1377results about "Ligases" patented technology

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 includes a mutant T7 DNA ligase or a biologically active fragment thereof, which has greater activity than wild type T7 DNA ligase. The mutant T7 DNA ligase, or the biologically active fragment, has one or more substitutions differing from the wild type, as described more fully in the Summary. The preferred mutant T7 DNA ligase has at least one of the following mutations: E63K (SEQ ID NO:4), K73E (SEQ ID NO:6), K137E (SEQ ID NO:7), K174E (SEQ ID NO:9), E182K (SEQ ID NO:11), K210E (SEQ ID NO: 13), E243K (SEQ ID NO:15), D245R (SEQ ID NO: 17), E268K (SEQ ID NO:19), E272K (SEQ ID NO:21), E289K (SEQ ID NO:23), K295E (SEQ ID NO:25) and D336R (SEQ ID NO:27).

The invention relates to a recombinant pichia pastoris strain capable of producing astaxanthin at high yield as well as a construction method and application of the recombinant pichia pastoris strain. The recombinant pichia pastoris strain is obtained by expressing ATP (adenosine triphosphate) citrate lyase (ACL) and acetyl-CoA synthase (ACS) or phosphoketolase (PK) and phosphotransacetylase (PTA), choline kinase (CK), inositol polyphosphate kinase (IPK) and vitreoscilla hemoglobin (VHB) in host bacteria. Wherein the host bacterium is a pichia pastoris gene modified strain PP-LC2. The astaxanthin production performance of the recombinant strain is verified on the basis of comparison of precursor supply pathways, IUP pathway construction and improvement of the oxygen supply capacity of the engineering strain, and the astaxanthin production capacity of the pichia pastoris is further improved. The construction method of the recombinant pichia pastoris is simple, the synthesis of astaxanthin can be better promoted, the engineering strain can efficiently synthesize the astaxanthin by utilizing methanol through amplification fermentation of a 5L fermentation tank, and industrial production is facilitated.

The invention relates to the technical field of dye synthesis, and particularly discloses a genetically engineered bacterium for synthesizing N-acetyl blue as well as a construction method and application of the genetically engineered bacterium. The supply capacity of precursor substances glutamic acid and glutamine of the genetically engineered bacterium is high, the fermentation process is simple, the production intensity of the precursor substances glutamine and N-acetylglutamine is improved by cutting off a consumption path, relieving negative feedback inhibition and enhancing the expression level of key genes through the genetically engineered bacterium, and finally the yield of N-acetylblue reaches 41.2 g / L.

The present application relates to a method of producing a protein comprising an unnatural amino acid (UAA), the method comprising culturing a host cell, where the host cell is a eukaryotic cell, together with a nucleotide sequence encoding a first recoding tRNA or a first recoding tRNA, where the first recoding tRNA comprises an anticodon complementarily paired with a first codon, wherein the first codon is a rare codon; a nucleotide sequence encoding a first aminoacyl-tRNA synthetase or a first aminoacyl-tRNA synthetase, the first aminoacyl-tRNA synthetase being capable of loading a first recoding tRNA with a first non-natural amino acid.

The invention discloses a novel application of an RNF6 gene, namely, an application of screening a medicine for preventing and / or treating Zika virus infection, aiming at inhibiting expression of the RNF6 gene or knocking out the RNF6 gene. A series of functional experiments prove that the RNF6 gene promotes the replication of the Zika virus, and the purpose of reducing the replication of the Zika virus can be achieved by using an expression inhibitor of the RNF6 gene or knocking out the RNF6 gene; experimental results show that the RNF6 gene can be used as a potential target for preventing and / or treating Zika virus infection, and a new direction is provided for development of drugs for preventing and / or treating Zika virus infection targeting the RNF6 gene in the future.

The present disclosure relates, generally, to methods for producing, purifying, and using circular RNA from a prokaryotic system.

The invention specifically discloses a composition for detecting NAD < + > and application thereof, and relates to the technical field of biological detection. According to the composition for detecting NAD < + >, the composition comprises an NAD < + > binding protein fragment A connected with an Fc tag, an NAD < + > binding protein fragment B and a connecting peptide, the detection result is accurate and reliable, and meanwhile the technical difficulty, uncertainty and high cost faced by antibody screening in a traditional method are avoided.

Described herein are programmable DNA proteolytic target chimera complexes that can be used both for the direct treatment of cancer by inhibiting biochemical pathways that are overexpressed in cancer cells, and for the indirect treatment of cancer by recruiting the E3 ligase complex to engage with a protein of interest or a mutant thereof and initiating proteolysis. Also described herein are methods of using the complexes in the treatment of cancer, as well as compositions comprising the complexes.

The invention relates to an L-valine production strain as well as a construction method and application thereof, the strain is obtained by transformation through a metabolic synthesis path of L-valine, and the construction method comprises the following steps: by taking Cornebacterium glutamicum ATCC 13032 (C.glutamicum) as an original strain, carrying out editing transformation such as gene knockout, gene overexpression, gene heterologous expression, gene double-copy expression and the like on a genome of the original strain by utilizing a metabolic engineering means. The L-valine production strain disclosed by the invention can be used for synthesizing L-valine from the beginning by taking glucose as a carbon source, has the advantages of high production rate, short fermentation period, high strain stability and low cost, and lays a foundation for realizing large-scale production of L-valine. According to the fermentation tank fermentation method, the L-valine is produced in a mechanical stirring type fermentation tank by using the L-valine production strain Va1-09, 86.4 g / L of L-valine is produced by fermenting for 36 hours, and the method has a good industrial prospect.

The invention discloses a tomato LACS2 gene promoter and application thereof, and belongs to the technical field of biology. On one hand, the invention provides the tomato LACS2 gene promoter, and on the other hand, the invention provides application of the tomato LACS2 gene promoter in promoting target gene expression in plants. According to the invention, a tomato LACS2 gene promoter fragment (788bp) is constructed to drive an EGFP-GUS (Enhanced Green Fluorescent Protein-Glucuronidase) gene fusion expression vector. GUS histochemical staining and laser confocal microscopy observation prove that the promoter fragment can guide normal expression of the reporter gene in the tomato epidermis. Therefore, the LACS2 promoter can be used for researching development, quality improvement and postharvest preservation of tomato peels and specific expression of exogenous genes in the tomato peels.

The disclosure provides, for example, double stranded DNA (dsDNA) molecules comprising one or more chemically modified nucleobases. In some embodiments, the dsDNA molecule is circular and comprises a first strand and a second strand, wherein the first strand comprises one or more chemically modified nucleobases, and the second strand is free of chemically modified nucleobases. In some embodiments, the dsDNA molecule comprises a promoter sequence and an effector sequence that encodes an effector.

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.

Disclosed herein are methods of increasing nitrogen fixation in a non-leguminous plant. The methods can comprise exposing the plant to a plurality of bacteria. Each member of the plurality comprises one or more genetic variations introduced into one or more genes or non-coding polynucleotides of the bacteria's nitrogen fixation or assimilation genetic regulatory network, such that the bacteria are capable of fixing atmospheric nitrogen in the presence of exogenous nitrogen. The bacteria are not intergeneric microorganisms. Additionally, the bacteria, in planta, produce 1% or more of the fixed nitrogen in the plant.

Proposed are recombinant entomopathogenic bacteria transformed using promoter replacement technique, a preparation method, and uses thereof for producing pesticides and controlling pests. Provided are recombinant entomopathogenic bacteria in which the promoter of the GXP synthase gene (gxpS gene) is replaced in the entomopathogenic bacteria with an arabinose inducible promoter using a promoter replacement technique. The recombinant entomopathogenic bacteria prepared in the present disclosure can produce large quantities of GXPs, which acts to suppress immunity of pests, by controlling the expression of the GXP synthetase gene (gxpS gene) through an addition of an inducer. The recombinant entomopathogenic bacteria prepared in the present disclosure can also effectively control pests through a response that suppresses the immunity of pests.

The application relates to an engineering bacterium for high yield of observation blue and a construction method and application thereof, and belongs to the technical field of biological synthesis of natural dyes. The engineering bacterium for high yield of observation blue expresses icd, bpsA, glnA Y405F and gdhA; the engineering bacterium knocks out acnR, yggB, glsK, aceA and ldh. By knocking out the yggB and aceA genes, performing site-directed mutation (Y405F) on the glnA gene, replacing the glsK gene with the glnA Y405F gene, replacing the acnR gene with the icd gene, replacing the ldh gene with the gdhA gene, and integrating the bpsA gene, the application can block the formation of by-products such as lactate and succinic acid in the observation blue synthesis process, improve the flow direction of the citric acid->isocitric acid->alpha-ketoglutaric acid->glutamic acid->glutamine path, and then improve the intracellular glutamine concentration, so that the yield of observation blue is improved.

The invention belongs to the field of biosynthesis, and particularly relates to sclareol synthase and engineered yeast for producing sclareol. In order to solve the problem that in the prior art, sclareol biosynthesis yield is generally low, sclareol synthase is mutated and optimized, an SsScs mutant containing at least one mutation of Y491F, G307L or N269F is obtained, 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 21.13 g / L and is greatly improved compared with the prior art.

The invention belongs to the field of plant seed biology and rice breeding, and discloses an application of an OsBTB05 gene or an OsBTB05 protein in regulation and control of storage resistance of rice seeds and a related breeding method. In particular discloses application of an OsBTB05 gene or an OsBTB05 protein in regulation and control of the storability of rice seeds, the nucleotide sequence of the OsBTB05 gene is shown as SEQ ID NO: 1 or SEQ ID NO: 2, and the amino acid sequence of the OsBTB05 protein is shown as SEQ ID NO: 3. The invention also discloses a molecular marker-assisted selection method for breeding the high-storage-resistance rice variety and a method for reducing the storage resistance of the rice seeds. The invention proves that the OsBTB05 gene is a key factor for regulating and controlling the storability of the rice seeds, and the expression level of the OsBTB05 gene is in significant positive correlation with the storability of the seeds. A clear molecular target and a brand-new gene resource are provided for improving the seed storage resistance from a genetic source.

The invention relates to recombinant saccharomyces cerevisiae for de novo synthesis of mevalonic acid as well as a construction method and application of the recombinant saccharomyces cerevisiae, and belongs to the technical field of genetically engineered bacteria. The technical problem to be solved by the invention is to provide the recombinant saccharomyces cerevisiae for de novo synthesis of mevalonic acid. According to the recombinant saccharomyces cerevisiae, saccharomyces cerevisiae is used as a chassis strain, and ACL, CTP1, RtME and MDH3 are over-expressed. A series of recombinant saccharomyces cerevisiae strains with high yield of mevalonic acid are constructed through a gene recombination method, the strains can be fermented in a conventional culture medium to prepare mevalonic acid, the fermentation process is simple, the cost is low, the yield is high, and the maximum yield of mevalonic acid can reach 5692.45 mg / L. According to the invention, the recombinant saccharomyces cerevisiae strain is used as a chassis strain, and recombinant genetically engineered bacteria for producing the santalene, the santalol and the patchouli alcohol are respectively obtained through gene recombination, so that the yields of the santalene, the santalol and the patchouli alcohol produced by fermentation are improved.

The invention discloses a recombinant halomonas for producing polyhydroxyalkanoate as well as a construction method and application of the recombinant halomonas. The recombinant halomonas comprises an exogenous gene, the exogenous gene comprises a combination of an aldD gene, a dhaT gene and an orfz gene, and the recombinant halomonas weakens the expression of an endogenous prpc gene. According to the invention, a P34HB synthesis route is successfully constructed through a synthesis route of exogenous introduction from 1, 4-butanediol (BDO) to 4-hydroxybutyryl coenzyme A, the proportion of each monomer in the copolymer can be effectively regulated and controlled, especially after exogenous addition of short-chain fatty acids (such as formic acid, acetic acid, propionic acid, butyric acid and the like), the synthesis of copolymers such as PHB, PHBV, P34HB, P34HB3HV and the like can be further improved, and the synthesis efficiency of the copolymer is improved. And the proportion of different monomers is adjusted, so that the diversity and yield of the copolymer are enhanced.

The invention discloses a glutathione bifunctional synthetase mutant with improved catalytic activity and application, and belongs to the field of gene engineering and fermentation engineering. A series of mutants with improved enzyme activity are obtained by carrying out site-specific combinatorial mutation on a site 61, a site 136, a site 485, a site 498 and a site 722 of the glutathione bifunctional synthetase GshFst, the specific enzyme activity reaches 13.58 U.mg <-1 >-27.34 U.mg <-1 >, the half-life period at 37 DEG C reaches 195-320 min, and the enzyme activity and the thermal stability are remarkably improved compared with those of a wild type. The enzyme mutant provided by the invention has a wide application prospect in the production of glutathione and the construction of genetically engineered microorganisms for producing glutathione.

The invention belongs to the technical field of gene engineering, and particularly relates to a biotin ligase mutant as well as a coding gene, an expression vector, a recombinant cell and application thereof. The amino acid sequence of the biotin ligase mutant is as shown in SEQ ID NO. 2. The biotin ligase (Bar A) mutant provided by the invention can be efficiently expressed in eukaryotes, the expression level of the biotin ligase (Bar A) mutant is obviously improved in mammalian cells compared with a wild type, and the biotin ligase (Bar A) mutant has good catalytic activity after expression, can effectively catalyze biotinylation of protein containing a biotin receptor sequence, and has good application prospects. The method has a wide application prospect in the fields of biotinylated protein preparation, and protein labeling, positioning, immunodetection, affinity purification, function research and the like based on a biotin-avidin system (BAS).

The invention relates to the technical field of fish genetic breeding, in particular to a method for improving grass carp reovirus resistance of grass carp. Four specific knockout targets of the herc4 gene are disclosed, the grass carp herc4 gene is specifically knocked out by utilizing a CRISPR / Cas9 gene editing technology, and a herc4 gene knockout individual, namely the high-resistance grass carp reovirus fish, is obtained. According to the invention, the function of the herc4 in the grass carp is verified for the first time, a new grass carp germplasm with improved grass carp reovirus resistance is rapidly obtained by knocking out the single gene of the herc4, and the herc4 has important industrial value.

The purpose of the present invention is to provide a method for producing a methyl compound. Provided is a method for producing a methyl compound using formic acid.

The invention discloses a Yarrowia lipolytica strain for synthesizing ergothioneine and application of the Yarrowia lipolytica strain, and belongs to the technical field of genetic engineering and bioengineering. According to the invention, a key enzyme TrEGT1 is subjected to semi-rational transformation, so that the ergothioneine yield of a strain expressing a mutant is increased by 240.9%, precursor metabolic network screening is further modularized, precursor flux is enhanced, a competitive approach of a precursor is knocked out and weakened, and strategies such as fermentation process optimization and the like are further optimized; and when the constructed strain is fermented in a 5L bioreactor, the yield of ergothioneine can reach 10.3 g / L. The highest-level ergothioneine synthesis reported at present is realized, the feasibility of synthesizing the ergothioneine by using the Yarrowia lipolytica as the chassis cell is verified, and a reference value is provided for efficiently synthesizing the ergothioneine by using other chassis cells.

The present disclosure provides guided microbial remodeling (GMR) methods for the rational improvement of plant-associated microbes to perform plant-beneficial functions. The GMR methods described herein allow for non-intergeneric genetic optimization of key regulatory networks within the microbes, which improve plant-beneficial functions over wild-type microbes but don't have the risks associated with transgenic approaches (e.g., unpredictable gene function, public and regulatory concerns, etc.). The present disclosure also provides remodeled microbes and compositions thereof. The utilization of remodeled microbes and compositions thereof will enable farmers to realize more productive and predictable crop yields without the nutrient degradation, leaching, or toxic runoff associated with traditional synthetically derived fertilizers.

The invention provides an application of a rice OsRMT1 gene in improving the salt tolerance of rice, and belongs to the technical field of plant genetic engineering.According to the invention, the OsRMT1 gene in rice is obtained through a designed primer, and by constructing an overexpression positive material and a knockout mutant material of OsRMT1, it is proved that OsRMT1 can negatively regulate the salt tolerance of rice, and the OsRMT1 gene can be used for improving the salt tolerance of rice. The method can be used for cultivating and screening salt-tolerant rice varieties. The OsRMT1 gene in the rice is knocked out, so that the content of OsMBL1 in the rice can be increased, and the salt tolerance of the rice is improved by increasing the content of OsMBL1.

The present invention belongs to the field of genetic engineering technology and discloses a plasmid-free genetically engineered bacterium that can efficiently synthesize γ-aminobutyric acid from scratch using a cheap carbon source as a substrate, a method and an application thereof. The engineered bacterium is based on wild-type E. coli MG1655, overexpresses the T7 RNA polymerase gene; deletes the genes gabT and puuE; overexpresses gadC and gad bm , gdh, gltA, pyc, and ppc genes; and a growth-coupled promoter was used to dynamically regulate the expression of the sucA and argA genes in the GABA production pathway in Escherichia coli. All genetic manipulations in this engineered bacterium were performed within the genome, leaving no plasmid residues and requiring no antibiotics or inducers. The resulting strain exhibited stable production performance and a simple fermentation process. Using the engineered bacterium under a staged pH control process for 38 hours, the yield of γ-aminobutyric acid reached 35.4 g / L, demonstrating promising industrial application potential.