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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 glutamicumwas 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 T1/2value of 288.79 min at 50 °C, showing a 3.29-fold increase compared to M0. Meanwhile, the Tmvalue 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.

3-ketosteroid Δ1-dehydrogenases (Δ1-KstDs) are FAD-dependent and substrate-inducing enzymes, which catalyze the introduction of double bonds between C1 and C2 atoms of the A ring of 3-ketosteroid substrates. They are essential in the initial stages of the steroid core's breakdown. Additionally, Δ1-KstDs are particularly intriguing for applications in pharmaceutical manufacturing, environmental bioremediation, and the etiology of infectious illnesses. A wide range of microorganisms, particularly bacteria from the phylum Actinobacteria, have Δ1-KstDs. Δ1-KstDs can be classified into at least seven separate groups based on the sequence data in NCBI, and the enzymes in each group exhibit unique structural and catalytic properties. Understanding these properties completely is crucial for utilizing and developing Δ1-KstDs in metabolic engineering and enzyme engineering. This review describes and explains the biochemical and enzymatic characteristics of Δ1-KstDs based on a phylogenetic tree. To assist in the selection of highly active enzymes for engineering applications, the three-dimensional structures of Δ1-KstDs associated with enzyme mechanisms are stressed. The biotechnological application of microbial Δ1-KstDs is also covered in this article, including genetic engineering based on metabolic strains and related genetic modification techniques for creating new productive industrial strains, the development and transformation of the heterologous expression system, the molecular modification and the optimization of catalytic conditions, and the use of microbial fermentation to increase product yield. Furthermore, we also highlight the recent development in the use of isolated Δ1-KstDs combined with a FAD cofactor regeneration system. We conclude by summarizing the concepts and techniques used in subsequent research and application development. All of these knowledge might serve as a guide for new mining and industrial applications in Δ1-KstDs.

With staggering progress on genetic manipulation strategies, Saccharomyces cerevisiaeis becoming an ideal cell factory for the de novo biosynthesis of lipid compounds. However, due to their hydrophobicity, lipids tend to be accumulated within intracellular spaces and cause a high burden on cell activity and induce product inhibition effect, which ultimately restricted the lipids biomanufacturing for industrial application. Herein, an oleic acid stress (OAS) model was applied for the long-time domestication of BY4741 cells, and a subclone of A-22 was obtained through a series of acclimation (0.1% glucose and 0.2% oleic acid), showing increased accumulation of both biomass and intracellular lipid droplets compared to WT. Comparative transcriptome analysis indicated that compared to fatty acid metabolism, most transcripts enriched in the pathways of glucose catabolism (glycolysis and citrate cycle) and lipid synthesis (phospholipid and sterol) were down-regulated under OAS. While interestingly, most the above transcripts tended to be ‘restored’ in adapted strain A-22. In addition, for physical adaptation, significant increase of phosphatidylcholines was identified by lipidomic analysis, which probably caused the subsequent subcellular expansion of peroxisomes and lipid droplets as observed in the adapted strain, since phosphatidylcholines are the major constituent of their membranes. The present study systematically investigated both the phenotype change and molecular mechanism on adaptation of S. cerevisiaetowards oily environment. Detailed information on functional transcripts may provide novel rational modification targets to reinforce the hydrophobic lipids biosynthesis within S. cerevisiaeengineered cell factory.

The d-amino acid oxidase (DAAO) from Rhodotorula taiwanensishas 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 RtwDAAO, 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 Tmapp. 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.

Microbial production of valuable compounds can be enhanced by various metabolic strategies. This study proposed combinatorial metabolic engineering to develop an effective Escherichia colicell 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.

Efficient formate dehydrogenase (FDH)-based cofactor regeneration systems are widely used for biocatalytic processes due to their ready availability, low reduction potential, and production of only benign byproducts. However, FDHs are usually specific to NAD+, and NADPH regeneration with formate is challenging. Herein, an FDH with a preference for NAD+from Azospirillum palustre(ApFDH) was selected owing to its high activity. By static and dynamic structural analyses, a beneficial substitution, D222Q, was identified for cofactor-preference switching. However, its total activity was substantially decreased by 90% owing to the activity–specificity trade-off. Subsequently, a semirational library was designed and screened, which yielded a variant ApFDHD222Q+A199G+H380Swith satisfactory activity and NADP+specificity. Our analysis of dynamical cross-correlations revealed a substitution combination that brought balance to the dynamical correlation network. This combination successfully overcame the activity–specificity–stability trade-off and resulted in a beneficial outcome. The substitution combination (D222Q-A199G/H380S-C256A/C146S) enabled the simultaneous improvement of activity, specificity, and stability and was successfully applied to other 17 FDHs. Finally, by employing engineered ApFDH, an NADPH regeneration system was developed, optimized, and utilized for the asymmetric biosynthesis of l-phosphinothricin.

The increasingly complex molecular structures and high requirements of advanced industries are triggering a transformation in chemical production modes. Bio-manufacturing provides efficient strategies and brings the advantages of high atomic economy, few side reactions, and strong adaptability to processes, as well as environmental friendliness, which can contribute toward global efforts against greenhouse effect and environmental pollution. The significance of bio-manufacturing can be specifically illustrated by examining the bio-manufacturing process from the scientific discovery of a key compound to its technological integration and engineering innovation. The development of statins—important drugs for hypercholesterolemia treatment—is a good example of the progress and application of bio-manufacturing. The production of the first-generation statins from microorganisms, the second-generation statins using bioconversion, and the third-generation statins through an evolution from total chemical synthesis to chemoenzymatic synthesis demonstrates the technological and engineering revolution of bio-manufacturing, which is of great importance for energy conservation, cost saving, and waste emission reduction. With advances in cutting-edge biotechnologies, as well as the integration of multiple disciplines, bio-manufacturing is expected to promote the advancement of more intelligent processes to realize sustainable and green industrial development.

Lipase-catalyzed stereoselective resolution of cis-(±)-dimethyl 1-acetylpiperidine-2,3-dicarboxylate (cis-(±)-1) is an attractive route for the synthesis of (S,S)-2,8-diazobicyclo[4.3.0]nonane, an important chiral intermediate of the fluoroquinolone antibiotic, moxifloxacin. In our previous study, a lipase from Sporisorium reilianum(SRL) was identified to possess excellent thermostability and pH stability. However, the low enzymatic activity of the SRL is a challenge that must be addressed. A rational design was initially employed for SRL tailoring according to the engineered Candida antarcticalipase B (CALB), resulting in a beneficial variant called SRL-I194N/V195L. Subsequently, two key amino acid residues in loop 6, L145 and L154, which might modulate the lid conformation between open and closed, were identified. A tetra-site variant, SRL-I194N/V195L/L145V/L154G (V13), with a significantly enhanced activity of 87.8 U∙mg−1was obtained; this value was 2195-fold higher than that of wild-type SRL. Variant V13 was used to prepare optically pure (2S,3R)-dimethyl 1-acetylpiperidine-2,3-dicarboxylate ((2S,3R)-1), resolving 1 mol∙L−1cis-(±)-1 with a conversion of 49.9% in 2 h and absolute stereoselectivity (E > 200). Excellent stability with a half-life of 92.5 h was also observed at 50 °C. Overall, the study findings reveal a lipase with high activity toward cis-(±)-1 at an industrial level and may offer a general strategy for enhancing the enzyme activity of other lipases and other classes of enzymes with a lid moiety.

Precise control of a biocatalytic system is highly important but remains a major challenge. Herein, we developed a light-controlled enzyme production-biocatalysis one-pot system by combination of photocatalysis and photogenetic-regulated enzyme production. As proof-of-principle, a photoenzyme in an Escherichia coli-based biocatalyst was produced and restored by gene expression in the dark for decarboxylation under illumination. The kinetic resolution of racemic phosphinothricin with an intermittent illumination model displayed higher conversion (49%) and excellent stereoselectivity (ee = 98%), compared to a continuous illumination model (conv. = 44%; ee = 71%).

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 ilvCwas 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-ilvCV412Areached 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.

A chemoenzymatic strategy was developed for the highly efficient synthesis of l-phosphinothricin employing a robust immobilized amidase. An enzymatic hydrolysis of 500 mM N-phenylacetyl-d,l-phosphinothricin resulted in 49.9% conversion and 99.9% eeof l-phosphinothricin within 6 h. To further evaluate the bioprocess for l-phosphinothricin production, the biotransformation was performed for 100 batches under a stirred tank reactor with an average productivity of 8.21 g L–1h–1. Moreover, unreacted N-phenylacetyl-d-phosphinothricin was racemized and subjected to the enzymatic hydrolysis, giving l-phosphinothricin with a 22.3% yield. A total yield of 69.4% was achieved after one recycle of N-phenylacetyl-d-phosphinothricin. Significantly, this chemoenzymatic approach shows great potential in the industrial production of l-phosphinothricin.

Sugar alcohols are the prominent alternatives of sugars in food, medical, and health industries. The ruthenium supported on multiwalled carbon nanotubes (Ru/MWCNTs) catalysts were prepared based on the Ru valence regulation strategy and applied for selective sugar hydrogenation to prepare various sugar alcohols including xylitol, arabinitol, sorbitol, mannitol, and galactitol for the first time, with high selectivity (>99.0%) and yield (>98.0%) under mild conditions (≤110 °C, 3.0 MPa H2pressure). The hydrogenation reaction of xylose was further optimized and under mild conditions (100 °C, 3.0 MPa H2pressure, and 500 rpm), which were lower than ever reported for high efficient synthesis of xylitol, 99.8% xylose conversion and 99.0% xylitol yield were achieved after 120 min of reaction.

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 (CpNAMPTY13G+Y15S+F76P) with a remarkable 5-fold increase in enzyme activity was obtained. By utilizing CpNAMPTY13G+Y15S+F76Pas a biocatalyst, the accumulation of β-nicotinamide mononucleotide reached as high as 19.94 g L–1within 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.

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.

A chemoenzymatic process combing nitrilase-catalyzed hydrolysis and continuous flow racemization was developed for highly efficient manufacturing of (S)-pregabalin. Nitrilase was immobilized on epoxy resin and employed for the biosynthesis of (S)-3-cyano-5-methylhexanoic acid ((S)-CMHA), the precursor of (S)-pregabalin. Isobutylsuccinonitrile (IBSN) (700 mM) was converted to (S)-CMHA with a conversion of 45.3% and eeof 99.5%. Meanwhile, the unreacted (R)-IBSN was continuously racemized by the γ-alumina-based solid base catalysts (ASBCs) in a packed bed reactor, and an outstanding space-time yield of 6.76 mol L–1day–1was achieved. Moreover, the ASBCs exhibited excellent operational stability in the racemization system and retained 85.9% catalytic activity after 20 days of continuous racemization operation. (S)-CMHA was subsequently separated and purified by a membrane separation system in 95% yield and was hydrogenated in one step to (S)-pregabalin in a yield of 88%. These obtained results exhibited the efficiency of the chemoenzymatic process and the great potential for industrialization of (S)-pregabalin.

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. tsukubaensisstrains 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. tsukubaensisscreening method has been established and optimized.

An efficient chemoenzymatic route has been developed for the synthesis of optically active pregabalin (PGB) from isobutylsuccinonitrile (IBSN). (S)-3-cyano-5-methylhexanoic acid ((S)-CMHA), a critical chiral intermediate of PGB, was synthesized using regio- and enantioselective hydrolysis of IBSN by immobilized Escherichia colicells harboring nitrilase BrNIT from Brassica rapa. The catalytic performances of immobilized cells were investigated, and high enantioselectivity (E> 150) and substrate conversion (>41.1%) were obtained at a substrate loading of 100 g/L by immobilized cells after 12 batches of reaction. The unreacted (R)-IBSN was recycled by racemization with a high yield of 94.5%, and the resultant (S)-CMHA was hydrogenated directly to the desired PGB with a high purity of 99.6% and optical purity of 99.4%. The input of raw materials and Efactor of this chemoenzymatic route were demonstrated to be much lower than those of the first- and second-generation routes for PGB synthesis.

Candida antarcticalipase B (CALB) is a robust biocatalyst for production of chemicals. In this study, an engineered lipase B (CALB-I189K) was covalently immobilized onto a glutaraldehyde activated amino resin (ESR-3) and used for the preparation of chiral moxifloxacin precursor though enzymatic resolution of cis-(±)-dimethyl 1-acetylpiperidine-2,3-dicarboxylate (cis-(±)-1). The immobilization conditions were optimized, and the immobilized CALB-I189K exhibited improved thermal stability, with a half-life of 247.5 h at 30 °C. It enables the resolution of 100 g L–1cis-(±)-1for 50 cycles in a stirred tank reactor, with an average productivity of 49.6 g L–1h–1. The resolution reaction was also performed in a recirculating packed bed reactor (RPBR) in a semicontinuous mode, and the average productivity reached 59.4 g L–1h–1under the optimal operating condition. Moreover, the RPBR bioconversion system has high operational stability and conserved over 85.7% of the initial conversion capacity after 50 cycles. This efficient bioconversion process demonstrated the potential of employing immobilized CALB-I189K for industrial production of chiral moxifloxacin precursor.

The production of echinocandin B (ECB) by Aspergillus nidulansCCTCC M2012300 was improved by integrating the temperature-shift and fed-batch control strategies. The kinetic characteristics of batch cultures were analyzed at different culture temperatures, and then a two-stage temperature control strategy was established. In the first 6 days, the temperature was maintained at 30 °C to obtain the maximal cell growth rate; subsequently, 25 °C was used to gain a high ECB formation rate. On the basis of temperature control, the ECB productivity was increased to 143.3 mg/(L day), which was a 1.3-fold improvement compared with the optimal constant-temperature cultivations. The influences of fed-batch cultures were further investigated. A maximal ECB productivity of 170.8 mg/(L day) was obtained through a three-stage mannitol pulse-feeding strategy, which was another 1.2-fold improvement than that of the batch fermentation. This is the first report of the use of a two-stage temperature control fed-batch strategy in ECB fermentation. This strategy was simple and economical to operate and may provide new guidance for the industrial-scale production of ECB.

Microbial fermentation for L-methionine (L-Met) production based on natural renewable resources is attractive and challenging. In this work, the effects of medium composition and fermentation conditions were investigated to improve L-Met production by genetically engineered Escherichia coliMET-3. Statistical optimization techniques including Plackett–Burman (PB) design and Box–Behnken design (BBD) were adopted first to optimize the culture medium. Results of PB-designed experiments indicated that the culture medium components including glucose, yeast extract, KH2PO4, and MgSO4.7H2O had significant effects on L-Met biosynthesis. With their best-predicted concentration established by BBD (glucose 37.43 g/L, yeast extract 0.95 g/L, KH2PO41.82 g/L, and MgSO4.7H2O 4.51 g/L), L-Met titer was increased to 3.04 g/L from less than 2.0 g/L. For further enhancement of L-Met biosynthesis, the fermentation conditions of batch cultivation carried out in a 5-L fermentor were optimized, and the optimum results were obtained at an agitation rate of 300 rpm, medium pH of 7.0, and induction temperature of 28 °C. Based on the optimization parameters, fed-batch fermentation with the modified medium was conducted. As a result, great improvement of L-Met titer (12.80 g/L) and yield (0.13 mol/mol) were achieved, with an increase of 38.53% and 30.0% compared with those of the basal medium, respectively. Furthermore, higher L-Met productivity of 0.261 g/L/h was obtained, representing 2.13-fold higher in comparison to the original medium. The results may provide a helpful reference for further study on strain improvement and fermentation control.

(R)-2-Hydroxy-4-phenylbutyric acid ((R)-HPBA) is a key intermediate for the synthesis of angiotensin-converting enzyme inhibitors. Asymmetric reduction of 2-oxo-4-phenylbutyric acid (OPBA) by keto acid reductases represents a promising approach for (R)-HPBA production due to its high atom economy and theoretical yield of up to 100%. However, the low activity of keto acid reductases restricts their industrial application. In this study, it is the first time that a keto acid reductase from Leuconostoc lactis(LlKAR) with superior activity was designed for the reduction of OPBA to (R)-HPBA through reconstruction of the substrate binding pocket. Several residues (Leu127, Thr129, Leu244, Leu245, and Ala250) were selected for mutagenesis. Through three-round mutagenesis, multiple positive mutants were picked out. Compared with wild-type LlKAR, the mutant L244G/A250G/L245R exhibited 3.5-fold improvement in specific activity and 6.80-fold improvement in catalytic efficiency (Kcat/Km). Using L244G/A250G/L245R coupled with Exiguobacterium sibiricumglucose dehydrogenase (GDH) for cofactor regeneration, the productivity of (R)-HPBA was up to 85.7 mM h−1with >99% enantiomeric excess in the reaction system. The engineered L244G/A250G/L245R represents a potential and competitive biocatalyst for practical application in the synthesis of (R)-HPBA.

O-succinyl-l-homoserine (OSH) is a promising platform chemical for the production of C4 chemicals with huge market potential which can be produced by fermentation from glucose. To construct a strain capable of producing OSH with high yield, the metJ(encodes transcriptional repressor) and metI(encodes a subunit of dl-methionine transporter) were deleted in Escherichia coliW3110 to obtain a strain E. coli∆JI. Then, overexpression of metL(encodes bifunctional aspartate kinase/homoserine dehydrogenase II) and inactivation of metB(encodes cystathionine γ-synthase) were implemented in one step, and the OSH titer of the resulting strain E. coli∆JIB*TrcmetLwas dramatically increased to 7.30 g/L. The feedback regulation was further relieved by progressively overexpressing metAfbr(encodes homoserine O-succinyltransferase), yjeH(encodes l-methionine exporter), and thrAfbr(encodes bifunctional aspartate kinase/homoserine dehydrogenase I) to increase the metabolic flux from aspartate to OSH. The 100% rationally designed strain E. coli∆JIB*TrcmetL/pTrc-metAfbr-Trc-thrAfbr-yjeHproduced 9.31 g/L OSH from 20 g/L glucose (0.466 g/g glucose) in batch fermentation, which represents the highest OSH yield from glucose reported to date. The culture profiles of the newly constructed strains were recorded to investigate their productive properties. The effects of l-methionine addition on the fermentation process of the optimal strain were also studied. Our results demonstrate that tuning the expression level of metL, inactivation of metB, and attenuation of feedback resistance of the crucial enzymes in the biosynthetic pathway are the key factors that impact the OSH production in E. coli.

Adaptable exploitation of the catalytic potential of membrane-bound d-sorbitol dehydrogenase (mSLDH) from Gluconobacter oxydansis desperately needed in the industrial-scale production of miglitol. In the present study, a carbonyl group-dependent colorimetric quantification method was developed for the assay of miglitol key intermediate 6-(N-hydroxyethyl)-amino-6-deoxy-α-l-sorbofuranose (6NSL), and a high-throughput screening process of positive mutants was processed. Combined with several rounds of ultraviolet irradiation mutagenesis and screening procedure, a positive mutant strain G. oxydansZJB16009 was obtained with significant increase in mSLDH catalytic activity by 1.5-fold, which exhibited an extremely accelerated uptake rate of d-sorbitol, and the fermentation time was significantly shortened from 22 to 11 h. In a 5-L biotransformation system, 60 g/L substrate N-2-hydroxyethyl glucamine (NHEG) was catalyzed by the resting cells of the mutant strain within 36 h and accumulated 53.6 g/L 6NSL, showing a 33.6% increase in the product yield. Therefore, it was indicated that the established high-throughput screening method could provide a highly efficient platform for the breading of G. oxydansstrain for the industrial biosynthesis of miglitol intermediate 6NSL.

Candida antarcticalipase B (CALB) is a versatile and robust lipase with high activity and enantioselectivity in the resolution of alcohols and amines. Poor specific activity toward carboxylic esters that are branched or substituted in close proximity to the carboxyl group limits the application of CALB. In this study, a semi-rational engineering approach was successfully applied to tailor CALB for efficient synthesis of the moxifloxacin chiral intermediate through enzymatic resolution of cis-(±)-dimethyl 1-acetylpiperidine-2,3-dicarboxylate. The combinatorial active-site saturation test was performed on CALB and two key residue sites, 189 and 190, were identified in the determination of hydrolytic activity. The specific activity and catalytic efficiency (kcat/Km) of the best variant mut-I189K were increased by 286- and 193-fold, respectively, without impacting its enantioselectivity. With 0.1 g L−1of purified mut-I189K loading, 1 M substrate was asymmetrically hydrolyzed with 49.9% conversion within 5 h, resulting in a space–time yield of 583.8 g L−1d−1. These results demonstrated that mut-I189K is a competitive biocatalyst for large-scale preparation of the moxifloxacin chiral intermediate.

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 colias 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 metAfrom Bacillus cereus(metXbc) and engineered an OAH-producing strain by combinatorial metabolic engineering. Initially, exogenous metXs/metAwere 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 metXbcwere carried out, accumulating 5.47 g/L OAH. Meanwhile, the homoserine pool was enriched by overexpressing metLwith 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.

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-4was 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.

Functional sugar alcohols are a kind of burgeoning food additives to substitute saccharose due to the prominent biofunctions including moderate sweetness, low calorific values and no influence on blood sugar fluctuation, thus applied widely in food, medical, health protection as well as chemical production. Traditional synthesis of functional sugar alcohols required high temperature and H2pressure, as well as leading to low reaction efficiency, side reactions and leaching of toxic metal ions. In the present work, Pt–Ni–Sn/MWCNTs catalyst was obtained using a liquid phase reduction method with the transition metals loading of Pt 3.4%, Ni 0.7%, Sn 0.4%, respectively. The catalyst catalyzed the preparation of xylitol, sorbitol, mannitol and galactitol with the high monosaccharide conversion (>99.5%) and yield (>99.1%) under relatively mild conditions (100 °C, 3.0 MPa H2), which were lower than ever reported. For the 13-batch reuse of Pt–Ni–Sn/MWCNTs, the total conversion of xylose was 99.4% and the total yield of xylitol was 99.2%, the productivity of 103.2 g xylitol per gram of catalyst was also obtained. Besides, no side reactions, no metal ions as well as no changes of pH were observed in the reaction process. Our trimetallic catalyst exhibited valuable potential in functional sugar alcohols production.

3-Hydroxypropionic acid (3-HP) is an important compound and precursor for a series of chemicals and polymeric materials. In this study, the 3-HP producing bacteria were constructed and studied for efficient synthesis of 3-HP. The results indicated that the instability of glycerol dehydratase (GDHt) affected the 3-HP production significantly, which was successfully solved by the expression of glycerol dehydratase reactivase (GdrB), with fivefold increase in 3-HP yield. Meanwhile, NAD+-regenerating enzymes GPD1 (glycerol-3-phosphate dehydrogenase) was expressed; however, the results showed 3-HP was significantly decreased from 56.73–4 mM, and malic acid was obviously increased. Analysis of the C flux distribution showed that the main reason for the results was the lack of NAD+. The addition of NAD+further increased the 3-HP production to 23.87 mM, demonstrating that the “regeneration of NAD+” was the major factor for enhancing 3-HP production.

We report a novel method for the seamless cloning of large DNA fragments (SCLF) of up to 44 kb or larger from Streptomyceschromosomal DNA. SCLF is based on homologous recombination in Streptomycesand is easy to perform. The strategy of SCLF is to flank the target sequence in the chromosomal DNA with two identical restriction sites by the insertion of plasmids containing that site at either end of the fragment, which is then isolated by plasmid rescue through the self-ligation of restriction digested genomic DNA. The method involves three steps: (i) placing a certain restriction site (CRS) at the 3′-end of the target sequence by insertion through homologous recombination of a plasmid containing the CRS; (ii) inserting through homologous recombination at the 5′-end of the target sequence a linearized self-suicide vector with the identical CRS; (iii) digesting the genomic DNA with the certain restriction enzyme followed by self-ligation in order to plasmid rescue the target fragment. SCLF can be applied to other Actinomycetales, and further optimizations may reduce the amount of time required to perform this technique.

Considering the essential influencing factors for the mechanical properties of the engineered cementitious composite (ECC), the polyvinyl alcohol (PVA) fiber was used and a batch of cube specimens and thin plate specimens of PVA-ECC were manufactured based on the variation of water cement ratio, amount of fly ash and amount of water reducer. The compressive strength test for the PVA-ECC cube specimens and the flexural strength test for the PVA-ECC thin plate specimens were conducted. A practical decision-making frame based on the Analytic Hierarchy Process (AHP) method was proposed to solve the problem of the optimal mix proportion of PVA-ECC, which can take into account the compressive strength, the flexural strength and the corresponding deformation capacity together.

Biotransformation of iminodiacetonitrile (IDAN) to iminodiacetic acid (IDA) was investigated with a newly isolated Alcaligenes faecalisZJUTBX11 strain showing nitrilase activity in the immobilized form. To reduce the mass transfer resistance and to increase the toleration ability of the microorganisms to the toxic substrate as well as to enhance their ability to be reused, encapsulation of the whole cells in alginate-chitosan-alginate (ACA) membrane liquid-core capsules was attempted in the present study. The optimal pH and temperature for nitrilase activity of encapsulated A. faecalisZJUTBX11 cells were 7.5°C and 35°C, respectively, which is consistent with free cells. Based on the Michaelis-Menten model, kinetic parameters of the conversion reaction with IDAN as the substrate were: Km= (17.6 ± 0.3) mmol L−1and Vmax= (97.6 ± 1.2) μmol min−1g−1of dry cell mass for encapsulated cells and (16.8 ± 0.4) mmol L−1and (108.0 ± 2.7) μmol min−1g−1of dry cell mass for free cells, respectively. After being recycled ten times, the whole cells encapsulated in ACA capsules still retained 90 % of the initial nitrilase activity while only 35 % were retained by free cells. Lab scale production of IDA using encapsulated cells in a bubble column reactor and a packed bed reactor were performed respectively.