Jiangnan University

Taowei Yang

Xian Zhang

Meijuan Xu

Zhiming Rao

Minglong Shao

Junxian Zheng

Jianing Zhang

Yu Liu

Zhenghong Xu

Song Liu

Shuqin Xu

Xu Li

Junping Zhou

Ziwei Wang

Zongyan Sha

Jingyi Hu

Peifeng Duan

Yaling Wang

Yan Chen

Jiamin Chen

Anqi Peng

Qi Chen

Shuping Xu

Meiqi Wu

Yetong Wang

CaiZhe Zhang

Yunlong Qi

Yuling Wu

Zaiwei Man

Renjie Gao

Meizhou Wang

The disclosure relates to a sucrose phosphorylase mutant with improved enzyme activity, and construction method thereof and use thereof, and belongs to the technical field of genetic engineering. The amino acid sequence of the mutant of the disclosure is as shown in SEQ ID NO: 1. The mutant of the disclosure is based on sucrose phosphorylase derived from, and subjected to site-directed mutagenesis to improve the enzyme activity of sucrose phosphorylase. The mutant is expressed inand used as a whole cell catalyst to produce 2-O-&#x3b1;-D-glycerol glucoside. At a 5 L fermentation tank level, a large quantity of 2-O-&#x3b1;-D-glycerol glucoside can be produced efficiently in a short time, which is conducive to expanding the prospect of industrial application of sucrose phosphorylase for the production of 2-O-&#x3b1;-D-glycerol glucoside and realizing its large-scale industrial application.

Sucrose phosphorylase mutant with improved enzyme activity and construction method thereof and use thereof

The disclosure discloses 17&#x3b2;-hydroxysteroid dehydrogenase mutants and application thereof, and belongs to the technical field of biology. The disclosure provides 17&#x3b2;-hydroxysteroid dehydrogenase mutants V107A, T155N, H164Y and V107A/T155N/H164Y with high specific enzyme activities, and the specific enzyme activities of the 17&#x3b2;-hydroxysteroid dehydrogenase mutants V107A, T155N, H164Y and V107A/T155N/H164Y are as high as 1.85, 1.93, 2.06 and 5.15 U/mg, respectively, which are 1.11, 1.16, 1.24 and 3.10 times larger than that of wild-type 17&#x3b2;-hydroxysteroid dehydrogenase (1.66 U/mg).

17&#x3b2;-hydroxysteroid dehydrogenase mutants and application thereof

The present invention discloses a method for producing a 1,2-amino alcohol compound by utilizing whole-cell transformation, and belongs to the technical field of gene engineering and microorganism engineering. According to the present invention, engineeredco-expresses epoxide hydrolase, alcohol dehydrogenase, &#x3c9;-transaminase and glutamate dehydrogenase, is capable of realizing whole-cell catalysis of an epoxide in one step to synthesize a 1,2-amino alcohol compound, and meanwhile, can realize regeneration of coenzyme NADPand an amino doner L-Glu; alcohol dehydrogenase expressed by the engineeredis RBS optimized alcohol dehydrogenase, and such RBS optimization can control the expression quantity of alcohol dehydrogenase, so that the catalysis rate of alcohol dehydrogenase and transaminase can achieve an optimum ratio, to eliminate influence caused by a rate-limiting step in a catalyzing course.

Method for producing 1,2-amino alcohol compound by whole cell transformation

The present disclosure discloses a method for producing 9&#x3b1;-hydroxy androstane-4-alkene-3,17-diketone by enzymatic conversion, and belongs to the fields of gene engineering and enzyme engineering. According to the present disclosure, oxidation subunit KshA, reduction subunit KshB and unknown active subunit KshC of 3-ketosteroid-9&#x3b1;-hydroxylase sourcing fromsp. Strain VKM Ac-1817D are successfully expressed inBL21, and KshC is identified as an oxidation subunit, the enzyme activity of which is far higher than that of KshA. BL21/pET-28a(+)-fdh constructed in the laboratory is used for expressing formate dehydrogenase (FDH), and by using crude enzyme liquid of KSH (KshB+KshC) and FDH engineering bacteria as a biocatalyst and a steroidal compound (AD) as a substrate, optimum reaction temperature is determined as 30&#xb0; C. and optimum pH is determined as 7.0. In optimum conditions, AD is converted to produce a product 9-OH-AD, and within 20 hours, the output of 9-OH-AD is 4.7 g/L, and the molar conversion rate reaches 96.7%. According to the present disclosure, in production of 9-OH-AD, coupling of a 3-ketosteroid-9&#x3b1;-hydroxylase hydroxylation system and a coenzyme recycling system is realized, and the method has the advantages of being high in efficiency, low in cost, green, environmentally friendly and the like.

Method for producing 9alpha-hydroxy androstane-4-alkene-3,17-diketone by enzymatic conversion

The present disclosure discloses a thermophilic L-asparaginase mutant and screening and fermentation methods thereof, and belongs to the field of gene engineering, enzyme engineering and fermentation engineering. In168, aCH1-derived L-asparaginase encoding gene is used as a template, and a mutation library is constructed by an error-prone PCR (epPCR) technology. A mutant strain with improved specific enzyme activity is screened through a high-flux screening method of synchronous cell disruption and enzyme activity measurement. Mutated residues included in a positive mutant are analyzed to construct a composite mutant strain S17G/A90S/R156S/K272A with improved specific enzyme activity and specific enzyme activity of 3108 U/mg. An expression quantity of the composite mutant strain in the168 is increased through measures of a strong promoter Pand RBS optimization. Finally, the168 with a gene of the L-asparaginase composite mutant strain is subjected to enzyme production fermentation in a 5 L fermentation tank through culture medium optimization and pH and feeding coupling strategies. The enzyme activity yield of the L-asparaginase is up to 6453+/&#x2212;127 U/mL.

Thermophilic L-asparaginase mutant and screening and fermentation methods thereof

The present invention provides an aspartase mutant, a recombinant expression vector and recombinant bacterium containing the aspartase mutant, and the use thereof, and belongs to the technical field of genetic engineering. The amino acid sequence of the aspartase mutant is as set forth in SEQ ID NO: 1. In the aspartase mutant of the present invention, on the basis of wild type aspartase (with an amino acid sequence as set out in SEQ ID NO: 3), glutamic acid at position 427 is mutated into glutamine. In the present invention, by mutating the amino acid residue at position 427 into glutamine, the polar environment near an active site is changed, and thus ammonia supply during substrate reaction is further facilitated, thereby improving an enzyme activity, enhancing the ability of the enzyme in synthesizing a &#x3b2;-amino acid, and providing a practical and effective strategy for industrial production of the &#x3b2;-amino acid.

Aspartase mutant, recombinant expression vector and recombinant bacterium containing aspartase mutant, and use thereof

The present invention discloses a-derived steroid C27-monooxygenase and an application thereof, which belong to the technical fields of genetic engineering and enzyme engineering. By the method of gene knockout and intensive expression, the present invention screens out three isoenzymes of a key enzyme SMO in the process of degrading sterol side chains from. The three isoenzymes are intensively expressed respectively in thefor the high yield of androsta-1,4-diene-3,17-dione (ADD), the yield of ADD is increased remarkably, wherein the effect of SMO2 is most remarkable. By overexpressing SMO2, the final ADD yield is increased from 5.2 g&#xb7;Lto 7.3 g&#xb7;L. The present invention provides a helpful guidance for the industrialization of the microbial fermentation method for increasing the ADD yield.

Mycobacterium neoaurum-derived steroid C27-monooxygenase and application thereof

The present invention discloses a single-cell factory for efficiently synthesizing &#x3b1;-aminobutyric acid and construction and application thereof, which belong to the technical field of microorganisms. The present invention expresses an L-threonine deaminase gene, an L-amino acid dehydrogenase gene and a dehydrogenase gene for providing cofactor NADH cycle in tandem to construct a recombinantsingle-cell factory which is used for efficiently synthesizing &#x3b1;-aminobutyric acid. The expression level of the L-threonine deaminase is optimized and controlled by an RBS sequence, so that the problem of transformation inhibition caused by the rapid accumulation of an intermediate product ketobutyric acid is solved, moreover, the expression level of the dehydrogenase for providing cofactor NADH cycle is optimized and controlled by a promoter and an RBS sequence, consequently, the NADH regeneration rate is increased, and ultimately, yield is increased. Utilizing the single-cell factory to carry out whole-cell transformation can reduce obstacles to substances getting in and out, increase the transformation rate and promote the intracellular cycle of cofactors without requiring exogenous addition, and the cost is low. Within 20 h, the yield of the recombinantsingle-cell factory in a 5 L fermentation tank is 204 g&#xb7;L, the space-time yield is 10.2 g&#xb7;L&#xb7;h, and a practical effective strategy is provided for industrialized production.

Single-cell factory for efficiently synthesizing &#x3b1;-aminobutyric acid and construction and application thereof

The invention discloses a method for increasing the yield of L-arginine by knocking out flavin reductases, and belongs to the technical field of amino acid production by microbial fermentation. Genes frd1 and frd2 for encoding hypothetic NADPH-dependent FMN reductase inSDNN403 are over-expressed inBL21 and are purified to form target proteins Frd181 and Frd188, and functions of the target proteins are identified to obtain a result showing that the proteins Frd181 and Frd188 both are NAD(P)H-dependent flavin reductases producing HO. By taking a genome of theSDNN403 as a template, frd1 and frd2 gene deletion fragments are obtained by overlap extension PCR; connecting pK18mobsacB to obtain knockout plasmids pK18mobsacB-&#x394;frd1 and pK18mobsacB-&#x394;frd2; carrying out electric shock to transform theSDNN403; and carrying out secondary screening to obtain recombinant strains 403&#x394;frd1 and 403&#x394;frd2. Found by flask shaking fermentation, the yield of L-arginine is obviously increased by knocking out the genes frd1 and frd2.

Method for increasing yield of L-arginine by knocking out Flavin reductases

The present invention relates to application of a novel signal peptide in L-arginine and its derivatives production from konjac powder, which belongs to the field of gene engineering, enzyme engineering and metabolism engineering. The present invention fused the signal peptide set forth in SEQ ID NO.1 with the &#x3b2;-mannanase ofCCTCC M 209200, and expressed the fused gene in the strain with high L-arginine yield. The recombinant strainCGMCC 0890/p MSPman had advantages on utilizing cheaper konjac powder as substrate, and after fermenting for 96 hours in a 5 L bioreactor, the L-arginine yield reached 45 g/L. Another two recombinant strains were constructed based onCGMCC 0890/pMSPman, and after fermenting for 96 hours in a 5 L bioreactor, the L-ornithine yield and L-citrulline reached 23.5 g/L and 26.3 g/L respectively.

Signal peptide, L-glutamic acid synthesized using konjac flour and methods of using same

The present invention relates to application of a novel signal peptide in L-glutamate and its derivatives production from konjac powder, which belongs to the field of gene engineering, enzyme engineering and metabolism engineering. The signal peptide which mediated secretion of &#x3b2;-mannanase was invented, and the recombinant strain with this signal peptide had advantages on utilizing konjac powder to produce related products, and its utilization efficiency of konjac powder, production efficiency, and yield were higher than other signal peptides. The recombinant strain possessing this new signal peptide had advantages on utilizing cheaper konjac powder as substrate to lower the process costs on L-glutamic acid and its high-value-added products.

Disclosed is a method for producing L-citrulline using recombinantcells as whole-cell biocatalysts. The present invention provides a recombinantthat expresses an exogenous arginine deiminase gene from. The recombinantSDNN403 is used as biocatalysts for converting L-arginine to produce L-citrulline. Using the method of the invention, the concentration of L-citrulline reached 301.4 g/L after a 48 hr conversion reaction, and the molar conversion rate reached 99.9%.

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Wuxi, China

Taowei Yang