488 results about "Isomerase" patented technology

The invention belongs to the technical field of biology, and relates to an escherichia coli engineering strain for producing L-isoleucine through a methyl malic acid way as well as a construction method and application of the escherichia coli engineering strain. According to the invention, Escherichia coli BW25113 is used as a chassis strain, and a methyl malic acid synthase high-activity mutant gene cimA3.7, an isopropyl malic acid isomerase gene GsleuCD, a 3-isopropyl malic acid dehydrogenase gene AfleuB and a leucine dehydrogenase gene Lsleudh are over-expressed; a methyl malic acid absorption protein gene is over-expressed, and a branched chain amino acid transport system is modified to improve the exosome of the L-isoleucine; all methyl malic acid pathway related genes are integrated to a genome of a chassis strain, and a plasmid-free L-isoleucine production strain is constructed; the copy number of a key gene on a genome is optimized, and the yield of the engineering strain L-isoleucine is increased by introducing a non-oxidative glycolysis approach. The yield of the L-isoleucine after the engineering strain is fermented for 34 hours in a fermentation tank is 56.6 g / L.

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 provides a keto-alcohol acid reductoisomerase mutant and application of the keto-alcohol acid reductoisomerase mutant in construction of genetically engineered bacteria for high yield of D-pantothenic acid. In order to solve the technical problem that in the prior art, the yield of D-pantothenic acid synthesized through microbial fermentation is not high, semi-rational design of enzyme is adopted, and a keto-alcohol acid reductoisomerase mutant with substrate specificity is obtained and applied to construction of genetically engineered bacteria for high yield of D-pantothenic acid. According to the invention, a genetically engineered bacterium with high yield of D-pantothenic acid is constructed by utilizing the keto-alcohol acid reductoisomerase mutant through a CRISPR-Cas9 gene editing technology, so that the yield of D-pantothenic acid is increased by about 12.04%, and compared with an original strain, the valine accumulation amount in obtained fermentation liquor is almost unchanged compared with that of a control, and the yield of D-pantothenic acid is greatly improved. However, the accumulation of branched chain amino acid isoleucine is less, so that the modification of the substrate specificity of the keto-alcohol acid reductoisomerase is achieved.

The invention discloses an inositol dehydrogenase mutant and a preparation method of D-chiral inositol, and belongs to the technical field of genetic engineering. The inositol dehydrogenase mutant is obtained by mutating an amino acid sequence as shown in SEQ ID NO.2 through the following mutations: the 200th amino acid is mutated into C from V, the 234th amino acid is mutated into Q from V, and the 256th amino acid is mutated into E from R. According to the invention, the amino acid sequence of wild type inositol dehydrogenase is mutated to obtain the inositol dehydrogenase mutant which is a high-activity mutant capable of catalyzing conversion of myo-inositol into 2-keto-myo-inositol; therefore, when the inositol dehydrogenase mutant and keto isomerase act together to prepare D-chiral inositol by taking myo-inositol as a substrate, the yield of the D-chiral inositol is effectively improved.

The specification relates to conjugates comprising one or more Dc and pharmaceutically acceptable salts thereof. The specification also relates the use of the conjugates for the treatment of diseases such as cancer, and intermediates useful for the synthesis of the conjugates.

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 keto-alcohol acid reductoisomerase mutant and application of the keto-alcohol acid reductoisomerase mutant in construction of genetically engineered bacteria for high yield of D-pantothenic acid. In order to solve the technical problem that in the prior art, the yield of D-pantothenic acid synthesized through microbial fermentation is not high, semi-rational design of enzyme is adopted, and a keto-alcohol acid reductoisomerase mutant with substrate specificity is obtained and applied to construction of genetically engineered bacteria for high yield of D-pantothenic acid. According to the invention, a genetically engineered bacterium with high yield of D-pantothenic acid is constructed by utilizing the keto-alcohol acid reductoisomerase mutant through a CRISPR-Cas9 gene editing technology, so that the yield of D-pantothenic acid is increased by about 12.04%, and compared with an original strain, the valine accumulation amount in obtained fermentation liquor is almost unchanged compared with that of a control, and the yield of D-pantothenic acid is greatly improved. However, the accumulation of branched chain amino acid isoleucine is less, so that the modification of the substrate specificity of the keto-alcohol acid reductoisomerase is achieved.

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 discloses an inositol dehydrogenase mutant as well as a preparation method and application thereof, and belongs to the technical field of gene engineering. The inositol dehydrogenase mutant is obtained by mutating an amino acid sequence as shown in SEQ ID NO.1, and the mutation is that the 247th amino acid is mutated from K to R, or the 266th amino acid is mutated from E to D. The inositol dehydrogenase mutant disclosed by the invention has higher enzyme activity, and when the inositol dehydrogenase mutant and ketoisomerase are catalyzed to synthesize D-chiral inositol by taking myo-inositol and NADP < + > as substrates, the reaction is more inclined to a forward reaction, so that the yield of the D-chiral inositol and the conversion rate of the substrate myo-inositol are further improved.

The invention relates to the technical field of synthetic biology, metabolic engineering and cell culture. The present invention provides a cell for the production of a negatively charged, preferably sialylated, oligosaccharide wherein the cell is genetically engineered to have or express, preferably overexpress, a hydrolytic UDP-N-acetyl-D-glucosamine-2-epimerase. The invention further provides the use of said cells in culture or incubation. Also described are methods of producing negatively charged, preferably sialylated oligosaccharides using the cells, and purification of the negatively charged, preferably sialylated oligosaccharides.

The invention discloses a genetically engineered bacterium for producing glucuronic acid as well as a construction method and application of the genetically engineered bacterium, and belongs to the technical field of genetic engineering. According to the genetically engineered bacterium for producing glucuronic acid, an inositol oxidase gene MMIOX is integrated on a site of a uronic acid isomerase gene uxaC. According to the invention, an inositol oxidase gene MMIOX is integrated on a genome of a genetically engineered bacterium for producing glucuronic acid, and a uronic acid isomerase gene uxaC is knocked out at the same time, so that the genetically engineered bacterium can efficiently catalyze inositol reaction to generate glucuronic acid through the integrated inositol oxidase gene MMIOX; the glucuronic acid isomerase gene uxaC is knocked out, so that isomerization reaction of glucuronic acid can be avoided, and the yield of glucuronic acid is further increased.

The invention belongs to the field of gene engineering, and discloses a sucrose isomerase mutant, a high-yield isomaltulose engineering bacterium and a construction method and application thereof. According to the sucrose isomerase mutant obtained through rational design of an enzyme molecular structure, the enzyme activity and the isomaltulose conversion rate of the sucrose isomerase mutant are remarkably improved, compared with a wild enzyme, the specific enzyme activity of the sucrose isomerase mutant is improved by 2.29 times, the isomaltulose conversion rate is improved to 98.9% from 91.1%, the impurity content is lower, and therefore the sucrose isomerase mutant is more suitable for synthesis of isomaltulose. A recombinant engineering bacterium of the high-yield isomaltulose engineering bacterium is further constructed; the recombinant engineering bacterium capable of efficiently expressing the sucrose isomerase is constructed by utilizing ubiquitin-like protein modified molecular genes to be fused with sucrose isomerase mutant coding genes, the enzyme activity of the prepared sucrose isomerase can reach 40.5 U / mg wet cells, and the recombinant engineering bacterium has a great application prospect in industrial production.

The invention discloses L-rhamnose isomerase and application thereof, and belongs to the technical field of genetic engineering. The amino acid sequence of the L-rhamnose isomerase is obtained by performing the following mutation on an amino acid sequence as shown in SEQ ID NO.1: the 258th amino acid is mutated from D to Q. Compared with the prior art, the L-rhamnose isomerase mutant D258Q is obtained by mutating wild type L-rhamnose isomerase, and compared with the wild type L-rhamnose isomerase, the L-rhamnose isomerase mutant D258Q is higher in enzyme activity and longer in half-life period, so that when the L-rhamnose isomerase mutant D258Q is used for catalyzing the conversion of D-psicose into D-allose, the L-rhamnose isomerase mutant D258Q has the advantages that the enzyme activity is higher, and the half-life period is longer; the catalytic capability is stronger and more durable, and the conversion efficiency of the reaction and the yield of D-allose are improved.

The invention relates to the technical field of gene engineering, in particular to an L-rhamnose isomerase mutant, a recombinant plasmid, a mutant strain and application. The amino acid sequence of the mutant is as shown in SEQ ID NO.4, and the nucleotide sequence of the coding gene is as shown in SEQ ID NO.3. A specific mutant gene sequence is designed, a recombinant plasmid pET28a (+)-L-Rhi is successfully constructed, the gene sequence is connected between enzyme cutting sites BamH I and Hind III of an escherichia coli expression vector pET-28a (+) to construct a mutant expression vector (the recombinant plasmid pET-28a (+)-L-Rhi), the mutant expression vector is transformed into an escherichia coli BL21 competent cell, a mutant strain is obtained, and the recombinant plasmid pET-28a (+)-L-Rhi is obtained. The obtained mutant strain can overexpress L-rhamnose isomerase, the conversion rate of D-allose is remarkably improved, the thermal stability of the enzyme is remarkably improved through the design of mutation sites, and the half-life period is effectively prolonged.

The present disclosure relates to recombinant microorganisms, such as bacteria, e.g., E. coli, engineered to have improved flux and / or yield of the DXP pathway. Cells can be engineered to comprise nucleotide sequences comprising dxs polypeptides (EC 2.2.1.7); dxr polypeptides (EC 1.1.1.267); ispD polypeptides (EC 2.7.7.60); ispE polypeptides (EC 2.7.1.148); ispF polypeptides (EC 4.6.1.12); ispG polypeptides (EC 1.17.7.1 or EC 1.17.7.3); ispH polypeptides (EC 1.17.7.4); and idi polypeptides (EC 5.3.3.2). Cells can be further engineered to express redox polypeptides, e.g., ferredoxins, flavodoxins, and / or flavodoxin / ferredoxin--NADP reductases (EC 1.19.1.1 or EC 1.18.1.2), e.g., to increase the activity of the ispG and / or ispH polypeptides. Some or all of the nucleotide sequences can be heterologous to the cells. The recombinant microorganisms can be used in methods of isoprenoid production.

The invention belongs to the field of biological pharmacy and biotransformation, and particularly relates to a tagatose-4-epimerase mutant as well as a coding gene 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 relates to the technical field of gene engineering, in particular to an L-rhamnose isomerase mutant, a recombinant plasmid, a mutant strain and application. The amino acid sequence of the mutant is as shown in SEQ ID NO. 2, and the nucleotide sequence of the coding gene is as shown in SEQ ID NO. 1. A specific mutant gene sequence is designed, a recombinant plasmid pET28a (+)-L-Rhi is successfully constructed, the gene sequence is connected between enzyme cutting sites BamH I and Hind III of an escherichia coli expression vector pET-28a (+) to construct a mutant expression vector (the recombinant plasmid pET-28a (+)-L-Rhi), the mutant expression vector is transformed into an escherichia coli BL21 competent cell, a mutant strain is obtained, and the recombinant plasmid pET-28a (+)-L-Rhi is obtained. The obtained mutant strain can overexpress L-rhamnose isomerase, the conversion rate of D-allose is remarkably improved, the thermal stability of the enzyme is remarkably improved through the design of mutation sites, and the half-life period is effectively prolonged.

The invention discloses a keto isomerase mutant as well as a preparation method and application thereof, and relates to the technical field of gene engineering. The keto isomerase mutant is obtained by performing the following mutations on an amino acid sequence shown as SEQ ID NO.2: the 34th amino acid is mutated from N to R; meanwhile, the 188th amino acid is mutated from I to K. The half-life period of the keto isomerase mutant obtained by mutating the amino acid sequence shown in SEQ ID NO.2 is prolonged, and the enzyme activity is improved, so that the yield of D-chiro-inositol is further improved.

The disclosure provides method of treating cancer in a patient in need thereof, comprising safely and efficaciously administering to the patient PLX038, a long lasting-PEGylated prodrug of the topoisomerase I inhibitor. The disclosure further provides combination therapies of PLX038 with inhibitors of the DNA damage response (DDR).

The invention discloses a production method of fucosylated oligosaccharide. The core of the method is as follows: the GDP-D-rhamnose is converted into the GDP-L-fucose by using the GDP-D-rhamnose-3, 5-epimerase; the GDP-D-rhamnose is obtained by converting the GDP-D-mannose-4, 6-dehydratase and the GDP-4-keto-6-deoxy D-mannose reductase by taking the GDP-D-mannose as a substrate, and the GDP-D-rhamnose is obtained by taking the GDP-D-mannose as a substrate. The invention relates to a method for producing a fucosylated oligosaccharide, which is characterized in that a GDP-D-mannose-4, 6-dehydratase, a GDP-4-keto-6-deoxyD-mannose reductase, a GDP-D-rhamnose-3, 5-epimerase and a fucosyltransferase are expressed in a genetically modified host cell, and the fucosylated oligosaccharide can be produced by the way mentioned above.

The application belongs to the technical field of genetic engineering, and discloses a kind of saccharomyces cerevisiae for producing rare ginsenoside by seaweed biomass and a construction method and application thereof.The saccharomyces cerevisiae has the following characteristics: overexpression of agarase, neojuncanohydrolase, hydroxymethylglutaryl coenzyme A reductase, isopentenyl diphosphate delta isomerase, dammaradienol synthase, protopanaxadiol synthase, cytochrome P450 reductase, protopanaxatriol synthase and glycosyltransferase.The application combines enzymolysis of seaweed biomass with fermentation of rare ginsenoside, which not only endows wild-type yeast with the ability to degrade seaweed biomass that it originally does not have, but also effectively improves the yield of squalene and downstream terpenes (rare ginsenoside Rh1) by overexpression of tHMG1 and IDI1.The saccharomyces cerevisiae can effectively utilize seaweed biomass to obtain squalene and rare ginsenoside Rh1, and has the characteristics of simplicity, economy and ecological friendliness, can convert cheap biomass into high-value products, and provides a way for the development of marine bioeconomy.

The application provides a recombinant Aspergillus niger with high trans-aconitic acid yield and a construction method and application thereof, and relates to the technical field of genetic engineering. Specifically, the recombinant Aspergillus niger heterogeneously expresses aconitate isomerase TbrA and / or Adi1. The application provides a new engineering strain for efficient production of trans-aconitic acid, solves the source predicament of needing plant extraction or needing chemical synthesis in traditional production of trans-aconitic acid, and greatly improves the yield of trans-aconitic acid.

Described is an antibody-drug conjugate (ADC) having the formula A-L, wherein A is an antibody or an antibody fragment and wherein L is a linker, said linker comprising: as a first payload a topoisomerase I inhibitor which is cell-permeable, e.g., a camptothecin cytotoxic molecule which is cell-permeable; as a second payload a topoisomerase I inhibitor which is not cell-permeable, e..g., a camptothecin cytotoxic molecule which is not cell-permeable; and as a as a third payload a toxin or a cytotoxin, e.g., an auristatin such asMMAE (Monomethyl auristatin E). Moreover, described is a pharmaceutical composition comprising said ADC and at least one pharmaceutically acceptable ingredient. Further, described method of treating a patient suffering from, being at risk of developing, and / or being diagnosed for a neoplastic disease.

The invention belongs to the technical field of gene engineering and protein engineering, and particularly relates to thermal-stable D-psicose 3-epimerase as well as a preparation method and application thereof. According to the invention, a series of D-psicose 3-epimerase DAE mutants are constructed, and six mutants which not only can retain good catalytic activity, but also have high thermal stability are obtained through screening. The catalytic temperature of the three mutants A19D / A223P / V241I is increased by 25 DEG C compared with that of a wild type, while the three mutants A19D / A223P / V241I still show the optimal thermal stability, the catalytic activity of the three mutants is improved by 17.0%, and the three mutants show good industrial suitability. The D-psicose 3-epimerase DAE mutant with improved thermal stability provided by the invention is beneficial to improving the production efficiency of the DAE in industrial application of D-psicose, and provides powerful industrial enzyme guarantee for large-scale and low-cost preparation of D-psicose.

The invention discloses an escherichia coli recombinant bacterium for whole-cell catalytic synthesis of rebaudioside M as well as a construction method and application of the escherichia coli recombinant bacterium. The Escherichia coli recombinant bacterium for the whole-cell catalytic synthesis of the rebaudioside M, which is constructed by the invention, contains a glycosyl transferase UGT76G1 coding gene mutant, a glycosyl transferase UGT91C1 coding gene mutant, a sucrose synthase atSuS1 gene and a polyphosphoglucokinase cgPPGK gene; a glucose-1-phosphate uridine transferase galU gene, a nucleoside diphosphate kinase ndk gene and a nucleoside diphosphate kinase ndk gene are over-expressed; meanwhile, an encoding gene araA gene of L-arabinose isomerase and an encoding gene ushA gene of 5 '-nucleotidase / UDG hydrolase are knocked out. According to the recombinant escherichia coli constructed by the invention, Reb D is prevented from being directly used as a synthetic substrate, so that the raw materials are easier to obtain, and the conversion rate of the substrate and the synthetic rate and yield of rebaudioside M are remarkably improved.

The invention belongs to the field of metabolic engineering, and discloses a genetically engineered bacterium for producing myo-inositol as well as a construction method and application of the genetically engineered bacterium. The genetically engineered bacterium takes escherichia coli as an original strain, and is obtained by performing the following gene editing on a genome of the escherichia coli: non-expressed lactose operon repressor protein, glucose phosphate dehydrogenase, acetokinase, glucose phosphate isomerase and glycerol repressor protein; and overexpressing inositol-1-phosphate synthase, inositol monophosphate, glucokinase, glucose permease and glycerol kinase. Compared with the prior art, the genetically engineered bacterium disclosed by the invention has remarkable advantages in the aspects of key enzyme expression rate, metabolism specificity, fermentation period and the like, and an efficient, stable, economical and feasible solution is provided for industrial production of myo-inositol.

The present invention discloses a class of rutaecarpine derivatives or their pharmaceutically acceptable salts with multi-target anti-tumor activity, and the general structural formula is shown as Formula I or Formula II: #imgabs0# Wherein: R1 is selected from C1-C5 alkoxy, hydroxyl, hydrogen; R2 is selected from hydrogen, halogen; R3 is selected from hydroxyl, #imgabs1# Through enzyme inhibition activity and in vitro anti-tumor activity tests, it was found that the compounds of the present invention have strong inhibitory activities against topoisomerase 1, topoisomerase 2 and c-MYC oncogene, opening up a new way and providing a new strategy for in-depth research and development of anti-tumor drugs with new structural types.

The invention discloses an enzyme preparation for catalytic synthesis of D-chiro-inositol and a preparation method of D-chiro-inositol, and belongs to the technical field of genetic engineering. The enzyme preparation for catalytic synthesis of D-chiral inositol has enzyme activity for catalyzing the reaction of muscle inositol and NADP < + > to generate D-chiral inositol, and comprises inositol dehydrogenase and keto isomerase, the amino acid sequence of the inositol dehydrogenase is shown as any one of SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5, and the amino acid sequence of the keto isomerase is shown as any one of SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8. The invention further discloses a preparation method of the enzyme preparation for catalytic synthesis of D-chiral inositol. The inositol dehydrogenase and the keto isomerase in the enzyme preparation are mutated on the basis of wild types, so that the enzyme activity of the inositol dehydrogenase and the keto isomerase is improved, and the conversion rate of D-chiral inositol is further improved.

The invention discloses a saccharomyces cerevisiae strain for de novo synthesis of fucoxanthine precursor neoxanthine and application of the saccharomyces cerevisiae strain, and belongs to the technical field of biology. According to the recombinant saccharomyces cerevisiae disclosed by the invention, the 3-hydroxy-3-methylglutaryl coenzyme A reductase, the isopentenyl pyrophosphate isomerase, the farnesyl pyrophosphate synthase and the endoplasmic reticulum size regulation factor are expressed in an enhanced manner; according to the method, geranyl geranyl diphosphate synthase, phytoene dehydrogenase, 15-cis-phytoene synthase, bifunctional lycopene cyclase / phytoene synthase, beta-carotene hydroxylase, zeaxanthin epoxidase truncated mutant and violaxanthin deep oxidase-like protein are subjected to heterologous expression, so that the zeaxanthin deep oxidase-like protein is obtained; the ROX1 and GAL80 genes in the saccharomyces cerevisiae are knocked out, and the expression of squalene synthase ERG9 is down-regulated. When the strain is subjected to shake flask fermentation, the contents of neoxanthine and violaxanthin of the strain respectively reach 1.78 mg / L and 26.77 mg / L, so that the strain has a wide application prospect.

The present invention relates to peptide conjugates of cytotoxins such as topoisomerase I inhibitors which are useful for the treatment of diseases such as cancer.