272 results about "Synthetic enzyme" patented technology

Provided are S-adenosylmethionine synthetase mutants and the use thereof, belonging to the technical field of biological enzyme engineering. Site-directed combinatorial mutations are performed on positions 189, 200, 234, 266 and 371 of S-adenosylmethionine synthetase MAT to obtain a series of mutants with improved enzyme activity, wherein the specific enzyme activity of I189V / V266H and V266H / N371R is increased by more than 10 times relative to the parent. The yields of S-adenosylmethionine synthesized by whole cells of recombinant bacteria expressing mutants I189V / V266H / N371R, V266H / Q234N / N371R and I189V / V266H / Q234N / N371R all reach 1550 mg / L or more. The provided enzyme mutants have broad application prospects in the production of S-adenosylmethionine and the construction of genetically engineered microorganisms for S-adenosylmethionine.

Disclosed in the present invention are a sucrose synthase mutant and a method for preparing RD using same. The sucrose synthase mutant is based on the amino acid sequence shown in SEQ ID NO: 2 and contains a mutation in at least one of the following sites: D296Q, R636Q, G514L, H531L, R567T, D569Q, E610N, A642N, E663N, L667T and T742R. By means of site-directed mutagenesis of sucrose synthase, the enzyme activity of the sucrose synthase mutant is improved compared with that of the wild type. Also, some mutants can still maintain a high conversion rate in a wide pH range.

The present application relates to a series of GDP-fucose synthetase polypeptides, which can be applied to the synthesis of human milk oligosaccharides (HMOs) precursor GDP-fucose, belonging to the field of enzyme engineering technology. In certain embodiments, the polypeptide having catalytic GDP-4-keto-6-deoxymannose converted into GDP-fucose comprises an amino acid sequence which is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100% identical to SEQ ID NO: 1 and / or to the polypeptide M1-14. The present application provides a series of polypeptides for synthesizing GDP-fucose.

The invention belongs to the technical field of plant genetic engineering, and particularly relates to a promoter of a pinus massoniana terpene synthase gene Pm TPS (-)-alpha-pinene and application of the promoter. According to the invention, a promoter fragment of a Pm TPS (-)-alpha-pinene gene and three homeopathic action elements with jasmonic acid response on the promoter fragment are obtained from pinus massoniana. A complete promoter fragment of the Pm TPS (-)-alpha-pinene gene of the pinus massoniana is transformed into arabidopsis thaliana by means of agrobacterium mediation, and the promoter can drive expression of a GUS gene in leaves of the arabidopsis thaliana or nicotiana benthamiana. GUS staining and GUS enzyme activity of transgenic Bensi tobacco leaves are reduced along with deletion of jasmonic acid response cis-acting elements on the promoter. Furthermore, the yield of pine resin terpenoids can be increased by utilizing the promoter and the homeopathic element thereof.

The invention belongs to the technical field of facility horticulture postharvest treatment, and particularly relates to a tomato postharvest quality regulation and control light supplementing system based on white light-metabolism key enzyme coupling activation and an application method thereof, and the tomato postharvest quality regulation and control light supplementing system comprises an enzyme activity targeting white light module, an enzyme activity feedback module and a fruit bearing regulation and control module. According to the postharvest tomato quality regulation and control light supplementing system based on white light-metabolic key enzyme coupling activation, a white light spectrum segmentation-key enzyme activity dynamic matching coupling mechanism is innovatively designed by combining the regulation and control rule of white light on the activity of key enzymes such as carotenoid synthetase and phenylpropane metabolic enzyme of tomato fruits; through an adjustable broad-spectrum white light source and an enzyme activity feedback module, directional accumulation of beneficial metabolites such as carotenoids and flavonoid compounds and efficient degradation of alkaloids are realized.

Provided herein are novel materials and methods for site-specific incorporation of phosphotyrosines into proteins. The novel methods of the invention encompass the use of a novel aminoacyl tRNA synthetase capable of charging compatible tRNAs with a phosphotyrosine precursor. The phosphotyrosine precursor is then incorporated, site-specifically, into a protein at sites where phosphotyrosine residues are desired. The phosphotyrosine precursors are subsequently treated to convert them into phosphotyrosine residues, yielding proteins with phosphotyrosines at selected sites. The scope of the invention encompasses novel aminoacyl tRNA synthetases, novel phosphotyrosine precursors, and methods of using these materials to create site-specific phosphorylated tyrosine residues in a protein.

This disclosure relates to modified tryptophan synthase and more particularly to modified beta-subunits of tryptophan synthase. The disclosure further relates to cells expressing such modified subunits and methods of producing non-canonical amino acids.

The invention discloses recombinant saccharomyces cerevisiae, a 11-seed oil fermentation product as well as a preparation method and application of the 11-seed oil fermentation product, and belongs to the technical field of synthetic biology and fermentation product preparation. According to the recombinant saccharomyces cerevisiae disclosed by the invention, a caffeic acid coenzyme A ligase 4CL coding gene, a chalcone isomerase CHI coding gene, a flavanone-3-hydroxylase F3H coding gene and a chalcone synthase CHS mutant coding gene are over-expressed, and the chalcone synthase CHS mutant is preferably Y69H / H71Y / Q161K. The preparation method of the 11-seed oil fermentation product comprises the following steps: fermenting a recombinant saccharomyces cerevisiae fermentation solution in the presence of a sophora flower bud extract and caffeic acid to obtain a first fermentation solution; fermenting the lactic acid bacteria fermentation liquor in the presence of 11 seed oil to obtain second fermentation liquor; and mixing the first fermentation liquid and the second fermentation liquid, continuing fermentation, and respectively collecting an oil phase and a water phase after fermentation is finished, so as to obtain a fermentation product. The product provided by the invention has multivitamin antioxidant activity.

The invention discloses a sucrose synthase mutant and a method for preparing RD (Reduced Disorder) by using the sucrose synthase mutant. The sucrose synthase mutant is obtained by mutation of at least one of the following sites on the basis of an amino acid sequence as shown in SEQ ID NO: 2: D296Q, R636Q, G514L, H531L, R567T, D569Q, E610N, A642N, E663N, L667T and T742R; site-directed mutagenesis is carried out on sucrose synthetase, so that the enzyme activity of the sucrose synthetase mutant is improved compared with that of a wild type, and meanwhile, a part of the mutant still maintains a relatively high conversion rate in a relatively wide pH range.

The application discloses a mutant of glutamate-cysteine synthetase and application of the mutant in synthesis of glutathione. Single-site mutation is carried out on amino acid residues at positions 150, 188, 235, 236, 241, 300, 301, 302 and 330 of wild-type glutamate-cysteine synthetase (Glutamate-cysteine ligase [EC:6.3.2.2]) from Citrobacter koseri (strain ATCC BAA-895), and mutant sites at positions 150, 236, 302 and 330 are screened. Mutants M1-M6 are obtained by combination mutation of the mutant sites screened above, and the enzyme activity of the mutant M4 with the highest activity is increased by 477% compared with that of the wild-type glutamate-cysteine synthetase. The mutant M4 is used to catalyze synthesis of glutathione, and the highest concentration reaches 23 g / L, which is increased by 4.2 times compared with the wild type, and the production cost is greatly reduced, so the mutant has important application value.

The invention relates to Corynebacterium glutamicum with high indigo yield as well as a construction method and application thereof, and belongs to the technical field of gene recombination fermentation. The recombinant corynebacterium glutamicum can be used for overexpression of the following genes: a D-glucose-6-phosphate dehydrogenase gene zwf, a II type deoxyarabinose type heptulose phosphate synthase gene aroG and a flavin-containing monooxygenase gene CcFMO. The recombinant corynebacterium glutamicum can weaken the following genes: a pyruvate kinase gene pyk and a tryptophan synthase gene trpB. The corynebacterium glutamicum is used for overexpressing a zwf gene, an aroG gene and a CcFMO gene, weakening a pyk gene and a trpB gene and enhancing the expression quantity of an indole synthetic pathway related enzyme gene, so that the yield of the corynebacterium glutamicum at a shake flask stage reaches 619mg / L in a fermentation production process of indigo under the condition of not additionally adding tryptophan. The invention provides the recombinant corynebacterium glutamicum with high indigo yield, which is low in production cost, good in stability and high in synthesis efficiency, and is suitable for practical popularization and application.

Disclosed herein are novel CBGA and CBGVA synthases and methods for improving their overall activity for synthesizing CBGA and CBGVA from their respective precursors, olivetolic acid (OA) or divaleric acid (DVA), and GPP. Fusion proteins for enhancing the synthesis of CBGA and CBGVA are also disclosed. The methods described herein also increase the titer and purity of CBGA and CBGVA produced by cells by: 1) reducing the formation of the by-products FCBGA and FCBGVA, which are synthesized from the prenylation of OA and DVA, respectively, with FPP; 2) increasing the intracellular availability of OA and DVA; and 3) increasing the rate of CBGA and CBGVA formation while reducing the accumulation of intermediates.

The invention relates to a non-natural cannabinoid synthase comprising at least one amino acid variation as compared to a wild type cannabinoid synthase Δ9-tetrahydrocannabinolic acid synthase (THCAS), comprising three alpha helices (αA, αB and αC) where a disulfide bond is not formed between alpha helix αA and alpha helix αC, wherein the non-natural cannabinoid synthase catalyzes the oxidative cyclization of cannabigerolic acid (CBGA) into a cannabinoid. The invention further relates to a non-natural Δ9-tetrahydrocannabinolic acid synthase (THCAS), a non-natural cannabidiolic acid synthase (CBDAS), and a non-natural cannabichromenic acid synthase (CBCAS) comprising at least one amino acid variation as compared to a wild type THCAS, CBDAS, or CBCAS, respectively, comprising three alpha helices (αA, αB and αC) and wherein a disulfide bond is not formed between alpha helix αA and alpha helix αC. The invention also relates to a nucleic acid, expression construct, and engineered cell for making the non-natural THCAS, CBDAS, and / or CBCAS. Also provided are compositions comprising the non-natural THCAS, CBDAS, and / or CBCAS; isolated non-natural THCAS, CBDAS, and / or CBCAS enzymes; methods of making the isolated enzymes; cell extracts comprising cannabinoids; and methods of making cannabinoids.

This invention relates to the field of oral care products technology, specifically to an artificial enzyme with antibacterial properties, its preparation method, and its application. This technical solution employs a chemical reduction method, where a palladium source reacts in an environment containing surfactants and reducing agents to obtain palladium nanozymes. The obtained palladium nanozymes exhibit uniform morphology, controllable particle size, good dispersibility in water, and good biocompatibility; they have no significant toxic side effects on human gingival fibroblasts, possess highly efficient catalase activity, and demonstrate strong antibacterial and bactericidal effects against oral pathogens, significantly reducing bacterial drug resistance; they can inhibit acid production by oral bacteria, preventing tooth decay; and they retain their original catalytic activity even after multiple cycles of use. Applying the palladium nanozymes of this solution to the manufacture of oral care products can solve the technical problems of unsatisfactory safety and easy development of drug resistance in existing oral care products using antibacterial and anti-caries methods, showing promising application prospects.

The invention discloses a method for synthesizing novel alkaloid by enzyme catalysis of tryptamine and alpha-ketoamide substances, and the method comprises the following steps: reacting a compound shown as a formula (A) with a compound shown as a formula (B) in the presence of isoviscin synthetase to obtain alkaloid shown as a formula (C). The method is mild in condition, free of pollution and simple in process route.

This invention relates to a recombinant Halomonas strain that produces polyhydroxyalkanoates (PHA) by overexpressing endogenous enzymes encoding ADP-dependent acetyl-CoA synthase (ADP-ACS). acd This gene not only enhances the recombinant bacterium's tolerance to acetic acid but also enables it to efficiently convert acetic acid or acetate substrates into acetyl-CoA, thereby participating in the tricarboxylic acid cycle (TCA cycle) and the synthesis of PHA. The present invention also relates to a method for producing PHA using the aforementioned recombinant Halomonas bacterium employing a pH-stat strategy.

The invention discloses a pinene synthase mutant and application thereof in pinene production, and belongs to the technical field of bioengineering. Isoleucine at the 409th site of pinene synthase is mutated into valine, phenylalanine at the 443rd site of pinene synthase is mutated into alanine, overexpression is carried out in serratia marcescens (HBQA7), and an engineering strain ST17 is constructed. Under the condition of shake flask fermentation, the pinene yield of the engineering strain ST17 reaches 0.80 g / L; the yield is obviously increased to 43.2 g / L through amplification culture in a 30 L fermentation tank. The invention provides an efficient, economic and environment-friendly novel method for industrial biosynthesis of pinene.

The present application relates to the field of bioengineering, and particularly to a mutant of an enzyme, a mutant of a glycosyltransferase and application thereof in preparing rhodiolin. The present application provides a mutant of an enzyme, which has the following amino acid site mutations on the basis of a wild-type glycosyltransferase: the 308th site is mutated from I to Q, the 333rd site is mutated from V to R, the 363rd site is mutated from W to S and the 386th site is mutated from S to G, and the amino acid sequence of the wild-type glycosyltransferase is shown as SEQ ID NO: 1. The present application obtains a glycosyltransferase and a sucrose synthase with improved thermal stability through structural rational design and deep learning modification, and can glycosylate tyramine to generate rhodiolin in the form of regenerating ADP-glucose, and the price of ADP is only about 20% of UDP, which has a cost advantage.

The invention belongs to the technical field of biological enzyme engineering, and particularly relates to a glucosamine-6-phosphate synthetase mutant and application thereof. A plurality of glucosamine-6-phosphate synthase mutants capable of remarkably improving the yield of N-acetylglucosamine are screened by performing single-point mutation or combined mutation on amino acid residues near a substrate binding pocket of the glucosamine-6-phosphate synthase or on the surface of protein of the glucosamine-6-phosphate synthase. According to the technical scheme, a foundation is laid for producing glucosamine by further transforming escherichia coli through metabolic engineering.

This invention discloses a mutant of glycolaldehyde synthase and its application in improving the thermostability of glycolaldehyde synthase, belonging to the field of biocatalysis application technology. The glycolaldehyde synthase mutant disclosed in this invention, based on the amino acid sequence shown in SEQ ID NO.1, contains the following mutations: A381P / E509F, A381P / K290P, A381P, S61A, A381P / K290P / E509F, A381P / C49A, A381P / T100V, A381P / S61A, or A381P / K290P / S61A. This mutant protein can catalyze the preparation of hydroxyacetaldehyde (glycoaldehyde) from formaldehyde. The mutant protein of this invention also improves the catalytic efficiency of the formaldehyde-to-glycoaldehyde conversion. Due to the improved thermostability, it is beneficial for continuous catalysis in actual industrial reactions, thus the mutant shows good prospects for industrial applications.

The invention provides a method for inserting an unnatural amino acid and application thereof, the method adopts a pyrrolysine aminoacyl-tRNA synthetase mutant as an orthogonal translation element to introduce the unnatural amino acid into a protein to obtain a protein containing the unnatural amino acid, the invention also provides an in-vitro cell-free protein synthesis system for inserting the non-natural amino acid, and the system can efficiently introduce the non-natural amino acid, especially the lysine analogue non-natural amino acid. The technical bottleneck that a natural translation system is low in non-natural amino acid recognition efficiency is solved, and an efficient and controllable technical tool is provided for protein function research and biological medicine development.

The application relates to the technical field of biotechnology, in particular to a dopamine reuptake inhibitor intermediate synthesis enzyme mutant and application, and amino acid sequences are shown in SEQ ID NO: 7-SEQ ID NO: 12. The stereoselectivity of asymmetric reduction catalysis of the dopamine reuptake inhibitor intermediate precursor is improved, the R-type dopamine reuptake inhibitor intermediate with high chirality purity is obtained, and the synthesis efficiency of the dopamine reuptake inhibitor intermediate is improved.

Provided are non-natural or variant β-ketoacyl-acyl carrier protein (ACP) synthase (KAS) IVa enzymes (KASIVa), polynucleotides encoding such variant KASIVa, host cells expressing such variant KASIVa, oils and oil products produced by such cells, and methods of making and using such variant KASIVa.

The invention discloses a gene CsANS1 for coding anthocyanin synthetase and application of the gene CsANS1, and belongs to the technical field of biology. The nucleotide sequence of the gene CsANS1 is as shown in SEQ ID NO. 1. The gene CsANS1 or related biological materials thereof can be used for synthesizing anthocyanin and quercetin in vitro. Recombinant engineering bacteria verify that the anthocyanin synthetase CsANS1 not only has activity of catalyzing colorless anthocyanin to generate anthocyanin, but also has relatively strong activity of catalyzing quercetin to generate, so that the limitation of anthocyanin and quercetin production in the prior art is effectively solved. The bifunctional catalytic characteristic of the gene CsANS1 widens the application prospect of ANS enzyme in metabolic engineering, and is helpful for promoting the development and utilization of natural active ingredients in the fields of food, medicine, cosmetics and the like.

The invention belongs to the technical field of biological catalysis, and particularly relates to a glycosyl transferase mutant and application thereof in catalytic synthesis of rebaudioside M. Engineering bacterium whole cells or broken enzyme liquid for co-expressing a glycosyl transferase G1 mutant, a glycosyl transferase C1 mutant and sucrose synthase SUS is used as a catalyst, Reb A is used as a substrate, sucrose is used as a glycoside donor, Reb M is synthesized under mild conditions, the space-time yield of the generated Reb M is up to 3.33 g / L / h, and the method has the advantages of being high in space-time yield, convenient to purify, green, environmentally friendly and the like, and is suitable for industrial production. The industrial application value is extremely high.

An engineered tyrosyl-tRNA synthetase / tRNA pair that co-translationally incorporates O-sulfotyrosine in response to UAG codons in E. coli and mammalian cells is described herein. This platform enables recombinant expression of eukaryotic proteins homogeneously sulfated at chosen sites.

The invention discloses a double-function (p) ppGpp synthetase or hydrolase mutant, a biological material and application of the double-function (p) ppGpp synthetase or hydrolase mutant to preparation of L-threonine, and belongs to the technical field of gene engineering. The technical problem to be solved by the invention is how to improve the yield of L-threonine. Relative to a wild type, the bifunctional (p) ppGpp synthetase or hydrolase mutant comprises one or more of the following mutations: K1) mutation of 174th amino acid of SEQ ID NO: 2 into cysteine, K2) mutation of 529th amino acid of SEQ ID NO: 2 into phenylalanine, and K3) insertion of histidine and aspartic acid between 84th amino acid and 85th amino acid of SEQ ID NO: 2. The bifunctional (p) ppGpp synthetase or hydrolase mutant provided by the invention can be used for improving the yield of L-threonine of Escherichia coli.

The invention belongs to the technical field of synthetic biology and fermentation engineering, and discloses an engineering bacterium for producing Indigoidine based on membrane vesicle engineering reinforced corynebacterium glutamicum and application of the engineering bacterium. According to the invention, the ncp1 gene of corynebacterium glutamicum is knocked out through a CRISPR-Cas12a gene editing technology, connection between cell walls and cell membranes is relieved, and a chassis strain of high-yield extracellular membrane vesicles (OMVs) is constructed; meanwhile, a recombinant expression vector is introduced, indigo synthetase BpsA is positioned and displayed on membrane vesicles by utilizing PorB anchoring protein, and activating enzyme Sfp is co-expressed. In fermentation production, a staged feeding and IPTG (isopropyl-beta-d-thiogalactoside) and Tween-80 dual induction strategy is adopted to promote thalli to express zymoprotein and release a large number of membrane vesicles at the same time. According to the method, the membrane vesicles are used as an extracellular microreactor, so that the'simultaneous synthesis and secretion 'of the indissolvable indigo is realized, the problems of cytotoxicity and metabolic inhibition caused by intracellular precipitation of the product are effectively solved, and the yield and extraction efficiency of the indigo are remarkably improved.

The application discloses a novel double-carrier expression system for protein expression and a construction method and application thereof. The novel double-carrier expression system for protein expression comprises a first carrier containing a nucleotide sequence for coding a first functional domain of glutamine synthetase and a nucleotide sequence of light chain and heavy chain of a protein to be expressed; and a second carrier containing a nucleotide sequence for coding a second functional domain of glutamine synthetase and a nucleotide sequence of a ScFv sequence of the protein to be expressed, and an amino acid sequence of the glutamine synthetase is composed of the first functional domain and the second functional domain. By using the expression system, the transfection efficiency and screening efficiency are greatly improved, and the expression amount of the target protein is obviously increased.

A modified pegRNA Cas9 nickase system that maintains potent on-target editing activity but has reduced off-target editing activity relative to wild-type Cas, and methods of production and use.