2211results about "Isomerases" patented technology

The present application relates to the field of biotechnology, and in particular to an epimerase for synthesizing D-tagatose and its application, wherein the epimerase produces a mutation at position 100, or at position 125, or at position 127, or at position 131, or at position 268, or at position 270, or at position 271, or at position 308, or at position 339, or at position 340, or at position 342, or at position 366, or at position 402, or at position 437, or at position 440 in the amino acid sequence shown in SEQ ID NO. 142; through the above-mentioned method, the epimerase exhibits excellent epimerization activity at the 4th carbon position of D-fructose, can efficiently convert D-fructose into D-tagatose, and is beneficial to improving the synthesis efficiency of D-tagatose.

The invention belongs to the technical field of bioengineering, and discloses a recombinant yarrowia lipolytica capable of efficiently and rapidly producing sclareol, which is characterized in that optimized sclareol synthase related coding genes ScLPPS, ScSCS, GGPPS1, GGPPS2, ERG20 and tHMGR coding genes are inserted into a yarrowia lipolytica genome; inserting optimized pentadienol utilization pathway coding genes such as IPK, THIM and IDI coding genes; and inserting an optimized ATP: citrate lyase coding gene ACLY to obtain the ATP: citrate lyase coding gene ACLY. The recombinant yarrowia lipolytica is based on knockout of a yarrowia lipolytica strain responsible for coding a non-homologous recombination gene ku70, so that the homologous recombination capability of the yarrowia lipolytica strain is enhanced, gene integration is realized through the homologous recombination function of the yarrowia lipolytica, the genetic stability of the introduced gene can be greatly improved, and the recombinant yarrowia lipolytica strain has a good application prospect. The construction method of the recombinant yarrowia lipolytica strain is efficient and simple to operate.

The invention belongs to the technical field of gene engineering, and particularly relates to mutants of murA and ygeA genes and application of the mutants in production of human milk oligosaccharides. According to the invention, murA and ygeA genes of escherichia coli are edited by using a gene editing technology, so that a genetic engineering chassis strain carrying murA and ygeA mutants is obtained. Further, the chassis strain is applied to the construction process of production strains of LNT II, LNnT, LNT, 3 '-SL and 6'-SL, and the yield of the LNT II, the LNnT, the LNT, the 3 '-SL and the 6'-SL is further increased.

The invention provides a genetically engineered bacterium, a preparation method thereof and application of the genetically engineered bacterium in synthesis of sclareol. The genetically engineered bacterium comprises a CcLPPS gene derived from Cistanus crenatus, and a CcLPPS gene derived from Cistanus crenatus, the genetically engineered bacterium is selected from yarrowia lipolytica. According to the invention, the stably expressed sclareol efficient de novo synthesis strain is constructed, and no organic reagent or any path intermediate substance is added in the whole fermentation process, so that the production cost is greatly reduced, and the subsequent separation and purification steps are simplified. The engineering yarrowia lipolytica strain provided by the invention is high in production intensity and simple in fermentation process, has a relatively good application prospect, and lays a foundation for producing sclareol by a microbial fermentation method.

According to the D-psicose 3-epimerase mutant and the application thereof, the D-psicose 3-epimerase mutant is obtained through protein C-terminal sequence modification, software analysis and sequence alignment of different sources of D-psicose 3-epimerase, the half-life period of the mutant enzyme at 60 DEG C, 70 DEG C and 80 DEG C is prolonged, and the activity of the mutant enzyme is improved. The substrate affinity and conversion efficiency of the enzyme to D-fructose as a substrate for synthesizing D-psicose are improved, and the Tm value of the mutant is 73.23 DEG C and is improved by 11.98 DEG C compared with that of a wild type; the half-life period at the temperature of 60 DEG C is 255.35 min and is 17.60 times that of a wild type, the problem that the half-life period of the wild type is too short at the high temperature is solved, the relative action time of the mutant enzyme is long, the yield of the product D-psicose is remarkably increased by applying the genetically engineered bacterium containing the modified enzyme for biotransformation, the product yield is increased by 4.3%, the equilibrium conversion rate reaches 33%, and the yield of the product is greatly increased. And a green, environment-friendly, sanitary and safe biological preparation method is provided for the preparation process of the D-psicose.

The invention relates to an aureobasidium pullulans strain for producing pullulan as well as a construction method and application of the aureobasidium pullulans strain, and belongs to the technical field of bioengineering. According to the aureobasidium pullulans strain constructed by the invention, alpha-phosphoglucose mutase PGM and uridine diphosphate glucose pyrophosphorylase UGP are continuously overexpressed, so that the yield of pullulan produced by fermentation of aureobasidium pullulans is remarkably improved, the fermentation titer of the strain is improved, the stability is improved, and the yield of pullulan produced by fermentation of aureobasidium pullulans is improved. An excellent strain is provided for preparing the pullulan by a microbial fermentation method, the separation cost in the downstream purification process of the pullulan is reduced, and an excellent strain is provided for industrial production of the pullulan.

The invention relates to a saccharomyces cerevisiae strain for efficiently synthesizing cordycepin as well as a construction method and application of the saccharomyces cerevisiae strain, and belongs to the technical field of microbial genetic engineering. The construction method provided by the invention comprises the following steps: carrying out multi-copy genome integrated expression on heterologous codon optimized 2 '-carbonyl-3'-deoxyadenosine reductase genes and 3 '-adenosine monophosphate phosphohydrolase genes, so as to increase the dosage of key enzymes; and supplementing the defective gene of the engineering strain to obtain the saccharomyces cerevisiae strain B5U3 with high yield of cordycepin. According to the obtained saccharomyces cerevisiae strain for efficiently synthesizing the cordycepin, the cordycepin yield reaches 2.92 g / L after shake flask fermentation, the cordycepin yield reaches 7.5 g / L after fermentation in a fermentation tank, and a foundation is laid for safer and more reliable industrial production of the cordycepin.

The invention discloses recombinant escherichia coli for producing D-psicose and a preparation method of the recombinant escherichia coli, and belongs to the field of genetic engineering. The technical problem to be solved in the invention is how to supply microorganisms for growth by using glycerol, acetic acid, fatty acid and the like as raw materials and prepare D-psicose by using glucose as a raw material. The invention provides recombinant Escherichia coli, which uses glycerol, acetic acid, fatty acid and the like as carbon sources to amplify (grow) the recombinant Escherichia coli, and uses D-glucose as a substrate to prepare D-psicose. According to the recombinant escherichia coli provided by the invention, glycerol, acetic acid, fatty acid and the like can be used as carbon sources for microbial growth, and D-psicose is synthesized by taking D-glucose as a raw material after the microbial growth reaches a certain biomass. Furthermore, a corresponding technical route for synthesizing the D-psicose is established.

The invention relates to the technical field of genetic engineering, and particularly discloses a genetic engineering bacterium for producing melanin as well as a construction method and application of the genetic engineering bacterium. According to the application, the yield of the melanin can be effectively increased by modifying a metabolic pathway of the genetically engineered bacterium, and the yield of the melanin reaches 6.2 g / L and 18.5 g / L respectively through shake-flask culture and 5L fermentation tank culture. Compared with the prior art, the yield of melanin produced by the genetically engineered bacterium is improved by 30% at the shake flask fermentation level, the fermentation yield of a fermentation tank is improved by 4.3 times, in addition, expensive tyrosine is changed into glucose as a fermentation precursor substance, the production cost is greatly reduced, and an efficient and feasible solution is provided for large-scale production of melanin.

The present disclosure concerns a fusion polypeptide for making acetyl-phosphate from glucose, a recombinant yeast host cell expressing the fusion polypeptide as well as methods of using the fusion polypeptide. The fusion polypeptide comprising (i) a glucose-6-phosphate isomerase moiety and (ii) a phosphoketolase moiety.

The invention belongs to the field of biological pharmacy and biotransformation, and particularly relates to a tagatose-4-epimerase mutant and application thereof. The tagatose-4-epimerase mutant disclosed by the invention is obtained by carrying out single mutation or combined mutation on an amino acid residue at a specific position of an amino acid sequence as shown in SEQ ID NO.1. Compared with wild type tagatose-4-epimerase, the mutant has the advantage that the catalytic activity is improved when the D-tagatose is prepared through conversion. The obtained tagatose-4-epimerase mutant has the advantages of being mild in reaction condition, green, environmentally friendly, single in product and easy to separate and purify, and the production cost is greatly reduced.

The invention belongs to the technical field of chondroitin sulfate biosynthesis, and discloses a recombinant strain for high-yield chondroitin sulfate, application of the recombinant strain and a preparation method of the chondroitin sulfate. The recombinant strain provided by the invention can be used for simultaneously expressing UDP-glucosamine isomerase, UDP-glucose dehydrogenase, chondroitin polymerase, glucosamine synthetase, phosphoglucosamine mutase, UDP-N-acetylglucosamine pyrophosphorylase, glucose-6-phosphate isomerase and glucose phosphate mutase; according to the present invention, glucose-1-phosphate uridyl transferase, ATP sulfonase, sulfotransferase, pyruvate kinase and APS kinase are adopted, and the enzyme molecules are matched with the original carbon metabolism pathway in the recombinant strain cell to jointly form the enzyme-linked pathway having the efficient CSA synthesis ability, such that the high-yield and high-purity preparation of the CSA can be achieved; the method has the advantages of simple operation, high biological safety of the obtained product, easy realization of large-scale production and the like, and has a good application prospect.

The present disclosure provides a recombinant yeast host cell having a plurality of native and / or heterologous enzymes that function in an engineered metabolic pathway to convert fructose-6-phosphate to acetyl-coenzyme A. The recombinant yeast host cell can be used, for example, in a process for making acetyl-coenzyme A, acetone and / or isopropanol. The plurality of native and / or heterologous enzymes is activated, upregulated, or overexpressed. The plurality of native and / or heterologous enzymes comprises a phosphoketolase; and optionally an acetate kinase; and / or a phosphotransacetylase. The recombinant yeast host cell comprises at least one of: at least two copies of a heterologous nucleic acid molecule encoding the phosphoketolase; a native and / or heterologous enzyme that functions in an engineered non-oxidative pentose phosphate pathway, wherein the native and / or heterologous enzyme is activated, upregulated, or overexpressed; and / or a native and / or heterologous protein that functions in an engineered metabolic pathway to convert pantothenate into acetyl-coenzyme A, wherein the native and / or heterologous protein is activated, upregulated or overexpressed and comprises at least one of: FEN2 or CAB1.

The invention discloses production of terpenoids and a strain, and belongs to the technical field of bioengineering. The serratia marcescens engineering bacteria for producing the semi-terpenoids or monoterpenoids are constructed, and can be used for producing linalool, isoprene, isopentenol, 1, 3, 4-triterpenes and 1, 3, 4-triterpenes. The composition is prepared from 1, 8-cineole, alpha-pinene, pinene, gamma-terpinene, geraniol, (+)-limonene, (-)-limonene, myrcene, beta-ocimene, sabinene, (-)-alpha-bisabolol, (-)-alpha-bisabolol, longifolene, wallenenes, beta-elemene, farnesene, patchouli alcohol, acyclic terpene and alpha-santalene. In a 30L fermentation tank, the yield of linalool produced by the serratia engineering bacteria is 40.72 g.L <-1 >.

The invention belongs to the field of biological pharmacy and biological conversion, and particularly relates to a tagatose-4-epimerase mutant and application of the tagatose-4-epimerase mutant in preparation of D-tagatose. The tagatose-4-epimerase mutant disclosed by the invention is obtained by carrying out single mutation or combined mutation on an amino acid residue at a specific position of an amino acid sequence as shown in SEQ ID NO.1. Compared with wild type tagatose-4-epimerase, the mutant has the advantage that the catalytic activity is improved when the D-tagatose is prepared through conversion. The obtained tagatose-4-epimerase mutant has the advantages of being mild in reaction condition, green, environmentally friendly, single in product and easy to separate and purify, and the production cost is greatly reduced.

The invention discloses a modified artificial'two-step 'terpene production way and high-yield rimane type terpenoids by using escherichia coli, hydrolase and drimenol synthase are subjected to fusion expression for the first time, and the yield is increased by 7.3 times. The phosphorylation efficiency is further improved by 3.6 times by combining a two-step method path PhoN with rational design, finally, the yield of drimenol is improved by 27 times by optimizing fermentation conditions and is improved to 398.4 mg / L from 15 mg / L, a high-yield system is successfully migrated to production of albicanol, and the yield of 3.5 g / L is obtained.

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 relates to the technical field of microbial genetic engineering, in particular to a method for improving cordycepin synthesized by saccharomyces cerevisiae and saccharomyces cerevisiae engineering bacteria. According to the invention, codon-optimized 2 '-carbonyl-3'-deoxyadenosine reductase gene and 3 '-adenosine monophosphate phosphohydrolase gene from cordyceps militaris are expressed by using plasmids, and then the plasmids are transformed into host cells to obtain the saccharomyces cerevisiae engineering bacteria. And inoculating a seed solution obtained by seed culture of the activated saccharomyces cerevisiae engineering bacteria into a fermentation culture medium containing a metabolic effector, and carrying out fermentation culture to synthesize cordycepin. Metabolic effectors (Cu < 2 + >, Fe < 2 + >, Mg < 2 + >, Zn < 2 + >, citric acid, cysteine, aspartic acid, glycine, VB1 and tea polyphenol) are added into a fermentation culture medium strain, and the variety and concentration of the metabolic effectors are optimized, so that the yield and synthesis efficiency of cordycepin are remarkably improved. By using the combination of the strain obtained by the invention and the optimal effector, the yield of cordycepin in a fermentation tank reaches 2.9 g / L.

The invention provides a preparation method of immobilized D-psicose-3-epimerase, which comprises the following steps: firstly aminating inorganic silicon nanoflowers, then activating by using a cross-linking agent glutaraldehyde, finally adding enzyme liquid, reacting in a shaking table for a period of time, and centrifuging and washing the reactant to obtain the product, namely the immobilized D-psicose-3-epimerase. The enzymatic activity and the stability of the immobilized enzyme are obviously improved, the specific enzyme activity of the immobilized enzyme is 446.44-1033.09 U / g protein, the activity of the immobilized enzyme is 163.09% and 181.53% of that of free enzyme after the immobilized enzyme is incubated for 6 h under the condition that the temperature is 30 DEG C or the pH value is 5, 63.18-70.78% of the initial activity is still reserved after the immobilized enzyme is repeatedly used for 10 times, and 46.27-54.38% of the initial activity is still reserved after the immobilized enzyme is stored for 15 days. The immobilized enzyme can catalyze fructose to generate psicose, and a green and efficient preparation technology is provided for production of psicose.

The invention discloses a genetically engineered bacterium for producing uridine diphosphate glucose as well as a construction method and application of the genetically engineered bacterium, and belongs to the technical field of genetic engineering. The following genes are overexpressed in the genetically engineered bacterium: a pyrimidine nucleoside operon gene, a carbamyl phosphate synthetase gene, an aspartate carbamyl transaminase gene, an orotic acid phosphoribose transferase gene, a phosphoribose pyrophosphate synthase gene, a uridylate kinase gene and a glucophosphate mutase gene. A uridine triphosphate glucose-1-phosphate uridine group transferase gene; the genetically engineered bacterium does not express the following genes: a purine nucleotide repressor protein gene, a deoxyribonucleoside triphosphate reductase gene, a nucleoside triphosphate pyrophosphohydrolase gene, a cytidine triphosphate synthase gene, a pyrimidine nucleotidase gene, a nucleotidase gene, an ornithine carbamoyltransferase gene, a UDPG dehydrogenase gene and a lacI repressor protein gene. The genetically engineered bacterium can be used for efficiently synthesizing UDPG by taking glucose as a substrate.

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 discloses a Sarcabomyces lipolytica strain for synthesizing (2S)-naringenin and application of the Sarcabomyces lipolytica strain, and belongs to the technical field of engineering and bioengineering. According to the invention, a key enzyme CHS is subjected to semi-rational transformation, so that the yield of the strain (2S)-naringenin expressing the mutant V196A / T132C is increased by 60%. According to the method, the modified CHS is further integrated into a yeast genome, conversion of p-coumaric acid to (2S)-naringenin is promoted by strengthening a shikimic acid pathway and enhancing supply of cofactors, so that the yield of the (2S)-naringenin reaches 776mg / L after the constructed strain is fermented in a 24-pore plate for 96 hours, and the yield of the constructed strain can reach 8.65 g / L after the strain is fermented in a 5L fermentation tank; the method lays a foundation for the subsequent biosynthesis of flavonoid compounds, and has potential value and significance for the development of synthetic biology.

The invention discloses a Yarrowia lipolytica strain for synthesizing delta-tocotrienol and application of the Yarrowia lipolytica strain, and belongs to the technical field of genetic engineering and bioengineering. According to the invention, a metabolic pathway of the delta-tocotrienol is constructed in the Yarrowia lipolytica, a mutant of a key enzyme, i.e., the uromelanic acid phytase, is screened, and the yield of the delta-tocotrienol is further increased by strengthening a shikimic acid pathway. The yield of delta-tocotrienol of the constructed engineering strain can reach 466.8 mg / L, a foundation is laid for subsequent biosynthesis of vitamin E compounds, and potential value and significance are achieved for development of synthetic biology.

The invention discloses a methanophilic genetically engineered bacterium for producing trehalose as well as a construction method and application of the methanophilic genetically engineered bacterium, and belongs to the technical field of industrial biology. The methanophilic genetically engineered bacterium capable of producing the trehalose is obtained by taking methanophilic bacteria methanophilic 5GB1, Alkalicicus glycogenes WONF2802 or methanophilic bacteria NG09 as host bacteria, introducing maltooligosyl trehalose synthase (MTS) into the host bacteria, and carrying out over-expression on maltooligosyl trehalose hydrolase (MTH), so as to obtain the methanophilic genetically engineered bacterium capable of producing the trehalose. Under the condition that methane or methanol serves as a carbon source, the yield of the produced trehalose can reach the gram level or above, a new low-carbon biosynthesis way can be provided for commercial production of the trehalose, and the methanophilic genetically engineered bacterium for producing the trehalose has a good industrial application prospect.

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 present application relates to ketol reductoisomerase variants, polynucleotides encoding the variants of the present disclosure, L-valine-producing microorganisms comprising the ketol reductoisomerase variants of the present disclosure or the polynucleotides encoding the variants, and methods of producing L-valine using the microorganisms of the present disclosure.

The present invention relates to a modified bacterium with anti-tumor activity, which comprises an essential gene expression cassette under the control of a strictly hypoxia inducible promoter and is deficient in at least one gene involved in or regulating the endogenous anti-oxidative stress response pathway or a functional expression product thereof. The present invention also relates to a pharmaceutical composition comprising the modified bacterium and anti-tumor use thereof.

The invention discloses L-arabinose isomerase, engineering bacteria and application, the amino acid sequence of the L-arabinose isomerase is shown as SEQ ID NO: 3, and the gene sequence for coding the L-arabinose isomerase is shown as SEQ ID NO: 2. On the basis of a genome of a Peribacillussp.S4 strain, a gene for coding the L-arabinose isomerase is excavated, and a stable prokaryotic expression system is constructed by performing codon optimization on the gene, so that the large-scale controllable production of the L-arabinose isomerase is realized, the yield of the enzyme is remarkably increased, the production cost of the enzyme is reduced, and the method is suitable for industrial production. A stable and economical enzyme source is provided for the production of D-tagatose; the L-arabinose isomerase expressed by the constructed engineering bacteria can significantly improve the yield of D-tagatose, and industrialization of D-tagatose is facilitated.

The invention particularly relates to a saccharomyces cerevisiae genetically engineered bacterium for de novo synthesis of 7-DHC as well as a construction method and application of the saccharomyces cerevisiae genetically engineered bacterium and belongs to the technical field of biology. According to the invention, a saccharomyces cerevisiae engineering bacterium SC9 is taken as an original strain, multiple times of multiple copies of tHMG1, DHCR24 and ERG1 are carried out on TY transposon, multiple copies of ERG2, POS5, ERG7, ERG11, IDI1 and ERG3 are carried out on TY transposon, and POS5, ERG3 and DHCR24 are singly copied on a genome, so that the content of intracellular reducing coenzyme II is increased, and a main path for synthesizing 7-DHC is enhanced. The genome single copy ACL, ACS1, ERG10, ERG19 and the like are used for enhancing the utilization of saccharomyces cerevisiae ethanol and dynamically regulating and controlling the synthesis of 7-DHC. The saccharomyces cerevisiae gene engineering strain constructed by the invention has high product accumulation, and lays a foundation for replacing a traditional multi-step chemical synthesis route and realizing industrial production.

The invention belongs to the field of biosynthesis, and in particular relates to LBD (Levandanenediol Pyrophosphate) synthase and engineered yeast for producing sclareol. In order to solve the problem that in the prior art, the sclareol biosynthesis yield is generally low, LSLPPs mutant containing at least one mutation of D730L, D374L, N674L or G379M is obtained by performing mutation and optimization on LSLPPs pyrophosphate synthase, and the mutant is used for sclareol synthesis. Meanwhile, in order to solve the problem of genetic instability caused by free plasmid expression of part of saccharomyces cerevisiae strains, a sclareol synthesis pathway is constructed in a yeast genome, and meanwhile, an acetyl coenzyme A synthesis pathway, an MVA pathway and a sclareol synthesis pathway are enhanced; chassis bacteria for synthesizing sclareol are modified in a manner of knocking out part of transcription inhibition factors, and the yield of a 5L fermentation tank reaches 26.11 g / L and is greatly improved compared with the prior art.