2295results about "Ligases" patented technology

The present disclosure provides methods and compositions for detecting and spatially locating analyte interactions and gene expression in a biological sample. For example, provided herein are methods of determining a location of at least one analyte in a biological sample using analyte-binding moieties, proximity ligation, and an array including capture probes.

Localized detection of RNA in a tissue sample that includes cells is accomplished on an array. The array include a number of features on a substrate. Each feature includes a different capture probe immobilized such that the capture probe has a free 3′ end. Each feature occupies a distinct position on the array and has an area of less than about 1 mm2. Each capture probe is a nucleic acid molecule, which includes a positional domain including a nucleotide sequence unique to a particular feature, and a capture domain including a nucleotide sequence complementary to the RNA to be detected. The capture domain can be at a position 3′ of the positional domain.

Localized detection of RNA in a tissue sample that includes cells is accomplished on an array. The array include a number of features on a substrate. Each feature includes a different capture probe immobilized such that the capture probe has a free 3′ end. Each feature occupies a distinct position on the array and has an area of less than about 1 mm2. Each capture probe is a nucleic acid molecule, which includes a positional domain including a nucleotide sequence unique to a particular feature, and a capture domain including a nucleotide sequence complementary to the RNA to be detected. The capture domain can be at a position 3′ of the positional domain.

Methods and systems are provided for generating and utilizing a genetically engineered bacterium comprising a modification in glnD, wherein said modification is selected from the group consisting of: deletion of the entire gene, deletion of substantially the entire gene, deletion of an ACT domain, deletion of more than 50% of an ACT domain, deactivation of an ACT domain, and deactivation of an UTase domain.

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 relates to the technical field of microbial fermentation, and discloses a genetically engineered bacterium of high-yield D-pantothenic acid as well as a preparation method and application of the genetically engineered bacterium. The D-PA yield of the engineering bacteria is greatly improved through the following effects that acetolactate synthase IlvB and IlvN shunted by pyruvic acid are subjected to overexpression, meanwhile, a transcription attenuation region is deleted, and valine feedback inhibition sites are mutated; the method comprises the following steps: overexpressing keto-alcohol acid reductoisomerase IlvC and dihydroxy acid dehydratase IlvD, and converting the IlvC coenzyme preference from NADPH into NADH, so as to break through the upstream of D-PA synthesis; overexpression of PanB is carried out, overexpression of a serine-glycine transport system is carried out, and synthesis of 5, 10-methylenetetrahydrofolic acid is promoted; meanwhile, the expression of ketopantoic acid reductase PanE is enhanced; and finally, over-expressing pantothenic acid synthetase PanC, and increasing the content of a cofactor ATP required by the PanC. According to the engineering bacteria provided by the invention, through overall reinforcement of the multiple modules, cell growth is improved due to balance of cofactors and increase of D-PA, and the yield of D-PA reaches an unprecedented height.

The invention relates to an engineering bacterium of high-yield ornamental blue as well as a construction method and application of the engineering bacterium, and belongs to the technical field of natural dye synthesis by a biological method. The engineering bacterium capable of highly producing the ornamental blue, disclosed by the invention, expresses icd, bpsA, glnAY405F and gd hA; acnR, yggB, glsK, aceA and ldh are knocked out from the engineering bacteria. According to the application, yggB and aceA genes are knocked out, site-directed mutagenesis (Y405F) is carried out on a glnA gene, a glsK gene is replaced by a glnAY405F gene, an acnR gene is replaced by an icd gene, an ldh gene is replaced by a gdhA gene, and a bpsA gene is integrated, so that the formation of by-products such as lactate and succinic acid in a blue-viewing synthesis process can be blocked; the flow direction of a passage from citric acid to isocitric acid to alpha-ketoglutaric acid to glutamic acid to glutamine is improved, so that the concentration of glutamine in cells is improved, and the yield of the ornamental blue is improved.

The invention discloses a streptomycete recombinant engineering strain for producing mycosporine-like metabolites, a gene expression cassette is integrated in a genome of the streptomycete recombinant engineering strain, the gene expression cassette is recombined to an attB site of a chromosome in a streptomycete chassis strain, the gene expression cassette is an operon controlled by a single promoter, and the operon is an operon controlled by a single promoter. The operon comprises 2-4 biosynthetic gene elements in MysA, MysB, MysC and MysD or mysDp334 in a synthetic pathway of mycosporine-like metabolites. The invention also discloses a construction method of the streptomyces recombinant engineering strain and an application of the streptomyces recombinant engineering strain in preparation of the mycosporine-like metabolites. A mycosporine-like metabolite is efficiently produced by heterologous expression of a mycosporine-like metabolite synthesis gene element in streptomyces, and four mycosporine-like metabolite products with high yield and strong ultraviolet absorption activity can be obtained by reconstruction of the biosynthesis gene element and recombinant expression in streptomyces pluvialis.

The present invention discloses a mutant strain of Streptomyces parvum and its application in producing glutathione, belonging to the field of genetic engineering. The present invention successfully constructs a mutant strain of Streptomyces parvum by overexpressing the glnA gene in Streptomyces parvum; wherein the nucleotide sequence of the glnA gene is shown in SEQ ID NO: 1. Experiments have shown that compared with the wild-type Streptomyces parvum, the mutant strain of Streptomyces parvum can significantly increase the content of glutathione and glucose in the fermentation product, and can also improve the utilization efficiency of ammonia nitrogen during the fermentation process. The present invention provides new strain resources and methods for mass production of bacterial-derived glutathione, and provides new agricultural antibiotic resources and strategies for preventing and controlling a variety of plant diseases.

The invention discloses construction and application of an L-malic acid producing issatchenkia orientalis strain, and belongs to the technical field of microorganisms and the technical field of microbial fermentation and metabolic engineering. Through a metabolic engineering means, a cytoplasm metabolic pathway of an issatchenkia orientalis strain is transformed, and a pyruvate decarboxylase coding gene, glycerol dehydrogenase and lactic dehydrogenase are knocked out; a pyruvate carboxylase gene derived from aspergillus flavus, a malate dehydrogenase gene derived from corynebacterium glutamicum and phosphoenolpyruvate carboxylase derived from escherichia coli are screened and integrated, so that a cytoplasm rTCA cycle synthesis pathway is enhanced; inserting a dicarboxylic acid external transporter gene from schizosaccharomyces cerevisiae at a neutral site GD21, and knocking out endogenous dicarboxylic acid internal transporter; the L-malic acid yield of the modified yeast strain in the fermentation process is remarkably increased and reaches 140.16 g / L. The engineering strain provided by the invention has good industrial application potential in efficient L-malic acid production.

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 discloses a wheat E3 ubiquitin ligase gene RFEL1 and application of the wheat E3 ubiquitin ligase gene RFEL1 in wheat disease resistance, and belongs to the technical field of plant genetic engineering. The invention discloses a nucleotide sequence of a wheat E3 ubiquitin ligase gene RFEL1 as shown in SEQ ID NO. 3. The invention further provides application of the wheat E3 ubiquitin ligase gene RFEL1 or related biological materials thereof. The application comprises any one of the following applications: A1, improving the resistance of wheat to stripe rust; a2, the resistance of wheat to powdery mildew is improved; and A3, the resistance of wheat to leaf rust is improved. The E3 ubiquitin ligase RFEL1 gene provided by the invention not only has broad-spectrum resistance to different stripe rust, but also has resistance to wheat leaf rust and powdery mildew. The E3 ubiquitin ligase RFEL1 gene disclosed by the invention is applied to broad-spectrum resistance breeding of wheat and has important application value.

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 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 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 belongs to the technical field of biology, and particularly relates to a difunctional gene cluster biosynthesized by phenol amine and salicylic acid and application of the difunctional gene cluster. The gene cluster comprises five genes, the five genes are S1EPS1, S1PBS3, S1CoAOMT2, S1CoOOMT3 and S1CoAOMT4, the nucleotide sequence of the S1EPS1 gene is as shown in SEQ ID No.1, the nucleotide sequence of the S1PBS3 gene is as shown in SEQ ID No.3, the nucleotide sequence of the S1CoAOMT2 gene is as shown in SEQ ID No.5, the nucleotide sequence of the S1CoOOMT3 gene is as shown in SEQ ID No.7, and the nucleotide sequence of the S1CoAOMT4 gene is as shown in SEQ ID No.9. The invention further discloses a preparation method of the gene cluster. The overexpressed gene cluster component SlEPS1 gene can significantly enhance accumulation of spermidine derivatives and salicylic acid of tomato plants so as to enhance the resistance to Pst DC3000, lays a foundation for genetic improvement in the aspect of tomato disease resistance by using the gene cluster, and also provides technical reference for excavation of related metabolic gene clusters in other crops and in the aspect of resistance breeding.

The invention discloses an application of a therapeutic target spot in preparation of a drug for treating targeted therapy resistance of kidney cancer. The therapeutic target spot is SLC27A3. The SLC27A3 comprises an SLC27A3 gene and a coding sequence thereof, an expression product of the SLC27A3 gene and a coding sequence thereof, and the SLC27A3 gene and an active fragment or derivative of the expression product thereof; the nucleotide of the SLC27A3 is as shown in SEQ ID NO. 1. According to the invention, a therapeutic target SLC27A3 is knocked out or silenced, and a compound Parishin for resisting the SLC27A3 protein is provided, so that the resistance of renal clear cell carcinoma cells to the pezopanib is remarkably improved, and the sensitivity of renal carcinoma cells to the pezopanib is enhanced.

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.

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 present invention provides for a genetically modified host cell reduced for expression of one of more endogenous enzyme(s) which enable the catabolism of propionyl-CoA and / or methylmalonyl-CoA, and comprising a methylmalonyl-CoA-dependent polyketide synthase (PKS).

Described and featured are compositions, a system and methods for identifying and selecting substrates of E3 ligases by ubiquitin biotinylation. The components of the compositions, system and methods are ubiquitin- and interaction-specific, thereby providing the enrichment and identification of endogenous or exogenous E3 ligase substrate molecules that are proximally ubiquitinated and biotinylated by components designed to interact both physically and functionally in the compositions, system and methods. The compositions, system and methods are useful and advantageous for identifying and selecting E3 ligase substrates (and / or associated molecules) that are modulated or induced by other agents, such as immunomodulatory imide agents (IMiDs), molecular glues and bifunctional proteolysis targeting chimeras (PROTAC®s).

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 relates to the technical production field of metabolic engineering and genetic engineering, and particularly provides a uracil production strain as well as a construction method and application thereof. According to the strain, a uridine kinase gene udk is knocked out on an E.coli W3110 genome, and meanwhile, a T7RNA polymerase gene is integrated; the uridine monophosphate kinase gene pyrH is weakened; the method comprises the following steps: overexpressing a carbamyl phosphate synthetase gene carAB, a ribose phosphate pyrophosphate kinase gene prsA, a pyrimidine-5 '-nucleoside enzyme gene ppnN, UMP phosphohydrolase genes umpH and umpG, a ribonucleoside hydrolase 1 gene rihA and a glutamine synthetase gene glnA; an orotic acid nucleoside monophosphate decarboxylase gene pyrF and an orotic acid ribosyltransferase gene pyrE of wild type bacillus subtilis B.subtilis 168 are introduced in a heterologous manner, so that an orotate nucleoside monophosphate decarboxylase gene pyrF and an orotate ribosyltransferase gene pyrE are obtained. The safe, efficient and low-cost production of the uracil is realized.

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.