9 results about "Substrate channeling" patented technology

The invention relates to the field of enzyme engineering, in particular to a mutant of nylon degrading enzyme and application of the mutant. The mutant of the nylon degrading enzyme is obtained by mutation at one or more key sites through stability modification guided by machine learning, substrate channel inlet loop region modification and tetramer interface modification on the basis of a wild type nylon degrading enzyme NylCp2-TS as shown in SEQ ID NO: 1; the mutant enzyme can efficiently degrade nylon materials in various forms, including films, particles and industrial fibers of nylon 6 and nylon 66, and has a wide application prospect in the field of nylon waste biodegradation.

The application discloses a kind of biological immobilized enzyme based on yeast surface display technology and the application of synthesis 2-phenylethanol, using yeast surface display technology, for the first time with Saccharomyces cerevisiae surface protein a-lectin as anchor protein, the key enzyme (transaminase, decarboxylase, ethanol dehydrogenase) involved in 2-PE producing Ehrlich pathway in Saccharomyces cerevisiae is separately or jointly carried out biological fixation, enhance substrate channel effect and proximity effect, in vitro one-step catalysis L-phenylalanine synthesis 2-PE, provide yield.The method for fixing multiple enzymes of Ehrlich pathway in the application can be used to reconfigure the biosynthesis pathway of other natural products in vitro, to provide feasible scheme for the rational design of cell factory.

The invention discloses a genetically engineered bacterium for producing D-pantothenic acid as well as a construction method and application of the genetically engineered bacterium, wild type C. glutamicum ATCC 13032 is taken as a chassis bacterium, an ilvE gene, an avtA gene, an ilvA gene and a pqo gene in an original strain genome are knocked out, ilvBNC from different sources and mutants thereof are screened, an optimized mutant ilvBNC-VWBGTG / ATG is determined, the gene cluster and promoters Ptuf and Pefu are connected and integrated to a strain DPAe-2, and the D-pantothenic acid is produced. An engineering strain DPAg-11 capable of efficiently accumulating KIV is obtained, then a promoter of an aceE gene is replaced by PdapA A16, KIV accumulated by the obtained engineering strain DPAg-15 reaches 3.99 g / L, panB, panC, panE or ilvC from different sources are screened, it is determined that BspanBC + EcpanE has the best effect, BspanBC + EcpanE is constructed on plasmids and then converted into various engineering strains, a promoter of an EcpanE gene is replaced by PdapB, and KIV accumulated by the engineering strain DPAg-11 and PdapA A16 reaches 3.99 g / L. The method has the advantages that the method is simple and easy to implement, a substrate channel can be built between dihydroxy acid dehydratase (DHAD) and KPR by polypeptide pair RIAD-RIDD, D-PA produced by the finally obtained strain reaches 3.21 g / L and is increased to 97.0 times that of DPA produced by a wild strain, and production of D-PA can be effectively promoted by the strategy of the built substrate channel.

The application discloses a cytochrome P450 enzyme mutant and a method for catalytically synthesizing betulinic acid, and relates to the technical fields of genetic engineering and biological enzyme catalysis.The reported RoCYP01 from Litsea cubeba is used as a mutation template, site-directed mutation is carried out, the substrate channel for the entry and exit of the enzyme catalytic center of a substrate and a product is changed, and a mutant with improved catalytic efficiency for the synthesis of betulinic acid from betulonic acid is obtained.Under the same catalytic conditions, the catalytic activity of the mutant is increased by 198% compared with the wild type, which has important guiding significance for the construction of a betulinic acid yeast engineering strain.

The invention provides a novel reverse aldolase and reductase cascaded Bi-BDO independent synthesis pathway, and relates to the technical field of bioengineering, the pathway constructs a non-natural metabolism module composed of a modified reverse aldolase RA variant, 4-hydroxybutyraldehyde reductase AHR and a coenzyme cyclic regeneration enzyme, the RA variant performs hydrophobic modification on a Lys146 site, and the reverse aldolase and reductase cascaded Bi-BDO is obtained. Aldol condensation of a non-phosphorylated substrate hydroxyacetaldehyde is realized to generate a C4 intermediate, RA and AHR are physically anchored by utilizing an artificially synthesized scaffold protein, toxicity of the intermediate is avoided through a substrate channel effect, the intermediate is instantly reduced into 1, 4-butanediol, engineering bacteria of aldA and gapA genes are knocked out, a precursor is generated by utilizing a xylose way, and the 1, 4-butanediol is obtained. The method has the advantages that the method is simple in operation, the reduction reaction is driven by in-situ regeneration of NADPH by adding formate, finally, the high-purity product is obtained through macroporous adsorption resin enrichment and vacuum rectification, the TCA circulation decarboxylation step is omitted, the carbon conversion rate is close to the theoretical limit, effective decoupling of production and growth is achieved, and the yield and purity of Bi-BDO are remarkably improved.

This invention discloses an engineered astaxanthin-producing probiotic yeast strain, its construction method, and its applications, belonging to the field of synthetic biology. The engineered strain uses β-carotene-producing *Saccharomyces cerevisiae* as the substrate cell. Its genome integrates a fusion gene selected through adaptability screening. This gene is composed of *BDC263crtW* from *Saccharomyces cerevisiae*, a flexible linker peptide, and *AspcrtZ* from *Alcaligenes aeruginosa*, linked sequentially. Simultaneously, based on systematic expression screening of multiple key genes, the endogenous acetyl-CoA synthesis key gene ACS1 was optimized and overexpressed, while the acetic acid metabolism negative regulatory gene YPL062W was knocked out. The construction method includes plasmid construction of the aforementioned fusion expression module and ACS1 expression module, and targeted integration into the genome. This invention significantly improves astaxanthin synthesis levels and enhances the engineered strain's tolerance to the simulated gastrointestinal environment and its antioxidant capacity through enzyme source optimization and substrate channel effects. This engineered strain can be directly used as a functional live bacteria preparation for food and feed additives, with broad prospects for industrial application.

The invention discloses a molecular docking-based glycosyl transferase substrate catalytic specificity multi-dimensional evaluation method, system, equipment and medium, and belongs to the technical field of protein structure simulation evaluation, and the method comprises the following steps: determining glycosyl transferase, and predicting a corresponding protein three-dimensional structure; performing molecular docking simulation based on the predicted protein three-dimensional structure; the catalytic specificity of different substrates is evaluated according to the difference between receptor substrate channels and ligands; and displaying a docking result through visual software. According to the method, a set of complete standardized operation program is formed, a clear methodology guide is provided for similar research, on the basis of traditional docking scoring, three-dimensional space size measurement analysis of the acceptor and the ligand is creatively used as a key screening index, multi-dimensional evaluation from the bonding strength to the space suitability is achieved, and the evaluation accuracy is improved. A new visual angle is provided for understanding an enzyme substrate recognition mechanism.

The invention discloses a recombinant Yarrowia lipolytica engineering strain for producing ergosterol and application of the recombinant Yarrowia lipolytica engineering strain, and belongs to the technical field of genetic engineering and bioengineering. According to the method, sterol synthesis route genes are screened, the speed limiting step is determined, high-yield ergosterol is preliminarily achieved through combined expression, the flow direction of carbon metabolic flow is further pulled through lipid droplet engineering and cell area chamber engineering, and the yield of ergosterol is increased. On the basis of enzyme modification engineering, the catalytic efficiency of the ERG11 on the lanosterol is improved through substrate channel engineering and proton-dependent catalytic path construction. Through multi-copy integration of the genes in the speed limiting step, the yield of ergosterol in a shake flask of the constructed engineering strain reaches 433.1 mg / L, and the yield of ergosterol in a 5L fermentation tank system reaches 4.58 g / L.

The invention belongs to the technical field of green chemistry, and particularly relates to a method for bi-directionally synthesizing chiral lactam through bi-enzyme cascade-chemical catalysis. According to a methylpiperidone synthesis route taking 2-methyl glutaronitrile as a raw material, nitrile hydratase is subjected to iterative saturated mutation, substrate channel residues and active site residues are subjected to semi-rational engineering transformation, and 4-cyano valeramide and 4-cyano-2-methyl butyramide are generated with high selectivity. And carrying out cascade reaction on the modified nitrile hydratase and amidase, so as to directionally generate the corresponding mono-cyano carboxylic acid. Then, a Raney nickel catalyst hydrogenation reaction is adopted to treat the whole-cell catalytic solution, and two regioselectivity products can be obtained, namely 5-methyl-2-piperidone (the yield is 96.9%, and the regioselectivity is 98.3%) and 3-methyl-2-piperidone (the yield is 67.9%, and the regioselectivity is 70.4%).