Publications by authors named "Zhiming Rao"

117 Publications

Vaccinium bracteatum Thunb. as a promising resource of bioactive compounds with health benefits: An updated review.

Food Chem 2021 Mar 31;356:129738. Epub 2021 Mar 31.

School of Food Science and Technology, State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China; National Engineering Research Center for Functional Food, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China. Electronic address:

Vaccinium bracteatum Thunb.(VBT) is well-known for many physiological and bioactivities in some ancient Chinese pharmacopeias and modern researches. The health benefits are related to the presence of various nutritional and bioactive compounds. This review aims to demonstrate an updated overview of VBT in respect of botanical characters, nutritional and bioactive composition, main biological activities, and current applications. Various studies have emphasized at promising health benefits of VBT against hyperglycemia, oxidative stress, inflammation, depressive disorder, and retinal damage. However, the applications of VBT are limited to some native traditional foods and Chinese medicine. The novel beneficial efficacy and applications are still needed to be investigated. In conclusion, more research is necessary to overcome these gaps between the in-depth insights of health benefits and potential industrial applications. This review will contribute in future research for developing the functional foods derived from VBT.
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http://dx.doi.org/10.1016/j.foodchem.2021.129738DOI Listing
March 2021

Enhanced production of L-arginine by improving carbamoyl phosphate supply in metabolically engineered Corynebacterium crenatum.

Appl Microbiol Biotechnol 2021 Apr 10;105(8):3265-3276. Epub 2021 Apr 10.

Key Laboratory of Industrial Biotechnology of the Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, China.

Carbamoyl phosphate is an important precursor for L-arginine and pyrimidines biosynthesis. In view of this importance, the cell factory should enhance carbamoyl phosphate synthesis to improve related compound production. In this work, we verified that carbamoyl phosphate is essential for L-arginine production in Corynebacterium sp., followed by engineering of carbamoyl phosphate synthesis for further strain improvement. First, carAB encoding carbamoyl phosphate synthetase II was overexpressed to improve the synthesis of carbamoyl phosphate. Second, the regulation of glutamine synthetase increases the supply of L-glutamine, providing an effective substrate for carbamoyl phosphate synthetase II. Third, carbamate kinase, which catalyzes inorganic ammonia synthesis carbamoyl phosphate, was screened and selected to assist in carbamoyl phosphate supply. Finally, we disrupted ldh (encoding lactate dehydrogenase) to decrease by-production formation and save NADH to regenerate ATP through the electron transport chain. Subsequently, the resulting strain allowed a dramatically increased L-arginine production of 68.6 ± 1.2 g∙L, with an overall productivity of 0.71 ± 0.01 g∙L∙h in 5-L bioreactor. Stepwise rational metabolic engineering based on an increase in the supply of carbamoyl phosphate resulted in a gradual increase in L-arginine production. The strategy described here can also be implemented to improve L-arginine and pyrimidine derivatives. KEY POINTS: • The L-arginine production strongly depended on the supply of carbamoyl phosphate. • The novel carbamoyl phosphate synthesis pathway for C. crenatum based on carbamate kinase was first applied to L-arginine synthesis. • ATP was regenerated followed with the disruption of lactate formation.
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http://dx.doi.org/10.1007/s00253-021-11242-wDOI Listing
April 2021

[Advances in stress tolerance mechanisms and synthetic biology for the industrial robustness of Corynebacterium glutamicum].

Sheng Wu Gong Cheng Xue Bao 2021 Mar;37(3):831-845

Key Laboratory of Industrial Biotechnology, Ministry, of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, Jiangsu, China.

As a model industrial host and microorganism with the generally regarded as safe (GRAS) status, Corynebacterium glutamicum not only produces amino acids on a large scale in the fermentation industry, but also has the potential to produce various new products. C. glutamicum usually encounters various stresses in the process of producing compounds, which severely affect cell viability and production performance. The development of synthetic biology provides new technical means for improving the robustness of C. glutamicum. In this review, we discuss the tolerance mechanisms of C. glutamicum to various stresses in the fermentation process. At the same time, we highlight new synthetic biology strategies for boosting C. glutamicum robustness, including discovering new stress-resistant elements, modifying transcription factors, and using adaptive evolution strategies to mine stress-resistant functional modules. Finally, prospects of improving the robustness of engineered C. glutamicum strains ware provided, with an emphasis on biosensor, screening and design of transcription factors, and utilizing the multiple regulatory elements.
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http://dx.doi.org/10.13345/j.cjb.200631DOI Listing
March 2021

Semi-quantitative activity assays for high-throughput screening of higher activity gamma glutamyl transferase and enzyme immobilization to efficiently synthesize L-theanine.

J Biotechnol 2021 Mar 24;330:9-16. Epub 2021 Feb 24.

The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu Province 214122, China. Electronic address:

The bio-production of theanine is currently of significant interest due to its wide applications in food and healthcare products. Gamma glutamyl transferase (GGT) has been widely applied in L-theanine synthesis, but L-theanine yields remain prohibitively low for commercial production. In this study, a robust high-throughput screening process for isolating GGT mutants was developed through a combination of error-prone PCR techniques and a colorimetric reaction. The co-expression of PrsA lipoprotein enhances the secretion of GGT, thus GGT could be obtained quickly and easily without crushing cells. Random mutations on ggt genes were introduced by using error-prone PCR kits to build a large mutant library. A colorless compound generated by the reaction between NH (released from L-theanine synthesis) and OPA was measured quantitatively by UV/visible spectroscopy when mixed with TCA and DMSO. Approximately 30 positive clones with improved color formation on the 96-well plates were identified, and mutants T413P and T463S with more than by 30 % higher transpeptidation activity versus the original GGT were isolated. To improve the operational stability and economical use, mutant GGT was immobilized on a prepared oxidized cellulose nanofiber membrane. The remaining activity of immobilized GGT was 88 % versus 72 % of free enzyme over 15 h. A fed-batch conversion was performed with the immobilized GGT, and over 70 g/L L-theanine could be accumulated within 18 h after feeding twice. Versus other studies, this is one of the best L-theanine synthesis systems using immobilized GGT.
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http://dx.doi.org/10.1016/j.jbiotec.2021.02.011DOI Listing
March 2021

Rational engineering of the Plasmodium falciparuml-lactate dehydrogenase loop involved in catalytic proton transfer to improve chiral 2-hydroxybutyric acid production.

Int J Biol Macromol 2021 May 22;179:71-79. Epub 2021 Feb 22.

The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu Province 214122, China. Electronic address:

l-lactate dehydrogenases (LDHs) has been widely studied for their ability to reduce 2-keto acids for the production of 2-hydroxy acids, whereby 2-hydroxybutyric acids (2-HBA) is among the most important fundamental building blocks for synthesizing pharmaceuticals and biodegradable materials. However, LDHs usually show low activity towards 2-keto acids with longer side chain such as 2-oxobutyric acid (2-OBA). Here rational engineering of the Plasmodium falciparum LDH loop with residue involved in the catalytic proton transfer was initially studied. By combining homology alignment and structure-based design approach, we found that changing the charge characteristics or hydrogen bond network interactions of this loop could improve enzymatic catalytic activities and stabilities towards 2-OBA. Compared with wild type, variant N197D showed 1.15 times higher activity and 2.73 times higher K/Km. The half-life of variant N197D at 40 °C increased to 77.9 h compared with 50.4 h of wild type. Furthermore, asymmetric synthesis of (S)-2-HBA with coenzyme regeneration revealed 95.8 g/L production titer within 12 h for variant N197D, 2.05 times higher than using wild type. Our study indicated the importance of loop with residues involved in the catalytic proton transfer process, and the engineered LDH would be more suitable for (S)-2-HBA production.
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http://dx.doi.org/10.1016/j.ijbiomac.2021.02.144DOI Listing
May 2021

Biochemical Characterization and Structural Insight into Interaction and Conformation Mechanisms of Lysine Decarboxylase (SmcadA).

Molecules 2021 Jan 29;26(3). Epub 2021 Jan 29.

The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 LiHu Boulevard, Wuxi 214122, China.

Inducible lysine decarboxylases (LDCs) are essential in various cellular processes of microorganisms and plants, especially under acid stress, which induces the expression of genes encoding LDCs. In this study, a novel LDC (SmcadA) was successfully expressed in , purified and characterized. The protein had an optimal pH of 6 and a temperature of 40 °C and phylogenetic analysis to determine the evolution of SmcadA, which revealed a close relation to sp., among others. The molecular weight of SmcadA was approximately 75 kDa after observation on SDS-PAGE and structural modeling showed the protein as a decamer, comprised of five interlinked dimers. The biocatalytic activity of the purified wild-type SmcadA (WT) was improved through site directed mutations and the results showed that the Arg595Lys mutant had the highest specific activity of 286.55 U/mg, while the Ser512Ala variant and wild-type SmcadA had 215.72 and 179.01 U/mg, respectively. Furthermore, molecular dynamics simulations revealed that interactions through hydrogen bonds between the protein residues and cofactor pyridoxal-5-phosphate (PLP) are vital for biocatalysis. Molecular Dynamics (MD) simulations also indicated that mutations conferred structural changes on protein residues and PLP hence altered the interacting residues with the cofactor, subsequently influencing substrate bioconversion. Moreover, the temperature also induced changes in orientation of cofactor PLP and amino acid residues. This work therefore demonstrates the successful expression and characterization of the purified novel lysine decarboxylase from and provided insight into the mechanism of protein-cofactor interactions, highlighting the role of protein-ligand interactions in altering cofactor and binding site residue conformations, thus contributing to improved biocatalysis.
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http://dx.doi.org/10.3390/molecules26030697DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7866039PMC
January 2021

Efficient single whole-cell biotransformation for L-2-aminobutyric acid production through engineering of leucine dehydrogenase combined with expression regulation.

Bioresour Technol 2021 Apr 7;326:124665. Epub 2021 Jan 7.

The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu Province 214122, PR China. Electronic address:

Leucine dehydrogenase (LDH) is widely used in the preparation of L-2-aminobutyric acid (L-2-ABA), however its wide application is limited by 2-ketobutyric acid (2-OBA) inhibition. Firstly, a novel high-throughput screening method of LDH was established, specific enzyme activity and 2-OBA tolerance of Lys72Ala mutant were 33.3% higher than those of the wild type. Subsequently, we constructed a single cell comprised of ivlA, Esldh, fdh and optimized expression through fine-tuning RBS intensity, so that the yield of E. coli BL21/pET28a-R3ivlA-Esldh-fdh was 2.6 times higher than that of the original strain. As a result, 150 g L-threonine was transformed to 121 g L-2-ABA in 5 L fermenter with 95% molar conversion rate, and a productivity of 5.04 g·L·h, which is the highest productivity of L-2-ABA currently reported by single-cell biotransformation. In summary, our research provided a green synthesis for L-2-ABA, which has potential for industrial production of drug precursors.
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http://dx.doi.org/10.1016/j.biortech.2021.124665DOI Listing
April 2021

Redistribution of Intracellular Metabolic Flow in Improves Carbon Atom Economy for High-Yield 2,5-Dimethylpyrazine Production.

J Agric Food Chem 2021 Mar 1;69(8):2512-2521. Epub 2021 Feb 1.

Key Laboratory of Industrial Biotechnology of the Ministry of Education, Laboratory of Applied Microorganisms and Metabolic Engineering, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu 214122, China.

2,5-Dimethylpyrazine (2,5-DMP) is an important pharmaceutical intermediate and an important essence. Conventional chemical synthesis methods are often accompanied by toxic substances as by-products, and the biosynthesis efficiency of 2,5-DMP is insufficient for industrial applications. In this study, the and genes were overexpressed to enhance enzymatic and nonenzymatic reactions in metabolic pathways, and was knocked out to block competitive branching carbon flow metabolic pathways. Finally, a genetically engineered strain with the highest carbon recovery rate (30.18%) and the highest yield reported to date was successfully constructed, and 9.21 g·L threonine was able to produce 1682 mg·L 2,5-DMP after 24 h. At the same time, an expression regulation strategy and whole-cell biocatalysis helped to eliminate the damage to cells caused by 2,5-DMP, aminoacetone, and reactive oxygen species generated by aminoacetone oxidase from , and the negative effect of 2-amino-3-ketobutyrate CoA ligase on the yield of 2,5-DMP in was also demonstrated.
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http://dx.doi.org/10.1021/acs.jafc.0c07408DOI Listing
March 2021

Identification of bottlenecks in 4-androstene-3,17-dione/1,4-androstadiene-3,17-dione synthesis by Mycobacterium neoaurum JC-12 through comparative proteomics.

J Biosci Bioeng 2021 Mar 8;131(3):264-270. Epub 2020 Dec 8.

Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China; Laboratory of Applied Microorganisms and Metabolic Engineering, School of Biotechnology, Jiangnan University, Wuxi 214122, China. Electronic address:

Intermediates such as 4-androstene-3,17-dione (AD) and 1,4-androstadiene-3,17-dione (ADD) have extensive clinical applications in the production of steroid pharmaceuticals. The present study explores the effect of two factors in the production of these intermediates in Mycobacterium neoaurum JC-12: the precursor, phytosterol and a molecule that increases AD/ADD solubility, hydroxypropyl-β-cyclodextrin (HP-β-CD). Differentially expressed proteins were separated and identified using 2D gel electrophoresis (2-DE) and matrix assisted laser desorption/ionization time-of-flight/time-of-flight tandem mass spectrometry (MALDI-TOF/TOF-MS/MS). In total, 31 proteins were identified, and improved expression levels of ten proteins involved in metabolism was induced by phytosterol and/or HP-β-CD, which strengthened the stress resistance of the strain. In the presence of phytosterol and/or HP-β-CD, five proteins involved in the synthesis of AD/ADD, acetyl-CoA acetyltransferase (AAT), alcohol dehydrogenase (ADH), enoyl-CoA hydratase (EH) and short-chain dehydrogenase 1 and 2, increased their expression levels. Reverse transcription-quantitative PCR (RT-qPCR) was used to verify the 2-DE results and the transcriptional level of these five proteins. This analysis identified AAT, ADH, EH, and electron transfer flavoprotein subunit α/β as the possible bottlenecks for AD/ADD synthesis in M. neoaurum JC-12, which therefore are suggested as targets for strain modification.
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http://dx.doi.org/10.1016/j.jbiosc.2020.10.006DOI Listing
March 2021

Integrated gene engineering synergistically improved substrate-product transport, cofactor generation and gene translation for cadaverine biosynthesis in E. coli.

Int J Biol Macromol 2021 Feb 7;169:8-17. Epub 2020 Dec 7.

The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 LiHu Boulevard, Wuxi 214122, Jiangsu, China. Electronic address:

Several approaches for efficient production of cadaverine, a bio-based diamine with broad industrial applications have been explored. Here, Serratia marcescens lysine decarboxylase (SmcadA) was expressed in E. coli; mild surfactants added in biotransformation reactions; the E. coli native lysine/cadaverine antiporter cadB, E. coli pyridoxal kinases pdxK and pdxY overexpressed and synthetic RBS libraries screened. Addition of mild surfactants and overexpression of antiporter cadB increased cadaverine biosynthesis of SmcadA. Moreover, expression of pdxY gene yielded 19.82 g/L in a reaction mixture containing added cofactor precursor pyridoxal (PL), without adding exogenous PLP. The screened synthetic RBS1, applied to fully exploit pdxY gene expression, ultimately resulted in PLP self-sufficiency, producing 27.02 g/L cadaverine using strain T7R1_PL. To boost SmcadA catalytic activity, the designed mutants Arg595Lys and Ser512Ala had significantly improved cumulative cadaverine production of 219.54 and 201.79 g/L respectively compared to the wild-type WT (181.62 g/L), after 20 h reaction. Finally, molecular dynamics simulations for WT and variants indicated that increased flexibility at the binding sites of the protein enhanced residue-ligand interactions, contributing to high cadaverine synthesis. This work demonstrates potential of harnessing different pull factors through integrated gene engineering of efficient biocatalysts and gaining insight into the mechanisms involved through MD simulations.
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http://dx.doi.org/10.1016/j.ijbiomac.2020.12.017DOI Listing
February 2021

[Effect of key notes of TCA cycle on L-glutamate production].

Sheng Wu Gong Cheng Xue Bao 2020 Oct;36(10):2113-2125

School of Biotechnology, Jiangnan University, Wuxi 214122, Jiangsu, China.

Glutamic acid is an important amino acid with wide range of applications and huge market demand. Therefore, by performing transcriptome sequencing and re-sequencing analysis on Corynebacterium glutamicum E01 and high glutamate-producing strain C. glutamicum G01, we identified and selected genes with significant differences in transcription and gene levels in the central metabolic pathway that may have greatly influenced glutamate synthesis and further increased glutamic acid yield. The oxaloacetate node and α-ketoglutarate node play an important role in glutamate synthesis. The oxaloacetate node and α-ketoglutarate node were studied to explore effect on glutamate production. Based on the integrated strain constructed from the above experimental results, the growth rate in a 5-L fermenter was slightly lower than that of the original strain, but the glutamic acid yield after 48 h reached (136.1±5.53) g/L, higher than the original strain (93.53±4.52) g/L, an increase by 45.5%; sugar-acid conversion rate reached 58.9%, an increase of 13.7% compared to 45.2% of the original strain. The application of the above experimental strategy improved the glutamic acid yield and the sugar-acid conversion rate, and provided a theoretical basis for the metabolic engineering of Corynebacterium glutamicum.
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http://dx.doi.org/10.13345/j.cjb.200038DOI Listing
October 2020

[Whole-cell biosynthesis of 2-O-α-D-glu-copyranosyl-sn-glycerol by recombinant Bacillus subtilis].

Sheng Wu Gong Cheng Xue Bao 2020 Sep;36(9):1918-1928

Key Laboratory of Industrial Biotechnology of Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, Jiangsu, China.

2-O-α-D-glu-copyranosyl-sn-glycerol is a high value-added product with prospective application in food, cosmetics, health products and pharmaceutical industries. However, industrial scale of 2-O-α-D-glu-copyranosyl-sn-glycerol has not yet been applied in China, and there are few related reports on 2-O-α-D-glu-copyranosyl-sn-glycerol synthesis. The purpose of this experiment is to develop a method for catalyzing the synthesis of food-grade 2-O-α-D-glu-copyranosyl-sn-glycerol using whole cells of "Generally Recognized as Safe" (GRAS) recombinant Bacillus subtilis. In our work, a recombinant B. subtilis 168/pMA5-gtfA that heterologously expressing Leuconostoc mesenteroides sucrose phosphorylase was constructed and used as a whole-cell catalyst to synthesize 2-O-α-D-glu-copyranosyl-sn-glycerol. Optimizing the culture temperature, time and whole cell transformation conditions has increased the yield of 2-O-α-D-glu-copyranosyl-sn-glycerol. The results showed that 1.43 U/mL of sucrose phosphorylase was achieved in B. subtilis 168/pMA5-gtfA after culturing for 20 h at 30 °C in fermentation medium. The highest conversion rate reached 75.1%, and the yield of 2-O-α-D-glu-copyranosyl-sn-glycerol was 189.3 g/L with an average transformation rate of 15.6 mmol/(L·h) after 48 hours whole-cell transformation with the sucrose concentration of 1 mol/L and the glycerol concentration of 2.5 mol/L at 30 °C, OD₆₀₀ 40 and pH 7.0. This is the highest yield of 2-O-α-D-glu-copyranosyl-sn-glycerol synthesized catalytically by recombinant B. subtilis that was ever reported, and this study provides the theoretical and experimental basis for the industrial production and application of 2-O-α-D-glucopyranosyl-sn-glycerol.
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http://dx.doi.org/10.13345/j.cjb.190588DOI Listing
September 2020

Regulator RcsB Controls Prodigiosin Synthesis and Various Cellular Processes in Serratia marcescens JNB5-1.

Appl Environ Microbiol 2021 01 4;87(2). Epub 2021 Jan 4.

Key Laboratory of Industrial Biotechnology of the Ministry of Education, Laboratory of Applied Microorganisms and Metabolic Engineering, School of Biotechnology, Jiangnan University, Wuxi, China

Prodigiosin (PG), a red linear tripyrrole pigment normally secreted by , has received attention for its reported immunosuppressive, antimicrobial, and anticancer properties. Although several genes have been shown to be important for prodigiosin synthesis, information on the regulatory mechanisms behind this cellular process remains limited. In this work, we identified that the transcriptional regulator RcsB encoding gene () negatively controlled prodigiosin biosynthesis in Disruption of conferred a remarkably increased production of prodigiosin. This phenotype corresponded to negative control of transcription of the prodigiosin-associated operon by RcsB, probably by binding to the promoter region of the prodigiosin synthesis positive regulator FlhDC. Moreover, using transcriptomics and further experiments, we revealed that RcsB also controlled some other important cellular processes, including swimming and swarming motilities, capsular polysaccharide production, biofilm formation, and acid resistance (AR), in Collectively, this work proposes that RcsB is a prodigiosin synthesis repressor in and provides insight into the regulatory mechanism of RcsB in cell motility, capsular polysaccharide production, and acid resistance in RcsB is a two-component response regulator in the Rcs phosphorelay system, and it plays versatile regulatory functions in However, information on the function of the RcsB protein in bacteria, especially in , remains limited. In this work, we illustrated experimentally that the RcsB protein was involved in diverse cellular processes in , including prodigiosin synthesis, cell motility, capsular polysaccharide production, biofilm formation, and acid resistance. Additionally, the regulatory mechanism of the RcsB protein in these cellular processes was investigated. In conclusion, this work indicated that RcsB could be a regulator for prodigiosin synthesis and provides insight into the function of the RcsB protein in .
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http://dx.doi.org/10.1128/AEM.02052-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7783331PMC
January 2021

Surface charge-based rational design of aspartase modifies the optimal pH for efficient β-aminobutyric acid production.

Int J Biol Macromol 2020 Dec 1;164:4165-4172. Epub 2020 Sep 1.

The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu Province, China. Electronic address:

β-Aminobutyric acid (BABA) can be widely used in the preparation of anti-tumor drugs, AIDS drugs, penicillin antibiotics, and plant initiators. However, the efficient, economical, and environmentally friendly production of BABA still faces challenges. Its important production enzyme, aspartase, catalyzes the substrate crotonic acid, and depends on harsh conditions, such as high temperatures and the presence of strong alkali. Here, we modified the surface charge of the enzyme to enable it to become more negatively charged (K19E, N87E, N125D, S133D, Q262E, and N451E; from -60 to -80), reducing its optimal pH from 9.0 to 8.0. The M20 enzyme showed improved specific activity (400.21 mU/mg at pH 8.0; 2.47-fold that of aspartase), and at pH 7.0, its activity increased 3-fold. The thermal stability of the enzyme was also improved. For the production of BABA, a 500 g/L whole-cell transformation was obtained with a 1.41-fold increase in yield, and the final production of BABA reached 556.1 g/L within 12 h. Our method provides a new strategy for modifying the characteristics of the enzyme through the modification of its surface charge, which also represents the first modification of the optimal pH for aspartase.
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http://dx.doi.org/10.1016/j.ijbiomac.2020.08.229DOI Listing
December 2020

Engineered disulfide bonds improve thermostability and activity of L-isoleucine hydroxylase for efficient 4-HIL production in 168.

Eng Life Sci 2020 Jan 9;20(1-2):7-16. Epub 2019 Oct 9.

The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology Jiangnan University Wuxi Jiangsu Province P. R. China.

4-Hydroxyisoleucine, a promising drug, has mainly been applied in the clinical treatment of type 2 diabetes in the pharmaceutical industry. l-Isoleucine hydroxylase specifically converts l-Ile to 4-hydroxyisoleucine. However, due to its poor thermostability, the industrial production of 4-hydroxyisoleucine has been largely restricted. In the present study, the disulfide bond in l-isoleucine hydroxylase protein was rationally designed to improve its thermostability to facilitate industrial application. The half-life of variant T181C was 4.03 h at 50°C, 10.27-fold the half-life of wild type (0.39 h). The specific enzyme activity of mutant T181C was 2.42 ± 0.08 U/mg, which was 3.56-fold the specific enzyme activity of wild type 0.68 ± 0.06 U/mg. In addition, molecular dynamics simulation was performed to determine the reason for the improvement of thermostability. Based on five repeated batches of whole-cell biotransformation, 168/pMA5- recombinant strain produced a cumulative yield of 856.91 mM (126.11 g/L) 4-hydroxyisoleucine, which is the highest level of productivity reported based on a microbial process. The results could facilitate industrial scale production of 4-hydroxyisoleucine. Rational design of disulfide bond improved l-isoleucine hydroxylase thermostability and may be suitable for protein engineering of other hydroxylases.
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http://dx.doi.org/10.1002/elsc.201900090DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6999076PMC
January 2020

Improved Prodigiosin Production by Relieving CpxR Temperature-Sensitive Inhibition.

Front Bioeng Biotechnol 2020 3;8:344. Epub 2020 Jun 3.

The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, China.

Prodigiosin (PG) is a typical secondary metabolite mainly produced by . CpxR protein is an OmpR family transcriptional regulator in Gram-negative bacteria. Firstly, it was found that insertion mutation of in JNB 5-1 by a transposon Tn5G increased the production of PG. Results from the electrophoretic mobility shift assay (EMSA) indicated that CpxR could bind to the promoter of the gene cluster and repress the transcription levels of genes involved in PG biosynthesis in JNB 5-1. In the Δ mutant strain, the transcription levels of the gene cluster and the genes involved in the pathways of PG precursors, such as proline, pyruvate, serine, methionine, and S-adenosyl methionine, were significantly increased, hence promoting the production of PG. Subsequently, a fusion segment composed of the genes , and , responsible for proline, serine, and methionine, was inserted into the gene in JNB 5-1. On fermentation by the resultant engineered , the highest PG titer reached 5.83 g/L and increased by 41.9%, relative to the parental strain. In this study, we revealed the role of CpxR in PG biosynthesis and provided an alternative strategy for the engineering of to enhance PG production.
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http://dx.doi.org/10.3389/fbioe.2020.00344DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7283389PMC
June 2020

Directed Evolution of Ornithine Cyclodeaminase Using an EvolvR-Based Growth-Coupling Strategy for Efficient Biosynthesis of l-Proline.

ACS Synth Biol 2020 07 8;9(7):1855-1863. Epub 2020 Jul 8.

The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China.

l-Proline takes a significant role in the pharmaceutical and chemical industries as well as graziery. Typical biosynthesis of l-proline is from l-glutamate, involving three enzyme reactions as well as a spontaneous cyclization. Alternatively, l-proline can be also synthesized in l-ornithine and/or l-arginine producing strains by an ornithine aminotransferase (OCD). In this study, a strategy of directed evolution combining rare codon selection and pEvolvR was developed to screen OCD with high catalytic efficiency, improving l-proline production from l-arginine chassis cells. The mutations were generated by CRISPR-assisted DNA polymerases and were screened by growth-coupled rare codon selection system. OCD from was identified with 2.85-fold increase in catalytic efficiency for the synthesis of l-proline. Furthermore, we designed and optimized RBS for the and coupling cascade using RedLibs, as well as sRNA inhibition of to moderate l-proline biosynthesis in l-arginine overproducing . The strain PS6 with best performance reached 15.3 g/L l-proline in the shake flask and showed a titer of 38.4 g/L in a 5 L fermenter with relatively low concentration of residual l-ornithine and/or l-arginine.
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http://dx.doi.org/10.1021/acssynbio.0c00198DOI Listing
July 2020

Significantly enhancing production of -4-hydroxy-l-proline by integrated system engineering in .

Sci Adv 2020 May 22;6(21):eaba2383. Epub 2020 May 22.

The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China.

4-hydroxy-l-proline is produced by -proline-4-hydroxylase with l-proline through glucose fermentation. Here, we designed a thorough "from A to Z" strategy to significantly improve -4-hydroxy-l-proline production. Through rare codon selected evolution, M1 produced 18.2 g L l-proline. Metabolically engineered M6 with the deletion of , , , and , and mutation focused carbon flux to l-proline and released its feedback inhibition. It produced 15.7 g L-4-hydroxy-l-proline with 10 g L l-proline retained. Furthermore, a tunable circuit based on quorum sensing attenuated l-proline hydroxylation flux, resulting in 43.2 g L-4-hydroxy-l-proline with 4.3 g L l-proline retained. Finally, rationally designed l-proline hydroxylase gave 54.8 g L-4-hydroxy-l-proline in 60 hours almost without l-proline remaining-the highest production to date. The de novo engineering carbon flux through rare codon selected evolution, dynamic precursor modulation, and metabolic engineering provides a good technological platform for efficient hydroxyl amino acid synthesis.
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http://dx.doi.org/10.1126/sciadv.aba2383DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7244267PMC
May 2020

Characterization of promising natural blue pigment from Vaccinium bracteatum thunb. leaves: Insights of the stability and the inhibition of α-amylase.

Food Chem 2020 Oct 4;326:126962. Epub 2020 May 4.

School of Food Science and Technology, State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China; National Engineering Research Center for Functional Food, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China. Electronic address:

A concise method of natural blue pigment from Vaccinium bracteatum thunb. leaves (VBTL) was proposed firstly in this research. The potential properties of new pigment source needed to be assessed for further application. During the shelf life and thermal-accelerated storage, the blue pigment was prone to stable in the mildly acidic system, which was observed retention of 35.85% for storage of 12 weeks in pH 4.0. The 65% of blue pigment retained after the in vitro simulated digestion. The blue pigment exhibited inhibition effect on the pancreatic α-amylase (IC = 2.915 mg/mL). The analysis of inhibition kinetics indicated the blue pigment was an uncompetitive inhibitor. Through fluorescence quenching and circular dichroism study, the blue pigment inhibited α-amylase activity via the structural unfolding of α-amylase molecule and changing the secondary structure. These findings provided the new evidence of the digestion resistibility of VBTL pigment and the functional food - "Wu mi".
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http://dx.doi.org/10.1016/j.foodchem.2020.126962DOI Listing
October 2020

Optimization of l-arginine purification from Corynebacterium crenatum fermentation broth.

J Sep Sci 2020 Jul 4;43(14):2936-2948. Epub 2020 Jun 4.

The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Jiangsu, P. R. China.

l-Arginine has many special physiological and biochemical functions, with wide applications in the food and pharmaceutical industry. Few studies on the purification of l-arginine from fermentation broth have been conducted; however, none of them were systematic enough for industrial scale-up. Therefore, it is necessary to develop a highly efficient and systematic process for the purification of l-arginine from fermentation broth. In this study, we screened out a cation exchange resin, D155, having high exchange capacity, high selectivity, and easy elution capacity, and analyzed its adsorption isotherm, thermodynamics, and kinetics using different models. Further, the process parameters of fixed-bed ion exchange adsorption and elution were optimized, and the penetration curve during the operation was modeled. Based on the fixed-bed ion-exchange parameters, a 30-column continuous ion-exchange system was designed, and the flow velocity in each zone was optimized. Finally, to obtain a high purity of l-arginine, the purification tests were conducted using anion exchange resin 711, and an l-arginine yield of 99.1% and purity of 98.5% was obtained. This effective and economical method also provides a promising strategy for separation of other amino acids from the fermentation broth, which is of great significance to the l-arginine fermentation industry.
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http://dx.doi.org/10.1002/jssc.202000067DOI Listing
July 2020

Cloning and Expression of a Novel Leucine Dehydrogenase: Characterization and L--Leucine Production.

Front Bioeng Biotechnol 2020 31;8:186. Epub 2020 Mar 31.

The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, China.

Among many genes encoding for amino acid dehydrogenase, a novel leucine dehydrogenase gene from (LeuDH) was isolated by using genome mining strategy. LeuDH was overexpressed in BL21 (DE3), followed by purification and characterization. The high thermostability of the enzyme confers its half-life up to 14.7 h at 50°C. Furthermore, the substrate specificity shows a broad spectrum, including many L-amino acids and aliphatic α-keto acids, especially some aryl α-keto acids. This enzyme coupled with recombinant formate dehydrogenase (FDH) was used to catalyze trimethylpyruvic acid (TMP) through reductive amination to generate enantiopure L--leucine (L-Tle). In order to overcome the substrate inhibition effect, a fed-batch feeding strategy was adopted to transform up to 0.8 M of TMP to L-Tle, with an average conversion rate of 81% and L-Tle concentration of 65.6 g⋅L. This study provides a highly efficient biocatalyst for the synthesis of L-Tle and lays the foundation for large-scale production and application of chiral non-natural amino acids.
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http://dx.doi.org/10.3389/fbioe.2020.00186DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7136578PMC
March 2020

Development of a Novel Biosensor-Driven Mutation and Selection System via Growth of for the Production of L-Arginine.

Front Bioeng Biotechnol 2020 13;8:175. Epub 2020 Mar 13.

Jiangnan University (Rugao) Food Biotechnology Research Institute, Rugao, China.

The high yield mutants require a high-throughput screening method to obtain them quickly. Here, we developed an L-arginine biosensor (ARG-Select) to obtain increased L-arginine producers among a large number of mutant strains. This biosensor was constructed by ArgR protein and promoter, and could provide the strain with the output of bacterial growth via the reporter gene ; strains with high L-arginine production could survive in 10% sucrose screening. To extend the screening limitation of 10% sucrose, the sensitivity of ArgR protein to L-arginine was decreased. SYPA5-5 and its systems pathway engineered strain Cc6 were chosen as the original strains. This biosensor was employed, and L-arginine hyperproducing mutants were screened. Finally, the HArg1 and DArg36 mutants of SYPA5-5 and Cc6 could produce 56.7 and 95.5 g L of L-arginine, respectively, which represent increases of 35.0 and 13.5%. These results demonstrate that the transcription factor-based biosensor could be applied in high yield strains selection as an effective high-throughput screening method.
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http://dx.doi.org/10.3389/fbioe.2020.00175DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7082233PMC
March 2020

PII Signal Transduction Protein GlnK Alleviates Feedback Inhibition of -Acetyl-l-Glutamate Kinase by l-Arginine in Corynebacterium glutamicum.

Appl Environ Microbiol 2020 04 1;86(8). Epub 2020 Apr 1.

Key Laboratory of Industrial Biotechnology of the Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, China

PII signal transduction proteins are ubiquitous and highly conserved in bacteria, archaea, and plants and play key roles in controlling nitrogen metabolism. However, research on biological functions and regulatory targets of PII proteins remains limited. Here, we illustrated experimentally that the PII protein GlnK (CgGlnK) increased l-arginine yield when was overexpressed in Data showed that CgGlnK regulated l-arginine biosynthesis by upregulating the expression of genes of the l-arginine metabolic pathway and interacting with -acetyl-l-glutamate kinase (CgNAGK), the rate-limiting enzyme in l-arginine biosynthesis. Further assays indicated that CgGlnK contributed to alleviation of the feedback inhibition of CgNAGK caused by l-arginine. analysis of the binding interface of CgGlnK-CgNAGK suggested that the B and T loops of CgGlnK mainly interacted with C and N domains of CgNAGK. Moreover, F11, R47, and K85 of CgGlnK were identified as crucial binding sites that interact with CgNAGK via hydrophobic interaction and H bonds, and these interactions probably had a positive effect on maintaining the stability of the complex. Collectively, this study reveals PII-NAGK interaction in nonphotosynthetic microorganisms and further provides insights into the regulatory mechanism of PII on amino acid biosynthesis in corynebacteria. Corynebacteria are safe industrial producers of diverse amino acids, including l-glutamic acid and l-arginine. In this study, we showed that PII protein GlnK played an important role in l-glutamic acid and l-arginine biosynthesis in Through clarifying the molecular mechanism of CgGlnK in l-arginine biosynthesis, the novel interaction between CgGlnK and CgNAGK was revealed. The alleviation of l-arginine inhibition of CgNAGK reached approximately 48.21% by CgGlnK addition, and the semi-inhibition constant of CgNAGK increased 1.4-fold. Furthermore, overexpression of in a high-yield l-arginine-producing strain and fermentation of the recombinant strain in a 5-liter bioreactor led to a remarkably increased production of l-arginine, 49.978 g/liter, which was about 22.61% higher than that of the initial strain. In conclusion, this study provides a new strategy for modifying amino acid biosynthesis in .
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http://dx.doi.org/10.1128/AEM.00039-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7117919PMC
April 2020

Blakeslea trispora Photoreceptors: Identification and Functional Analysis.

Appl Environ Microbiol 2020 04 1;86(8). Epub 2020 Apr 1.

The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, People's Republic of China.

is an industrial fungal species used for large-scale production of carotenoids. However, light-regulated physiological processes, such as carotenoid biosynthesis and phototropism, are not fully understood. In this study, we isolated and characterized three photoreceptor genes, , , and , in Bioinformatics analyses of these genes and their protein sequences revealed that the functional domains (PAS/LOV [Per-ARNT-Sim/light-oxygen-voltage] domain and zinc finger structure) of the proteins have significant homology to those of other fungal blue-light regulator proteins expressed by and The photoreceptor proteins were synthesized by heterologous expression in The chromogenic groups consisting of flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) were detected to accompany BTWC-1 proteins by using high-performance liquid chromatography (HPLC) and fluorescence spectrometry, demonstrating that the proteins may be photosensitive. The absorbance changes of the purified BTWC-1 proteins seen under dark and light conditions indicated that they were light responsive and underwent a characteristic photocycle by light induction. Site-directed mutagenesis of the cysteine residual (Cys) in BTWC-1 did not affect the normal expression of the protein in but did lead to the loss of photocycle response, indicating that Cys represents a flavin-binding domain for photon detection. We then analyzed the functions of BTWC-1 proteins by complementing , , and into the counterpart knockout strains of for each gene. Transformation of the complement into knockout strains restored the positive phototropism, while the addition of complement remedied the deficiency of carotene biosynthesis in the knockout strains under conditions of illumination. These results indicate that and are involved in phototropism and light-inducible carotenogenesis. Thus, genes share a conserved flavin-binding domain and act as photoreceptors for control of different light transduction pathways in Studies have confirmed that light-regulated carotenogenesis is prevalent in filamentous fungi, especially in mucorales. However, few investigations have been done to understand photoinduced synthesis of carotenoids and related mechanisms in , a well-known industrial microbial strains. In the present study, three photoreceptor genes in were cloned, expressed, and characterized by bioinformatics and photoreception analyses, and then functional analyses of these genes were constructed in The results of this study will lead to a better understanding of photoreception and light-regulated carotenoid synthesis and other physiological responses in .
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http://dx.doi.org/10.1128/AEM.02962-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7117921PMC
April 2020

A Negative Regulator of Carotenogenesis in .

Appl Environ Microbiol 2020 03 2;86(6). Epub 2020 Mar 2.

The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, People's Republic of China.

As an ideal carotenoid producer, has gained much attention due to its large biomass and high production of β-carotene and lycopene. However, carotenogenesis regulation in still needs to be clarified, as few investigations have been conducted at the molecular level in In this study, a gene homologous to carotenogenesis regulatory gene () was cloned from the mating type (-) of , and the deduced CrgA protein was analyzed for its primary structure and domains. To clarify the -mediated regulation in , we used the strategies of gene knockout and complementation to investigate the effect of expression on the phenotype of In contrast to the wild-type strain, the null mutant (Δ) was defective in sporulation but accumulated much more β-carotene (31.2% improvement at the end) accompanied by enhanced transcription of three structural genes (, , and ) for carotenoids throughout the culture time. When the wild-type copy of was complemented into the null mutant, sporulation, transcription of structural genes, and carotenoid production were restored to those of the wild-type strain. A gas chromatography-mass spectrometry (GC-MS)-based metabolomic approach and multivariate statistical analyses were performed to investigate the intracellular metabolite profiles. The reduced levels of tricarboxylic acid (TCA) cycle components and some amino acids and enhanced levels of glycolysis intermediates and fatty acids indicate that more metabolic flux was driven into the mevalonate (MVA) pathway; thus, the increase of precursors and fat content contributes to the accumulation of carotenoids. The zygomycete is an important strain for the production of carotenoids on a large scale. However, the regulation mechanism of carotenoid biosynthesis is still not well understood in this filamentous fungus. In the present study, we sought to investigate how influences the expression of structural genes for carotenoids, carotenoid biosynthesis, and other anabolic phenotypes. This will lead to a better understanding of the global regulation mechanism of carotenoid biosynthesis and facilitate engineering this strain in the future for enhanced production of carotenoids.
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http://dx.doi.org/10.1128/AEM.02462-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7054107PMC
March 2020

Loss of Serine-Type D-Ala-D-Ala Carboxypeptidase DacA Enhances Prodigiosin Production in .

Front Bioeng Biotechnol 2019 3;7:367. Epub 2019 Dec 3.

Key Laboratory of Industrial Biotechnology of the Ministry of Education, Laboratory of Applied Microorganisms and Metabolic Engineering, School of Biotechnology, Jiangnan University, Wuxi, China.

, a gram-negative bacterium, found in a wide range of ecological niches can produce several high-value products, including prodigiosin, althiomycin, and serratamolide. Among them, prodigiosin has attracted attention due to its immunosuppressive, antimicrobial, and anticancer properties. However, the regulatory mechanisms behind prodigiosin synthesis in remains limited. Here, a transposon mutant library was constructed to identify the genes related to prodigiosin synthesis, and gene encoding a peptidoglycan synthesizing enzyme D-Ala-D-Ala carboxypeptidase DacA was found to negatively regulates prodigiosin synthesis. Quantitative measurements revealed that disruption of increased prodigiosin production 1.46-fold that of the wild-type strain JNB5-1 in fermentation medium. By comparing differences in cell growth, gene expression level, cell morphology, membrane permeability, and intracellular prodigiosin concentration between wild-type strain JNB5-1 and mutant SK4-72, results revealed that the mechanism for hyper-producing of prodigiosin by the mutant was probably that disruption enhanced prodigiosin leakage, which in turn alleviated feedback inhibition of prodigiosin and increased expression of gene cluster. Collectively, this work provides a novel insight into regulatory mechanisms of prodigiosin synthesis and uncovers new roles of DacA protein in regulating cell growth, cell morphology, and membrane permeability in . Finally, this study offers a new strategy for improving production of high-value compounds in .
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http://dx.doi.org/10.3389/fbioe.2019.00367DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6901396PMC
December 2019

LysR-Type Transcriptional Regulator MetR Controls Prodigiosin Production, Methionine Biosynthesis, Cell Motility, HO Tolerance, Heat Tolerance, and Exopolysaccharide Synthesis in Serratia marcescens.

Appl Environ Microbiol 2020 02 3;86(4). Epub 2020 Feb 3.

Key Laboratory of Industrial Biotechnology of the Ministry of Education, Laboratory of Applied Microorganisms and Metabolic Engineering, School of Biotechnology, Jiangnan University, Wuxi, China

Prodigiosin, a secondary metabolite produced by , has attracted attention due to its immunosuppressive, antimicrobial, and anticancer properties. However, information on the regulatory mechanism behind prodigiosin biosynthesis in remains limited. In this work, a prodigiosin-hyperproducing strain with the gene disrupted (ZK66) was selected from a collection of TnG transposon insertion mutants. Using real-time quantitative PCR (RT-qPCR) analysis, β-galactosidase assays, transcriptomics analysis, and electrophoretic mobility shift assays (EMSAs), the LysR-type regulator MetR encoded by the gene was found to affect prodigiosin synthesis, and this correlated with MetR directly binding to the promoter region of the prodigiosin-synthesis positive regulator PigP and hence negatively regulated the expression of the prodigiosin-associated operon. More analyses revealed that MetR regulated some other important cellular processes, including methionine biosynthesis, cell motility, HO tolerance, heat tolerance, exopolysaccharide synthesis, and biofilm formation in Although MetR protein is highly conserved in many bacteria, we report here on the LysR-type regulator MetR exhibiting novel roles in negatively regulating prodigiosin synthesis and positively regulating heat tolerance, exopolysaccharide synthesis, and biofilm formation., a Gram-negative bacterium, is found in a wide range of ecological niches and can produce several secondary metabolites, including prodigiosin, althiomycin, and serratamolide. Among them, prodigiosin shows diverse functions as an immunosuppressant, antimicrobial, and anticancer agent. However, the regulatory mechanisms behind prodigiosin synthesis in are not completely understood. Here, we adapted a transposon mutant library to identify the genes related to prodigiosin synthesis, and the gene encoding the LysR-type regulator MetR was found to negatively regulate prodigiosin synthesis. The molecular mechanism of the mutant hyperproducing prodigiosin was investigated. Additionally, we provided evidence supporting new roles for MetR in regulating methionine biosynthesis, cell motility, heat tolerance, HO tolerance, and exopolysaccharide synthesis in Collectively, this work provides novel insight into regulatory mechanisms of prodigiosin synthesis and uncovers novel roles for the highly conserved MetR protein in regulating prodigiosin synthesis, heat tolerance, exopolysaccharide (EPS) synthesis, and biofilm formation.
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http://dx.doi.org/10.1128/AEM.02241-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6997736PMC
February 2020

Intracellular Environment Improvement of for Enhancing Androst-1,4-Diene-3,17-Dione Production by Manipulating NADH and Reactive Oxygen Species Levels.

Molecules 2019 Oct 25;24(21). Epub 2019 Oct 25.

The Key Laboratory of Industrial Biotechnology of Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China.

As one of the most significant steroid hormone precursors, androst-1,4-diene-3,17-dione (ADD) could be used to synthesize many valuable hormone drugs. The microbial transformation of sterols to ADD has received extensive attention in recent years. In a previous study, JC-12 was isolated and converted sterols to the major product, ADD. In this work, we enhanced ADD yield by improving the cell intracellular environment. First, we introduced a nicotinamide adenine dinucleotide (NADH) oxidase from to balance the intracellular NAD availability in order to strengthen the ADD yield. Then, the catalase gene from was also over-expressed to simultaneously scavenge the generated HO and eliminate its toxic effects on cell growth and sterol transformation. Finally, using a 5 L fermentor, the recombinant strain JC-12 produced 9.66 g/L ADD, which increased by 80% when compared with the parent strain. This work shows a promising way to increase the sterol transformation efficiency by regulating the intracellular environment.
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http://dx.doi.org/10.3390/molecules24213841DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6864555PMC
October 2019

Lys-Arg mutation improved the thermostability of Bacillus cereus neutral protease through increased residue interactions.

World J Microbiol Biotechnol 2019 Oct 31;35(11):173. Epub 2019 Oct 31.

The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 LiHu Boulevard, Wuxi, 214122, Jiangsu, China.

Neutral proteases have broad application as additives in modern laundry detergents and therefore, thermostability is an integral parameter for effective production of protein crystals. To improve thermostability, the contribution of individual residues of Bacillus cereus neutral protease was examined by site-directed mutagenesis. The Lys11Arg and Lys211Arg mutants clearly possessed improved thermostabilities (T were 63 and 61 °C respectively) compared to the wild-type (T was 60 °C). MD simulations further revealed that the mutants had low RMSD and RMSF values compared to wild-type BCN indicating increased stability of the protein structure. Lys11Arg mutant particularly possessed the lowest RMSD values due to increased residue interactions, which resulted in enhanced thermostability. The mutants also displayed strong stability to most inhibitors, organic solvents and surfactants after incubation for 1 h. This study demonstrated Lys-Arg mutation enhanced thermostability of BCN and thus provides insight for engineering stabilizing mutations with improved thermostability for related proteins.
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http://dx.doi.org/10.1007/s11274-019-2751-5DOI Listing
October 2019

Synthetic engineering of Corynebacterium crenatum to selectively produce acetoin or 2,3-butanediol by one step bioconversion method.

Microb Cell Fact 2019 Aug 6;18(1):128. Epub 2019 Aug 6.

The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, Jiangsu, China.

Background: Acetoin (AC) and 2,3-butanediol (2,3-BD) as highly promising bio-based platform chemicals have received more attentions due to their wide range of applications. However, the non-efficient substrate conversion and mutually transition between AC and 2,3-BD in their natural producing strains not only led to a low selectivity but also increase the difficulty of downstream purification. Therefore, synthetic engineering of more suitable strains should be a reliable strategy to selectively produce AC and 2,3-BD, respectively.

Results: In this study, the respective AC (alsS and alsD) and 2,3-BD biosynthesis pathway genes (alsS, alsD, and bdhA) derived from Bacillus subtilis 168 were successfully expressed in non-natural AC and 2,3-BD producing Corynebacterium crenatum, and generated recombinant strains, C. crenatum SD and C. crenatum SDA, were proved to produce 9.86 g L of AC and 17.08 g L of 2,3-BD, respectively. To further increase AC and 2,3-BD selectivity, the AC reducing gene (butA) and lactic acid dehydrogenase gene (ldh) in C. crenatum were then deleted. Finally, C. crenatumΔbutAΔldh SD produced 76.93 g L AC in one-step biocatalysis with the yield of 0.67 mol mol. Meanwhile, after eliminating the lactic acid production and enhancing 2,3-butanediol dehydrogenase activity, C. crenatumΔldh SDA synthesized 88.83 g L of 2,3-BD with the yield of 0.80 mol mol.

Conclusions: The synthetically engineered C. crenatumΔbutAΔldh SD and C. crenatumΔldh SDA in this study were proved as an efficient microbial cell factory for selective AC and 2,3-BD production. Based on the insights from this study, further synthetic engineering of C. crenatum for AC and 2,3-BD production is suggested.
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http://dx.doi.org/10.1186/s12934-019-1183-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6683508PMC
August 2019