170 results about "Pichia pastoris" patented technology

The invention discloses a recombinant pichia pastoris strain as well as a construction method and application thereof, and belongs to the field of microbial fermentation. The construction method specifically comprises the following steps that pGAPZB is used as a plasmid skeleton, a recombinant expression plasmid pGAPZB-ACSec containing an ACSec gene is constructed, and the sequence of the ACSec gene is shown as SEQ ID No.1; linearizing the recombinant expression plasmid, and transforming the linearized recombinant expression plasmid into a pichia pastoris host; and screening a pichia pastoris strain capable of expressing the ACSec gene. According to the invention, the ability of pichia pastoris to grow and metabolize by utilizing acetic acid is improved, and the application potential of pichia pastoris in fermentation production and synthesis of chemical products taking acetic acid as a precursor by utilizing a cheap carbon source is shown.

The invention provides pichia pastoris capable of efficiently expressing TFF3 fusion protein as well as a construction method and application of the pichia pastoris, and belongs to the technical field of engineering bacteria. The preservation number of the pichia pastoris is CCTCC (China Center For Type Culture Collection) NO: M 2025013. The pichia pastoris constructed by the invention can obviously improve the expression quantity of the TFF3, the shake flask expression quantity is up to 391 mg / L, and the pichia pastoris has a wide application prospect in large-scale industrial production of the TFF3.

The invention discloses a non-specific peroxidase HecUPO derived from Hypoxylon sp. (strain EC38), wherein the amino acid sequence of the non-specific peroxidase HecUPO is as shown in SEQ ID NO: 1. The enzyme can efficiently catalyze testosterone to generate 16 alpha-hydroxylation reaction to generate C16 alpha-hydroxytestosterone, the conversion rate reaches 70%, the separation yield reaches 82%, no by-product is generated, and the C16 alpha-hydroxytestosterone has excellent regioselectivity. The invention also discloses a gene for coding the HecUPO, a recombinant plasmid pPICZA-HecUPO containing the gene, a recombinant pichia pastoris engineering bacterium and an application of the recombinant pichia pastoris engineering bacterium in steroid hydroxylation. The non-specific peroxidase HecUPO disclosed by the invention has the advantages of being high in catalytic efficiency, specific in regioselectivity, simple in reaction system, green, environmentally friendly and the like, and a new enzymology resource is provided for fixed-point hydroxylation modification of steroid drugs.

The application discloses a recombinant collagen type XVII and a preparation method thereof, and relates to the technical field of genetic engineering. The application provides a recombinant collagen type XVII. Compared with the prior art, the recombinant collagen type XVII provided by the application has high yield of Pichia pastoris engineering bacteria, is not easy to be degraded, and has a stable triple helix structure.

The invention discloses a potato protein with good emulsibility, gelling property and foamability, and belongs to the fields of biology and genetic engineering. According to the invention, the designed potato protein realizes recombinant expression in pichia pastoris, and it is verified that the recombinant expressed protein can form a secondary structure and can form hydrogel at a concentration of 60 g / L. The invention also provides an application of the prepared potato protein as a food additive, shows emulsifying and gelling capabilities superior to those of commercial potato protein, and has huge value and potential in the fields of food and tissue engineering.

The invention provides a strain of pichia pastoris SIVE9110 and an application of the pichia pastoris SIVE9110 in Morisseria wine. The strain SIVE9110 is preserved in the China Center for Type Culture Collection (CCTCC for short) on September 17, 2025; the address is Wuhan University, Wuchang District, Wuhan City, Hubei Province; the postal code is 430072, and the preservation number is CCTCC (China Center For Type Culture Collection) NO: M 20252052; the Latin name is Pichia occidentalis, and the Latin name is Pichia occidentalis; the strain SIVE9110 and saccharomyces cerevisiae are sequentially fermented, so that the wind and soil characteristics of the maisseria wine can be highlighted; and the prepared maisseria wine is softer and mellow in mouth feel.

The invention specifically discloses a fermentation process suitable for III-type collagen expression, and relates to the technical field of biological fermentation of recombinant genetic engineering bacteria. According to the method disclosed by the invention, a double-carbon-source mixed feeding strategy of alanine and methanol is used, so that the problems that when pichia pastoris expresses exogenous target protein by taking methanol as a unique carbon source and energy source, cell growth and protein expression fight for the carbon source and the energy source together, the metabolic burden of cells is increased, and the expression efficiency is low are solved; through a three-step methanol fed-batch strategy, the expression efficiency is improved, and the toxic action of methanol on cells is reduced, so that the yield of the III-type collagen is further improved. The fermentation process provided by the invention solves the problem of reducing the production cost while improving the high-efficiency and high-quality expression of the target product under the condition of high-density thallus fermentation.

The present application belongs to the field of microbial metabolic engineering and synthetic biology technology application, in particular to a high methanol tolerance yeast chassis strain, a high 3-hydroxypropionic acid yield engineering bacterium and application. The high methanol tolerance yeast chassis strain takes wild type Pichia pastoris or fatty acid producing Pichia pastoris as a starting strain, is modified according to at least one of the following conditions (a)-(h), and a high methanol tolerance strain is obtained, wherein (a) MIOX gene knockout; (b) DMA2 gene knockout; (c) MET17 gene knockout; (d) BUB2 gene knockout; (e) CLB4 gene knockout; (f) UBP12 gene knockout; (g) ZFP gene knockout; (h) AOX1 gene mutation. The high methanol tolerance yeast chassis strain is further modified to obtain a high 3-hydroxypropionic acid yield engineering bacterium. The present application combines rational and non-rational metabolic engineering strategies, realizes efficient biosynthesis of fatty acids and 3-hydroxypropionic acid with methanol as the only carbon source, and lays an important foundation for low-carbon green biological manufacturing of chemicals.

The invention discloses a method for producing recombinant I-type mussel mucin rich in dopa, and belongs to the technical field of genetic engineering. According to the invention, Ala-Lys-Pro-Ser-Tyr-Pro-Pro-Thr-Tyr-Lys decapeptide of a natural type I mussel mucin sequence is taken as a repetitive unit, a gene expression vector for tandem expression of peptide fragments with different repetitive numbers is constructed, and pichia pastoris host bacteria are transformed, so that type I mussel mucin precursors with different molecular weights are obtained; in vitro, a precursor is subjected to dopa modification by high-activity tyrosinase obtained through screening in a mixed system containing ascorbic acid ethyl ether, copper sulfate and the like, and the recombinant I-type mussel mucin rich in dopa groups is obtained after purification. The recombinant I-type mussel mucin rich in dopa obtained by the invention has the advantages of high purity and high dopa modification rate, and has wide application prospects in the fields of biomedicine, surface chemistry and the like.

The invention particularly discloses a fermentation method and application of XVII type recombinant collagen, and relates to the technical field of biological fermentation. The fermentation method provided by the invention comprises a fermentation methanol induced expression stage, continuous fed-batch of amino acids of different types and different proportions, regulation of growth metabolism of pichia pastoris and improvement of the synthesis rate of recombinant collagen, the fermentation yield can be improved by 69.8%, and the fermentation time can be shortened by 12-24 h.

The invention discloses a recombinant ribitol dehydrogenase strain and application thereof, and belongs to the technical field of bioengineering. According to the recombinant ribitol dehydrogenase strain disclosed by the invention, pichia pastoris GS115 is taken as a starting strain, a recombinant ribitol dehydrogenase carrier is transformed, and the ribitol dehydrogenase gene sequence is shown as SEQ ID. NO 1. After fermentation in a 50L fermentation tank, the fermentation liquor is centrifuged, the supernate contains recombinase RDH with the purity of 95% or above, and the enzyme activity can reach 1059U / mL. The recombinase catalyzes a redox reaction between D-psicose and D-psilol to produce D-psilol, and the conversion rate can reach 56% after the reaction is balanced. The enzyme has good heat resistance, does not need metal ions to participate in a catalytic reaction, is low in product separation and purification difficulty, can keep the maximum enzyme activity of 80% or above in a wide pH (6-9) range, and can be applied to large-scale industrial production of rare sugar D-psilol.

The invention relates to the field of genetic engineering, in particular to a non-methanol-induced chitin deacetylase pichia pastoris expression vector and engineering bacteria as well as a construction method and application of the non-methanol-induced chitin deacetylase pichia pastoris expression vector and engineering bacteria. According to the invention, a carrier containing a constitutive strong promoter is adopted as a skeleton, a chitin deacetylase gene coding sequence is inserted into the carrier, methanol is not required to be used as an inducer when the obtained recombinant plasmid is applied, and the produced chitin deacetylase is efficient, green and environment-friendly, and can be applied to the fields of beauty industry, agriculture, biomedicine and the like. The invention establishes a non-methanol-induced chitin deacetylase pichia pastoris expression vector and engineering bacteria, which are used for high-efficiency expression of chitin deacetylase, and solves the problem of low enzyme yield of chitin deacetylase from natural sources.

The invention discloses a pichia pastoris recombinant strain for producing astaxanthin as well as a construction method and application of the pichia pastoris recombinant strain. According to the method, pichia pastoris serves as an operation platform, astaxanthin serves as a target product, a beta-carotene high-yield strain is obtained by increasing the dosage of beta-carotene synthesis genes, then astaxanthin is synthesized through pichia pastoris by integrating astaxanthin synthetase genes, and the astaxanthin yield is improved through optimization of a carotenoid synthesis module. In addition, the synthesis of the recombinant strain astaxanthin is promoted through multienzyme assembly of key nodes in an upstream path of an astaxanthin synthesis path and weakening of a squalene synthesis path in a branch path. On the basis, recombination strain cofactor synthesis is optimized, astaxanthin synthesis is improved, the astaxanthin yield is further increased, and the astaxanthin high-yield strain is obtained. According to the invention, the yield of astaxanthin reaches 4.75 g / L by using a pichia pastoris combined regulation strategy, which is the highest level in current microbial synthesis.

The application provides a uric acid oxidase secretion signal peptide and application thereof, and relates to the technical field of genetic engineering. In view of the problem that uric acid oxidases reported in the current literature are difficult to secrete in Pichia pastoris, the endogenous signal peptide of Pichia pastoris is screened, a signal peptide with strong secretion capacity is obtained, and the signal peptide is optimized in a targeted manner, a signal peptide mutant with strong secretion capacity is obtained, uric acid oxidase can be better secreted to the extracellular, and then the extracellular enzyme activity of uric acid oxidase is improved.

The invention discloses a whitening and brightening composition and a cosmetic thereof. The whitening and brightening composition is prepared from ascorbyl tetraisopalmitate, tranexamic acid, Brigtenyl, LightSKN CN, a resveratrol fermentation product, fermented lotus essence and super vitamin A, brigtenyl is a mixture composed of a first solvent, glycerin and diglucosyl gallic acid; lightSKN CN is a mixture composed of a second solvent, 1, 3-propylene glycol, xanthophyll, lecithin, sodium ascorbate and vitamin E; the resveratrol fermentation product is a mixture composed of a third solvent, a pichia pastoris fermentation lysate filtrate and resveratrol; the fermented lotus seed essence is a mixture composed of a fourth solvent, butanediol and a lotus seed extract; the super vitamin A is a mixture of isosorbide dimethyl ether and hydroxyl pinacolone retinoate. The whitening and brightening composition not only can inhibit melanin generation, reduce melanin transfer and accelerate melanin metabolism, but also can remove excessive free radicals in skin, so that the effects of long-acting skin whitening and skin brightening are achieved.

The invention relates to the technical field of genetic engineering, in particular to recombinant acidic pectinase GsPG3 and application thereof. The amino acid sequence of the recombinant acidic pectinase GsPG3 is as shown in SEQ ID NO: 4, the optimum pH of the recombinant acidic pectinase GsPG3 is 3.5, the optimum temperature of the recombinant acidic pectinase GsPG3 is 65 DEG C, and the recombinant acidic pectinase GsPG3 has excellent stability under an acidic condition (pH is 3-5) and can be activated by Ca < 2 + >. The enzyme can be efficiently expressed in pichia pastoris, the shake flask enzyme activity reaches 7235.4 U / ml, and the fermentation activity in a 30L fermentation tank reaches 91,150.3 U / ml. According to the method, the GsPG3 enzyme is specifically applied to beet pulp enzymolysis for the first time, the optimal process conditions are obtained through system optimization: the pH is 3.0, the temperature is 50-55 DEG C, the enzyme concentration is 2,000 U / ml, the substrate concentration is 0.25 g / ml, the time is 3h, and the pectin degradation rate of the beet pulp can reach 45.4% under the conditions.

The invention belongs to the technical field of biology, and provides endoglucanase FsCel45B of a GH45 family, a related biological material and a high-efficiency expression method of the endoglucanase FsCel45B. The GH45 family endoglucanase FsCel45B comprises the following A1) or A2): A1) a protein with an amino acid sequence as shown in SEQ ID NO: 1; a2) a protein which is obtained by substitution and / or deletion and / or addition of amino acid residues on the amino acid sequence shown in SEQ ID NO: 1 and has the same function as the protein shown in A1). The invention further provides an expression optimization strategy in pichia pastoris, based on a pichia pastoris expression vector pPICZ alpha A, an AOX1 promoter and an alpha-MF signal peptide sequence are replaced and co-expressed with disulfide bond folding isomerase (PDI) and a vesicle transport related gene Ssa4, the expression quantity is improved by combining with the increase of the gene copy number, and the expression optimization strategy has industrial production potential.

The invention relates to the technical field of bioengineering and fermentation engineering, and particularly discloses a method for improving expression of recombinant human epidermal growth factors by pichia pastoris, which comprises the step of adding carboxymethylation modified passion fruit peel polysaccharide derivatives into a culture medium in a pichia pastoris fermentation culture process. According to the method for improving the expression of the recombinant human epidermal growth factor by the pichia pastoris, a proper amount of carboxymethylation-modified passion fruit peel polysaccharide derivative (C-WPEP) is added into a pichia pastoris expression system, so that the expression quantity, stability and activity of the rhEGF can be remarkably improved, the operation is simple and convenient, the cost is low, and the method is suitable for industrial large-scale production of the rhEGF.

The application discloses a Pichia pastoris engineering bacterium for converting methanol to synthesize bakuchiol from scratch as well as construction and application of the bacterium. In different chromosomal neutral sites of the Pichia pastoris, a heterologous p-coumaric acid synthesis pathway is introduced, key genes of tyrosine and phenylalanine synthesis pathways of a heterologous shikimic acid pathway are knocked out, overexpressed or heterologously expressed, a high-yield p-coumaric acid strain is obtained, on the basis of the strain, a heterologous bakuchiol biosynthesis pathway is introduced, bakuchiol synthetase is overexpressed, key genes of a MVA pathway and genes of an exogenous acetyl coenzyme A supply pathway are endogenously overexpressed or heterologously expressed, after optimization of a methanol concentration and back complementation of a His4 gene of the strain, the yield of the bakuchiol is effectively increased to 91.2 mg / L, which is 59.8 times higher than that of an initial strain, and the yield of the bakuchiol in a 15L fermenter reaches 692.8 mg / L. The application has the characteristics of high conversion efficiency, low production cost, easy preparation, wide industrial application prospect and the like.

The invention relates to cellulase and a preparation method thereof. The preparation method of the cellulase comprises the following steps: inoculating activated pichia pastoris into a seed culture medium, and carrying out seed culture to prepare a seed solution; inoculating the seed solution to a fermentation culture medium, carrying out fermentation culture, in the fermentation culture process, feeding glycerol for supplementing materials, carrying out fermentation culture until the OD600 of the obtained fermentation liquor is 400-600, and stopping glycerol supplementing so as to keep thalli in a starvation state; and methanol is fed at a flow rate of 0.5 mL.L <-1 >. H <-1 > to 12 mL.L <-1 >. H <-1 > for supplementary feeding, and conversion is carried out to prepare a conversion solution containing cellulase.

The present application relates to the Pichia pastoris fermentation medium and fermentation method for expressing recombinant bovine lactoferrin, and belongs to the technical field of biochemistry, which solves the technical problem of improving the expression amount of bovine lactoferrin in the process of industrialized and large-scale preparation of recombinant bovine lactoferrin. The components and their proportions in the Pichia pastoris fermentation medium aqueous solution for expressing recombinant bovine lactoferrin are as follows: yeast extract 10-30 g / L, phosphoric acid aqueous solution 25-35 g / L, calcium sulfate 1.0-3.0 g / L, potassium sulfate 18-22 g / L, potassium hydroxide 4.0-5.0 g / L, glycerol 30-50 g / L, magnesium sulfate heptahydrate 7-13 g / L, ammonium chloride 4-5 g / L, PTM1 trace element solution 4-6 ml / L, and defoaming agent 1-2 ml / L. The high-density fermentation method of the present application is realized by controlling the technical parameters such as the dissolved oxygen amount, pH value, temperature and feeding medium rate in the fermentation process, so that the expression amount of bovine lactoferrin is improved to 4-5 g / L.

The invention belongs to the technical field of composite strains, particularly relates to a high-lignin straw fermentation composite microbial agent, and a preparation method and a fermentation process thereof, and aims to solve the problems of low lignin degradation efficiency, long period and high cost of a single microorganism in the prior art. According to the invention, a composite synergistic system of coriolus versicolor, bacillus subtilis and pichia pastoris is innovatively adopted, and efficient degradation is realized through a mechanism that the coriolus versicolor secretes a lignin degrading enzyme system, the bacillus subtilis assists cellulose decomposition and the pichia pastoris optimizes a microenvironment; the preparation method of the complex microbial inoculant comprises the following steps: culturing various strains in a proper culture medium to a specific growth state, and mixing according to an optimized volume ratio; the fermentation process relates to high-lignin straw pretreatment, inoculation, sealed fermentation under control conditions and regular ventilation, the lignin degradation rate can be increased, the feed nutritional value can be improved, the relative feeding value and quality can be improved, and economic and environment-friendly technical support is provided for resource utilization of agricultural by-products.

This application relates to the field of biotechnology, specifically disclosing a *Pichia pastoris* yeast, its construction method, and its applications. This application involves knocking out the *gas1* gene, which is related to β-glucan synthesis, in *Pichia pastoris*. By deleting the *gas1* gene, which encodes β-glucan in the cell wall of *Pichia pastoris*, this application reduces cell size, decreases the transfer distance from the cell surface to the nucleus, improves oxygen transfer and product secretion during high-density culture, and enhances fermentation efficiency. Furthermore, the *Pichia pastoris* yeast constructed using this application enhances the synthesis of the intracellular product bisabolol and also enhances its secretion capacity. This application, by reducing cell size and decreasing the carbon flow to cell wall polysaccharide synthesis while increasing the carbon source flow to target product synthesis, has significant implications for industrial production.

This invention relates to the fields of molecular cloning and protein expression, and particularly to a Pichia pastoris engineered strain expressing full-length human type III collagen and its applications. The engineered Pichia pastoris strain is obtained by cloning the enzymatically digested human type III collagen gene sequence into an expression vector to obtain a recombinant expression vector, which is then transformed into Pichia pastoris. This engineered Pichia pastoris strain was deposited at the China General Microbiological Culture Collection Center (CGMCC) on November 6, 2024, with accession number CGMCC NO.32510. The Pichia pastoris engineered strain obtained by this invention can efficiently produce the desired human type III collagen through fermentation, exhibiting excellent characteristics such as high yield and absence of endotoxins.

The invention relates to the technical field of microbial fermentation and genetic engineering, and particularly discloses a yeast engineering bacterium for efficiently producing plastic depolymerases and application of the yeast engineering bacterium. The engineering yeast takes pichia pastoris as an expression host, a molecular chaperone is co-expressed, a vacuole sorting receptor gene is knocked out or knocked down, in addition, a yeast cell factory for efficiently expressing the plastic depolymerases is constructed by further combining regulation and control element optimization and target gene copy number regulation, and the engineering yeast has the advantages that the engineering yeast can be used for efficiently expressing the plastic depolymerases; the regulatory element comprises any one or a combination of more of a strong promoter, a signal peptide and a C-terminal tag. The engineering yeast provided by the invention can realize efficient accumulation and secretion of the plastic depolymerases, obviously improve the enzyme yield and reduce the production cost of unit enzyme activity, and provides a stable and efficient yeast expression system for industrial and low-cost production of the plastic depolymerases; the method has an important application prospect in the fields of biodegradation and resource utilization of waste plastics.

The invention discloses a recombinant three-type collagen raw material and a medical beauty repair anti-aging product, and relates to the technical field of genetic engineering. The similarity between the amino acid sequence of the recombinant III-type collagen provided by the invention and the sequence shown in SEQ ID NO: 1 is greater than 99%, and the recombinant III-type collagen has the following characteristics that the soluble expression proportion is not less than 80% in an escherichia coli, pichia pastoris, bacillus or bacillus subtilis expression system. Through accurate sequence design and optimization, early translation termination and misfolding are effectively reduced, the effect of efficient expression in different expression systems (such as escherichia coli, pichia pastoris, bacillus and bacillus subtilis) is achieved, the soluble expression proportion is higher than 80%, the downstream purification difficulty and production cost are directly reduced, and the method is suitable for industrial production. And a foundation is laid for large-scale industrial production. Experiments prove that the recombinant three-type collagen provided by the invention has a good effect of promoting cell proliferation, and can be applied to preparation of drugs or medical equipment products for promoting cell proliferation or skin repair.

Unglycosylated lysostaphin variant protein, nucleic acid molecule, vector and host cell, as well as a method for production of unglycosylated lysostaphin variant protein in a yeast expression system are provided. The proteins are produced in a Pichia pastoris expression system and have been shown to have activity equivalent to wild-type lysostaphin. The lysostaphin variant proteins can be used as therapeutic proteins for treatment of diseases such as Staphylococcus aureus infection.

The present application provides a Pichia pastoris engineering bacterium and a kit and application thereof, a method for obtaining the Pichia pastoris engineering bacterium, a method for improving glucose oxidase yield of the Pichia pastoris, and a method for producing glucose oxidase, wherein the Pichia pastoris engineering bacterium carries a glucose oxidase gene and a pyruvate kinase gene. The Pichia pastoris engineering bacterium of the present application can realize high glucose oxidase yield and high enzyme activity, and lays a good foundation for large-scale production of glucose oxidase.

The invention relates to the technical field of genetic engineering and microbial fermentation, in particular to a surfactant-tolerant amylase mutant and application thereof. The 2-39 amino acid sequences of the amylase as shown in SEQ ID No.1 are deleted, the obtained amylase has higher surfactant resistance, and the enzymatic activity of the amylase is still maintained at 80% or above of the initial activity after the amylase is stored at room temperature for 6 months in the presence of 25% of a surfactant. The amylase mutant is efficiently expressed in pichia pastoris, and through further fermentation optimization, the production cost of the amylase is remarkably reduced, and the yield is increased by 176%, so that wide application of the amylase in the detergent industry is promoted.

The invention discloses a construction method and application of alpha-farnesene-producing pichia pastoris, and belongs to the technical field of biology. The invention discloses a novel pichia pastoris engineering strain which has the capability of efficiently synthesizing alpha-farnesene. According to the strain, through metabolic pathway optimization and key enzyme high expression, the carbon flow distribution efficiency of a substrate to alpha-farnesene is remarkably improved. The yield of an excellent strain pichia pastoris mut008 obtained through ARTP mutagenesis and multiple rounds of screening reaches 3.05 g / L under the level of a 5L fermentation tank when methanol is used as a substrate, and the yield preliminarily reaches the level required by industrial production of C1 substrate utilization; when glucose is used as a substrate, the yield reaches 30.66 g / L, and the industrial production cost can be reduced.