1247results about "Lyases" patented technology

The invention belongs to the technical field of gene engineering and enzyme engineering, and particularly relates to an ornithine decarboxylase mutant as well as a coding gene, an expression vector, a recombinant strain and application thereof. The ornithine decarboxylase mutant is obtained by mutating wild ornithine decarboxylase ODC10 of which the amino acid sequence is shown as SEQ ID NO.1, the catalytic activity of the obtained mutant is improved, and the biosynthesis efficiency of 1, 4-butanediamine is favorably improved. The recombinant strain for expressing the ornithine decarboxylase mutant constructed by the invention can efficiently synthesize 1, 4-butanediamine, and the molar conversion rate of ornithine is remarkably improved, so that the production benefit is improved, and the production cost is reduced.

The invention discloses a mesophilic carotenoid-producing recombinant yarrowia lipolytica strain as well as a construction method and application thereof, the recombinant strain exogenously expresses mesophilic original fatty acid desaturase (FAD3) and isocitrate dehydrogenase (IDH2) mutants from phaffia rhodozyma, and efficient synthesis under the condition of 30 DEG C is realized; secondly, a metabolic pathway of the recombinant strain is optimized based on a metabolic flow push-pull strategy, and isopentenyl pyrophosphate isomerase (IDI), farnesyl pyrophosphate synthase (ERG20), plant alkene dehydrogenase (CarB) and bifunctional plant alkene synthase (CarRP) are overexpressed, so that the flux of a precursor is increased; finally, gene multi-copy of astaxanthin synthase (crtS) and cytochrome P450 reductase (crtR) from phaffia rhodozyma is carried out, and the carotenoid production capacity of yarrowia lipolytica is further improved. The construction method of the mesophilic yarrowia lipolytica is simple, efficient synthesis of carotenoid can be better achieved at the higher temperature, the fermentation cost is greatly reduced, and the industrial production potential is achieved.

The invention discloses an arginine decarboxylase diaA enzyme mutant and application thereof in preparation of butanediamine, and belongs to the field of bioengineering. According to the invention, rational charge overturning transformation is simultaneously carried out on a pentamer meridian oligomeric interface and a dimer latitudinal oligomeric interface, so that stable assembly and efficient catalysis of the decamer under the condition of neutral to alkaline pH (7.0-9.0) are realized. Wherein positive charges are introduced into a meridian interface to weaken electrostatic repulsion, and negative charges are introduced into a latitudinal interface to enhance dimer compactness and substrate transfer efficiency. The specific enzyme activity of the representative double mutant AdiAD110K / H736E at pH 8.0 is about 35 times that of a wild type, and the representative double mutant AdiAD110K / H736E keeps a complete decamer state in a pH range of 7.0-9.0. The yield of butanediamine is up to 145.9 g / L under the whole-cell catalysis of the mutant.

The present invention provides a novel process for the production of aliphatic unsaturated C10-aldehyde compounds catalyzed by lipoxygenase (LOX) from a polyunsaturated fatty acid (PUFA) source. The invention also relates to the isolation and characterization of novel, preferably bifunctional LOXs from different algae sources, and the identification of structural and / or functional related LOXs from different bacterial sources. The invention also relates to the provision of enzyme mutants derived from said newly identified enzymes. Another aspect of the invention relates to the corresponding coding sequences of said enzymes, recombinant vectors and recombinant host cells, which are suitable for the production of such LOXs and for carrying out a new process for the production of aliphatic unsaturated C10-aldehyde compounds. Another aspect of the invention relates to the use of a specific aldehyde or aldehyde mixture obtained according to the invention as a flavoring ingredient or an ingredient of a food or feed composition.

The present invention relates to strategies for combining elements of non-ribosomal peptide synthase (NRPS) and polyketide synthase (PKS) into functional enzymes to promote the production of peptides and polyketide of any sequence. The present invention is based on the use of conserved sequence motifs within the T domain (or T domain-like) gene of the NRPS / PKS, which is used according to the invention as fusion sites for the production of new (and artificial) NRPS / PKS enzymes that can produce any peptide or polyketones of interest. The present invention provides artificial NRPS / PKS of the invention, methods and means for generating them, and nucleic acid libraries encoding various modules useful for designing NRPS / PKS for any peptide sequence of interest.

The invention discloses a rational copolymerization strategy for improving the stability of an oligomeric structure of acid-induced high-order oligomeric decarboxylase AdiA in a neutral to alkaline environment and application of the rational copolymerization strategy, and belongs to the field of bioengineering. According to the strategy, rational charge overturning transformation is carried out on a pentamer radial oligomeric interface and a dimer weft-wise oligomeric interface at the same time, and stable assembly and efficient catalysis of a decamer under the condition that the pH value is from 7.0 to 9.0 from neutral to alkaline are achieved. Wherein positive charges are introduced into a meridian interface to weaken electrostatic repulsion, and negative charges are introduced into a latitudinal interface to enhance dimer compactness and substrate transfer efficiency. The specific enzyme activity of the representative double mutant AdiAD471K / E467K / H736E is 45.5 times that of a wild type when the pH value is 8.0, and the representative double mutant AdiAD471K / E467K / H736E keeps a complete decamer state when the pH value is 7.0-9.0. The yield of butanediamine is up to 156.5 g / L by using the mutant to catalyze whole cells.

The invention discloses a high-efficiency lysine decarboxylase mutant and an application of the high-efficiency lysine decarboxylase mutant in synthesis of pentamethylene diamine. A series of mutants including a single mutant (such as N218G, T222V, C244P and the like), a double mutant and a three mutant are obtained by carrying out multi-site mutation on wild type lysine decarboxylase (SEQ ID NO.2) from Klebsiella grimontii. The catalytic efficiency of the mutant is remarkably improved, and the activity of the mutant is improved by 5.0-10.2 times compared with that of a wild type enzyme. According to the method disclosed by the invention, the substrate conversion rate of 99% or above can be realized within 2 hours through an optimized process of constructing an expression vector and a genetically engineered bacterium, inducing expression of lysine decarboxylase and synthesizing 1, 5-pentanediamine by utilizing a whole-cell catalysis technology. Compared with a traditional chemical method and a natural enzyme method, the method disclosed by the invention has the advantages of simplified process, high catalytic efficiency, low production cost and the like, and is suitable for producing bio-based 1, 5-pentamethylene diamine and nylon 56 salt.

The technology described herein relates to novel CRISPR / Cas systems and components thereof, vectors, proteins, fusion proteins, ribonucleoprotein complexes and methods of using the novel systems and components. The new system can be used in CRIS PRi applications and can be fused with any number of effector domain patterns, such as nuclease, base editor, leader editor, and the like, to conduct a variety of DNA modifications.

The invention belongs to the technical field of molecular biology, and particularly relates to sphingomonas paucimobilis recombinant engineering bacteria as well as a construction method and application thereof. The engineering strain is obtained by knocking out a metabolic bypass gene from sphingomonas paucimobilis, and the metabolic bypass gene is composed of a crtI gene or a phaC gene. The production amount of gellan gum can be increased by the delta phaC knockout strain and reaches 8.9 g / L. In addition, overexpression of energy metabolism related genes pgi or clpB or ycaJ is carried out, and the yield of a strain with the overexpressed pgi gene is increased to the maximum and reaches 9.5 g / L. Therefore, the pgi gene is overexpressed on the basis of the delta phaC knockout strain, the yield of gellan gum is further increased to 10.0 g / L, and the production efficiency is improved.

The invention discloses a tryptophan enzyme mutant and application thereof. The tryptophan enzyme mutant is obtained by mutation of at least one site of the 50th amino acid L, the 394th amino acid V and the 395th amino acid E of a tryptophan enzyme parent. The tryptophan enzyme mutant disclosed by the invention has good substrate bias, particularly has better selectivity and catalytic efficiency on 6 '-halogenated tryptophan, and not only simplifies the production process, but also improves the production efficiency when being used for biosynthesis production of the tylosin.

The invention discloses saccharomyces cerevisiae FNFHSc18 and application thereof, and belongs to the technical field of microorganisms. On one hand, the invention provides a saccharomyces cerevisiae engineering strain FNFHSc18 for synthesizing immune nutrients-beta-carotene and astaxanthin, and the preservation number of the saccharomyces cerevisiae engineering strain is CGMCC (China General Microbiological Culture Collection Center) No.24838; on the other hand, the invention provides application of the saccharomyces cerevisiae, the strain takes soybean meal as a solid state fermentation substrate, the content of crude protein and amino acid of the obtained product fermented soybean meal is remarkably increased, anti-nutritional factors such as oligosaccharide and urease are greatly reduced, more importantly, the fermented soybean meal is rich in beta-carotene and astaxanthin, and the content of the beta-carotene and the astaxanthin is greatly reduced. The soybean meal protein obtains new functional immune nutrition, so that the overall nutritional value of the soybean meal is improved, and the soybean meal reaches a leading level in the field of developing functional fermented soybean meal by utilizing saccharomyces cerevisiae.

The invention provides a glycolaldehyde synthetase mutant and application thereof in ethylene glycol synthesis, the protein of the mutant can catalyze formaldehyde to prepare hydroxyacetaldehyde (glycolaldehyde), meanwhile, the mutant does not depend on divalent metal ions to maintain the catalytic activity any more, and the activity is further improved in the presence of Co < 2 + > and Fe < 2 + >. The invention further provides a cofactor replacement strategy. The mutant protein provided by the invention can effectively improve the catalytic efficiency of formaldehyde to glycolaldehyde, and can be carried out under mild conditions.

The invention relates to the field of biological engineering and technology, in particular to an enzyme for synthesizing glabridin, a biological synthesis method of glabridin and a bacterial strain. The invention discloses a series of glabridin synthesizing enzymes (phenylalanine ammonialyase, cinnamic acid-4-hydrogenase, tyrosine ammonialyase, p-coumaric acid coenzyme A ligase, chalcone synthase, chalcone reductase, chalcone isomerase, isoflavone synthase, 2-hydroxyisoflavone dehydratase, 4 '-oxymethyltransferase, 4'-hydroxymethyltransferase and 4 '-hydroxymethyltransferase) derived from glycyrrhiza glabra for the first time. ) can be used in the preparation of a compound (e.g., isoflavone 2 '-hydrogenase, isoflavone reductase, Vissisoketoreductase, isoprenyltransferase, pterocarpin reductase, oxidative cyclase, demethylase, cytochrome P450 reductase, and the like). The de novo synthesis of the glabridin in a microbial cell factory is realized for the first time, and the application prospect of the glabridin biological manufacturing industry is greatly promoted.

The invention provides a kit for rapidly detecting MRSA and drug-resistant gene nucleic acid POCT of the MRSA based on a phage lyase mutant. The kit comprises nucleic acid extraction and purification and triple PCR rapid amplification detection reagents. The staphylococcus aureus bacteriophage lyase mutant nucleic acid release liquid containing the amino acid sequence as shown in SEQ ID NO: 1 is adopted, wall breaking can be rapidly conducted on staphylococcus aureus, and nucleic acid is released. The effect of the nucleic acid release liquid acting for 5 minutes is equivalent to or even better than that of the lysozyme acting for 60 minutes; according to the present invention, with the combination of the MRSA and drug-resistant gene triple PCR rapid amplification detection reagent, the total detection time is only 35 min, the time is saved by 55 min or more than 55 min compared with the lysozyme method, and the MRSA and drug-resistant gene nucleic acid POCT rapid detection can be achieved;

The present invention provides a 4-vinylguaiacol oxygenase mutant for synthesizing vanillin, and an engineered bacterium thereof. The 4-vinylguaiacol oxygenase mutant is an enzyme having the sequence as shown in SEQ ID NO. 1, which exhibits a double-site mutation of D27P / T216M, D27P / M351F, D27P / Y156D, M351F / Y156D, T216M / Y156D or T216M / M351F, or a single-site mutation of T216M, T216L, S280M, G307P or M351F, and can be used for catalyzing 4-vinylguaiacol to generate vanillin.

The invention relates to an Escherichia coli engineering strain for producing 2, 3-pentanediol as well as a construction method and application of the Escherichia coli engineering strain, and the method comprises the following steps: constructing recombinant plasmids pACYC-IlvA *-ThrA * BC for expressing an IlvA * gene of threonine deaminase, a ThrA gene of asparaginic acid kinase I, a Thr B gene of homoserine kinase and a ThrC gene of threonine synthetase; the method comprises the following steps: constructing a recombinant plasmid pRSF-Ilv2C-Aro10 for expressing an Ilv2C gene of acetohydroxy acid synthetase and an Aro10 gene of propiophenone dehydrogenase; and chemically converting the recombinant plasmid pACYC-IlvA *-ThrA * BC and the recombinant plasmid pRSF-Ilv2C-Aro10, and transferring the recombinant plasmid pACYC-IlvA *-ThrA * BC and the recombinant plasmid pRSF-Ilv2C-Aro10 into escherichia coli so as to obtain the escherichia coli engineering strain for producing the 2, 3-pentanediol. According to the escherichia coli engineering strain, the biosynthesis of 1.61 g / L of 2, 3-pentanediol is realized by using L-threonine and 40 g / L of glucose.

Provided is a variant of an RNA polymerase, the variant being characterized in that an amino acid corresponding to arginine at position 386 in the amino acid sequence of a wild-type T7 RNA polymerase represented by SEQ ID NO: 1 is substituted with an aromatic amino acid.

The invention discloses saccharomyces cerevisiae MBPSc21 and application thereof, and belongs to the technical field of microorganisms. On one hand, the invention provides a saccharomyces cerevisiae engineering strain MBPSc21 for expressing immune nutrient-beta-carotene, and the preservation number of the saccharomyces cerevisiae engineering strain MBPSc21 is CGMCC No.32663; on the other hand, the invention provides application of the saccharomyces cerevisiae, the strain takes soybean meal as a solid-state fermentation substrate, the crude protein content of the obtained product fermented soybean meal is remarkably increased, anti-nutritional factors such as oligosaccharide and urease are greatly reduced, more importantly, the fermented soybean meal is rich in beta-carotene, and the content of the beta-carotene is reduced. The soybean meal protein is endowed with new functional immune nutrition, so that the overall nutritional value of the soybean meal is improved, and a leading level is achieved in the field of developing functional fermented soybean meal by utilizing saccharomyces cerevisiae.

The invention relates to ulva polysaccharide lyase JpPL40A and application thereof, and belongs to the technical field of functional enzymes, the amino acid sequence of the ulva polysaccharide lyase JpPL40A is as shown in SEQ ID NO.1, and the coding gene of the ulva polysaccharide lyase JpPL40A is as shown in SEQ ID NO.2 or SEQ ID NO.3. The invention further discloses a preparation method of the ulva polysaccharide lyase JpPL40A. The ulva polysaccharide lyase JpPL40A disclosed by the invention can be used for hydrolyzing ulva to obtain unsaturated ulva disaccharide, tetrasaccharide, hexasaccharide and / or octasaccharide.

The invention provides a preparation method of Keemun camellia seed fermented oil, and belongs to the technical field of vegetable oil production. The preparation method comprises the following steps: S1, preparing recombinant saccharomycetes; s2, jointly fermenting the antarctic torulopsis, the recombinant saccharomycetes and the Keemun camellia seed oil; s3, separating an oil phase from a water phase, and taking the oil phase; and S4, dehydrating the oil phase to obtain the Keemun camellia seed fermented oil. Preferably, according to the preparation method of the recombinant saccharomycetes in the step S1, phenylalanine deaminase, cinnamate hydroxylase, 4-coumaric acid CoA ligase, chalcone synthetase, chalcone reductase and peroxidase are expressed in saccharomyces cerevisiae. According to the Keemun camellia seed fermented oil prepared by the method disclosed by the invention, the characteristic components of the Keemun camellia seed oil can be remarkably improved, the antioxidant capacity of the Keemun camellia seed oil is enhanced while the skin feeling of the Keemun camellia seed oil is improved, and the application value of the Keemun camellia seed oil in cosmetics is improved.

The invention discloses recombinant escherichia coli for producing N-acetylneuraminic acid as well as a construction method and application of the recombinant escherichia coli, and belongs to the technical field of genetic engineering. According to the invention, 6-phosphofructokinase is knocked out, glycerol kinase is introduced into a glycerol kinase mutant, a diffusion transporter is promoted by glucose to replace PTS phosphotransferase I, and a promoter is optimized, so that recombinant escherichia coli can grow by double carbon sources of glucose and glycerol, N-acetylneuraminic acid is synthesized, and meanwhile, the generation amount of acetic acid is extremely low. On the basis, a 6-phosphofructokinase coding gene pfkB is knocked out, a heterologous ED pathway from pseudomonas mobilis is introduced, and the yield under the pore plate level is further increased to 9.44 g / L through copy number optimization and RBS strength optimization.

The invention provides a method for producing borneol, the method comprising culturing the transgenic microorganism in a culture medium, and the microorganism comprises or expresses borneol diphosphate synthetase and phosphatase.

The present invention relates to an improved process for producing a product comprising a protein of interest (TP) capable of crystallizing under salt soluble conditions by crystallization of said TP. The present invention uses at least two different TP-containing protein feeds, which are mixed to produce a supersaturated protein solution in which TP crystallizes. One of the protein feeds must have a pH at least plus 0.1 to the pH (pHMCY, TP) providing the maximum crystallization yield of the TP, and one of the protein feeds must have a pH at most minus 0.1 to the pHMCY, TP.

The invention relates to the field of bioengineering, in particular to an enzyme composition, a product and application of the enzyme composition in preparation of Ectoine. The invention provides an enzyme composition. The enzyme composition comprises a mutant of 2-hydroxy-4-aminobutyric acid oxidase, a mutant of keto transaminase, a mutant of hydroperoxide hydrolase, a mutant of Ectoine synthetase, a mutant of pyruvic acid-CoA oxidase and lactobacillus helveticus lactic dehydrogenase. According to the method disclosed by the invention, a plurality of mutant enzymes are utilized for combined transformation for the first time, and cheap 2-hydroxy-4-aminobutyric acid is taken as a starting raw material to quickly and efficiently produce Ectoine; the enzyme method has the characteristics of easiness in control, short conversion time, few generated impurities and the like, so that the method has unique advantages in the aspects of low-cost and large-scale mass production of Ectoin.

The invention provides a saccharomyces cerevisiae engineering strain HYB05 capable of releasing astaxanthin in a controllable self-splitting manner. The saccharomyces cerevisiae engineering strain HYB05 is constructed by taking a basic strain for producing astaxanthin as a starting strain, introducing a coding sequence comprising cell wall lyase Lyc, a coding sequence of a signal peptide mutant F8A and a recombinant nucleic acid construct for expressing a regulatory element, and knocking out an SSD1 gene to relieve a translation inhibition effect. The cell wall lyase Lyc is found and disclosed by the inventor for the first time, and the amino acid sequence is shown as SEQ ID NO: 6; the amino acid sequence of the signal peptide mutant F8A is as shown in SEQ ID NO: 8. According to the engineering strain HYB05, by introducing the recombinant nucleic acid construct, the activation of the catalytic activity of cell wall lyase under specific conditions is realized, so that the cell wall of the saccharomyces cerevisiae is broken, and the astaxanthin synthesized by the saccharomyces cerevisiae is released. Compared with the prior art, the astaxanthin is produced through fermentation of the strain, the extraction steps are simplified, extraction of the astaxanthin in the yeast is achieved to the maximum extent, the yield of the astaxanthin is remarkably increased, and the technical effect is remarkable.

The invention relates to pectin lyase as well as a mutant, a recombinant expression vector and application thereof. The nucleotide sequence of the pectin lyase is as shown in SEQ ID NO.1, the full length of the gene is 1056 bp, 352 amino acids are coded in total, and the amino acid sequence is as shown in SEQ ID NO.2; the pectin lyase mutant is formed by site-directed mutagenesis on the basis of an amino acid sequence of pectin lyase, a mutated nucleotide sequence is as shown in SEQ ID NO.3, and the amino acid sequence is as shown in SEQ ID NO.4; compared with the existing pectin lyase, the pectin lyase and the mutant thereof provided by the invention have the characteristics of high activity and temperature stability, and can be widely applied to fruit juice extraction, fruit juice clarification and fruit and vegetable juice beverage processing.

The invention discloses a method for efficiently producing ergothioneine by metabolic engineering modified escherichia coli and application, and belongs to the technical field of biology. By adopting the recombinant escherichia coli EGT-1-B * provided by the invention, glucose can be converted into ergothioneine; therefore, the raw materials are cheap and easy to obtain, additives and heavy metals which are harmful to human health are avoided, a good foundation is laid for later product purification, and the requirements of commercial application can be met. Furthermore, genes related to metabolism of histidine, methionine, cysteine and S-adenosylmethionine are further overexpressed in the recombinant escherichia coli EGT-1-B *, so that the content of precursor amino acid synthesized by the ergothioneine is increased, and meanwhile, after the gene yqaB is knocked out, the yield of the ergothioneine is further increased; the utilization rate of glucose to ergothioneine can be further improved, and the yield of ergothioneine can reach 140.29 mg / L.

An engineered bacterium for improving tryptophan yield, a construction method therefor, and a use thereof. A strain capable of tolerating a high concentration of tryptophan was obtained by screening. Genome sequencing and protein sequence analysis of the strain revealed that some proteins in the strain had undergone point mutations, and these mutants can improve the tryptophan yield. In order to further improve the tryptophan yield, modifications are made to a protein sequence expressed by a fadR gene or a protein sequence expressed by a pepD gene in a starting bacterium. Such modifications enable the obtained engineered bacterium to have a higher tryptophan yield than the starting bacterium. In the case of large-scale production, in a 5 L fermentation tank, the tryptophan yield can reach 62.38±5.80 g / L, and the sugar-to-acid conversion rate can reach 24.1%. Compared with the original bacterium, the tryptophan yield is increased by 1.48 folds, and the sugar-to-acid conversion rate is increased by 1.26 folds. The biological material and the use thereof relate to the technical field of molecular biology and have wide practical application value.

The invention discloses a method for preparing inositol, particularly relates to a method for converting glucose into inositol through in-vitro multi-enzyme catalysis, and belongs to the field of enzyme catalysis preparation of inositol. According to the invention, the phosphoglucose synthase capable of realizing phosphorylation of glucose by using inorganic phosphate is screened, and the inositol can be generated through conversion by taking glucose as a substrate, so that a novel method for synthesizing the inositol through enzyme catalytic conversion is provided. In addition, the invention further provides hexokinase capable of improving the yield of inositol, and the conversion efficiency of glucose is greatly improved. Furthermore, the invention provides a multi-enzyme molecular machine containing the glucose phosphate synthase and / or hexokinase, the multi-enzyme molecular machine is applied to preparation of inositol, and the reaction steps are simple, green and pollution-free.

The invention discloses construction and application of a saccharomyces cerevisiae engineering strain for synthesizing vanillin by taking ferulic acid as a substrate, and belongs to the technical field of genetic engineering. The invention provides a recombinant saccharomyces cerevisiae, which is characterized in that a series of genes for producing vanillic acid and vanillyl alcohol from vanillin through oxidation and reduction are knocked out, and feruloyl coenzyme A synthetase FCS and p-hydroxycinnamyl coenzyme A hydratase ECH which are obtained by screening from a metagene library in a West Sichuan hot spring extreme environment are overexpressed. According to the engineering strain VAN002 with high yield of vanillin, the accumulation of vanillin reaches 16.8 g / L, and the molar conversion rate of catalyzing ferulic acid to synthesize vanillin reaches 93%, which is the highest level reported by synthesizing vanillin by saccharomyces cerevisiae at present.