates was characterized in sterol over-produced strain and the extracellular export of 7-DHC developed in present study significantly improved the cell biosynthetic capacity, which offered a novel modification idea for 7-DHC de novo biosynthesis by S. cerevisiae cell factory., (© 2023 Wiley-VCH GmbH.)

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 (de p value from 47.6% to >99.5%). Moreover, M6 exhibited a 6.3-fold increase in half-life (t 1/2 ) 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., (© 2023 Wiley Periodicals LLC.)

Importance: The adenosine 5'-triphosphate (ATP) regeneration system can significantly reduce the cost of many biocatalytic processes. Numerous studies have endeavored to utilize the ATP regeneration system based on Cytophaga hutchinsonii PPK ( Ch PPK). However, the wild-type Ch PPK enzyme possesses limitations such as low enzymatic activity, poor stability, and limited substrate tolerance, impeding its application in catalytic reactions. To enhance the performance of Ch PPK, we employed a semi-rational design approach to obtain the variant Ch PPK/A79G/S106C/I108F/L285P. The enzymatic kinetic parameters and the catalytic performance in the synthesis of nicotinamide mononucleotide demonstrated that the variant Ch PPK/A79G/S106C/I108F/L285P exhibited superior enzymatic properties than the wild-type enzyme. All data indicated that our engineered ATP regeneration system holds inherent potential for implementation in biocatalytic processes., Competing Interests: The authors declare no conflict of interest.

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 K m and V max 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., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Vibegron is a novel, potent, highly selective β 3 -adrenergic receptor agonist for the treatment of overactive bladder with higher therapeutic capacity and lower side effects. Methyl(2S,3R)-2-((tert-butoxycarbonyl)amino)-3-hydroxy-3-phenylpropanoate ((2S,3R)-aminohydroxy ester) is a key chiral intermediate for the synthesis of Vibegron. A novel carbonyl reductase from Exiguobacterium sp. s126 (EaSDR6) was isolated using data mining technology from GenBank database with preferable catalytic activity. Hydrogen bond network regulation was performed using site-directed saturation mutagenesis and combination mutagenesis. The mutant EaSDR6 A138L/S193A was obtained with the activity improvement by 4.58 folds compared with the wild type EaSDR6. The K m of EaSDR6 A138L/S193A was decreased from 1.57 mM to 0.67 mM, k cat was increased by 2.17 folds, and the overall catalytic efficiency k cat /K m was increased by 5.07 folds. The organic-aqueous biphasic bioreaction system for the asymmetric synthesis of (2S,3R)-aminohydroxy ester was constructed for the first time. Under the substrate concentration of 150 g/L, the yield of (2S,3R)-aminohydroxy ester was > 99.99%, the e.e. was > 99.99%, and the spatiotemporal yield was 1.55 g/(L·h·g DCW ) after 12 h reaction. While the substrate concentration was increased to 200 g/L and the reaction lasted for 36 h, the yield of (2S,3R)-aminohydroxy ester was > 99.99%, the e.e. was > 99.99% and the spatiotemporal yield was 1.05 g/(L·h·g DCW ). The substrate concentration and spatiotemporal yield were higher than ever reported., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Thermophilic composting (TC) can effectively shorten maturity period with satisfactory sanitation. However, the higher energy consumption and lower composts quality limited its widespread application. In this study, hyperthermophilic pretreatment (HP) was introduced as a novel approach within TC, and its effects on humification process and bacterial community during food waste TC was investigated from multiple perspectives. Results showed that a 4-hour pretreatment at 90 °C increased the germination index and humic acid/fulvic acid by 25.52% and 83.08%, respectively. Microbial analysis demonstrated that HP stimulated the potential functional thermophilic microbes, and significantly up-regulated the genes related to amino acid biosynthesis. Further network and correlation analysis suggested that pH was the key factor affecting bacterial communities, and higher HP temperatures help to restore bacterial cooperation and showed higher humification degree. In summary, this study contributed to a better understanding of the mechanism towards the accelerated humification by HP., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

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 (K m  = 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 Ym d-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., (© 2023 Wiley Periodicals LLC.)

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., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Microbial production of valuable compounds can be enhanced by various metabolic strategies. This study proposed combinatorial metabolic engineering to develop an effective Escherichia coli cell factory dedicated to L-cysteine production. First, the crucial regulatory modes that control L-cysteine levels were investigated to guide metabolic modifications. A two-stage fermentation was achieved by employing multi-copy gene expression, improving the balance between production and growth. Subsequently, carbon flux distribution was further optimized by modifying the C1 unit metabolism and the glycolytic pathway. The modifications of sulfur assimilation demonstrated superior performance of thiosulfate utilization pathways in enhancing L-cysteine titer. Furthermore, the studies focusing on cofactor availability and preference emphasized the vital role of synergistic enhancement of sulfur-carbon metabolism in L-cysteine overproduction. In a 5 L bioreactor, the strain BW15-3/pED accumulated 12.6 g/L of L-cysteine. This work presented an effective metabolic engineering strategy for the development of L-cysteine-producing strains.

Background: Biocatalytic production of L-phosphinothricin (L-PPT) is currently the most promising method. In this work, we use an Escherichia coli strain coexpressing of D-amino acid oxidase and catalase (E. coli DAAO-CAT) to oxidation biocatalytic D-PPT to PPO, then use the second E. coli strain coexpressing glutamate dehydrogenase and formate dehydrogenase (E. coli GluDH-FDH) to reduce biocatalytic PPO to L-PPT., Main Methods and Major Results: We compared the effects of different concentrations of IPTG or lactose on protein expression and enzyme activity in 5 L fermenter. The best induction conditions for E. coli DAAO-CAT were 0.05 mM IPTG, induction for 18 h at 28°C. The specific enzyme activities of DAAO and CAT were 153.20 U g -1 and 896.23 U g -1 , respectively. The optimal induction conditions for E. coli GluDH-FDH were 0.2 mM IPTG, induction for 19 h at 28°C. The specific enzyme activities of GluDH and FDH were 41.72 U g -1 and 109.70 U g -1 , respectively. The 200 mM D-PPT was biocatalyzed by E. coli DAAO-CAT for 4 h with space-time yield of 9.0 g·L -1 ·h -1 and conversion rate of over 99.0%. Then 220 mM PPO was converted to L-PPT by E. coli GluDH-FDH for 3 h with space-time yield of 14.5 g·L -1 ·h -1 and conversion rate of over 99.0%. To our knowledge, this is the most efficient biocatalytic reaction for L-PPT production., Conclusions and Implications: We found that IPTG has advantages compared with lactose in the enzyme activity and biomass of E. coli DAAO-CAT and E. coli GluDH-FDH, and IPTG is more environmentally friendly. Our data implicated that IPTG can replace lactose in terms of economic feasibility and effectiveness for scaled-up industrial fermentations., (© 2023 Wiley-VCH GmbH.)

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., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Enantiopure β-nitroalcohols, as an important class of nitro-containing compounds, are essential building blocks in pharmaceutical and organic chemistry, particularly for the synthesis of β-adrenergic blockers. In this study, we present the successful protein engineering of halohydrin dehalogenase HHDHamb for the enantioselective bio-nitration of various phenyl glycidyl ethers to the corresponding chiral β-nitroalcohols, using the inexpensive, commercially available, and safer nitrite as a nitrating agent. The chiral (R)- and (S)-1-nitro-3-phenoxypropan-2-ols were synthesized by the several enantiocomplementary HHDHamb variants through the whole-cell biotransformation, which showed good catalytic efficiency (up to 43% isolated yields) and high optical purity (up to >99% ee). In addition, we also demonstrated that the bio-nitration method was able to tolerate the substrate at a high concentration of 1000 mM (150 g/L). Furthermore, representative synthesis of two optically active enantiomers of the β-adrenergic blocker metoprolol was successfully achieved by utilizing the corresponding chiral β-nitroalcohols as precursors., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

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 α 8 /α 9 loop exhibited longer half-lives at 65-75 °C as well as lower K m and higher k cat /K m 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., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The effects of the co-addition of fungal agents and biochar on physicochemical properties, odor emissions, microbial community structure, and metabolic functions were investigated during the in-situ treatment of food waste. The combined addition of fungal agents and biochar decreased cumulative NH 3 , H 2 S, and VOCs emissions by 69.37%, 67.50%, and 52.02%, respectively. The predominant phyla throughout the process were Firmicutes, Actinobacteria, Cyanobacteria, and Proteobacteria. Combined treatment significantly impacted the conversion and release of nitrogen from the perspective of the variation of nitrogen content between different forms. FAPROTAX analysis revealed that the combined application of fungal agents and biochar could effectively inhibit nitrite ammonification and reduce the emission of odorous gases. This work aims to clarify the combined effect of fungal agents and biochar on odor emission and provide a theoretical basis for developing an environmentally friendly in-situ efficient biological deodorization (IEBD) technology., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Ltd.)

We developed a synthetic route for producing 3-amino-2-hydroxy acetophenone (3AHAP) from m-nitroacetophenone (3NAP) using an in vitro approach. Various reaction systems were evaluated, and a direct reaction method with crude enzyme and supersaturated substrates for optimal catalytic efficiency was chosen. The reaction system included three enzymes and was enhanced by adjusting enzyme molar ratios and optimizing ribosomal binding sites. We performed substrate docking and alanine scanning to identify key sites in the enzymes nitrobenzene nitroreductase (nbzA) and hydroxylaminobenzene mutase (habA). The optimal mutant was obtained through site-directed mutagenesis, and incorporated into the reaction system, resulting in increased product yield. After optimization, the yield of 3AHAP increased from 75 mg/L to 580 mg/L within 5 hours, the highest reported yield using biosynthesis. This work provides a promising strategy for the efficient and sustainable production of 3AHAP, which has critical applications in the chemical and pharmaceutical industries., (© 2023 Wiley-VCH GmbH.)