458 results about "Pichia pastoris" patented technology

The invention belongs to the technical application field of microbial metabolic engineering and synthetic biology, and particularly relates to a pichia pastoris engineering strain for high-yield sclareol as well as a construction method and application of the pichia pastoris engineering strain. The method comprises the following steps: constructing a biosynthetic pathway of sclareol in a host strain, and optimizing an intracellular mevalonic acid metabolic pathway and a central metabolic pathway to obtain an engineering strain A; the host strain is pichia pastoris; or overexpressing or knocking out a metabolism regulation factor in the obtained engineering bacterium A to obtain an engineering bacterium strain B; or, in the obtained engineering bacterium B, a synthetic pathway is targeted to peroxisome and optimized by adopting a cell region compartment strategy, and an engineering bacterium strain C is obtained. The invention provides a pichia pastoris chassis cell for synthesizing a diterpenoid sclareol and an engineering strain for synthesizing sclareol. By introducing and optimizing the expression of a sclareol synthesis pathway, the fermentation yields of sclareol in shake flask batch and bioreactor batch fed-batch fermentation are respectively 631.6 mg / L and 10.5 g / L.

The invention discloses a compound microbial agent for enhancing production of beta-phenethyl alcohol in a white spirit brewing process as well as a preparation method and application of the compound microbial agent, and belongs to the technical field of microorganisms. The compound microbial agent is prepared from aspergillus niger, pichia membranaefaciens, Kazakstan monospora, saccharomyces cerevisiae and sporospora delbrueckii; in the compound microbial agent, the final concentration OD600 of the aspergillus niger, the final concentration OD600 of the pichia membranaefaciens, the final concentration OD600 of the Kazakstein monospora, the final concentration OD600 of the saccharomyces cerevisiae and the final concentration OD600 of the sporospora delbrueckii are 0.8-1.5, 0.8-1.5, 0.5-1, 0.8-1.5 and 0.5-1 respectively. After the compound microbial agent provided by the invention is applied to a solid-state fermentation system of Luzhou-flavor liquor, the yield of the aroma substance beta-phenethyl alcohol can be obviously increased by adjusting the eukaryotic microbial community structure of the fermentation system; the method can effectively solve the problem that the stability of the method for producing beta-phenethyl alcohol in the process of intensifying Baijiu fermentation by a single strain is poor.

The invention belongs to the technical field of protein engineering and genetic engineering, and particularly relates to recombinant IV-type humanized collagen as well as a preparation method and application thereof. A section of brand new recombinant collagen is designed and obtained through methods such as screening, replacement and splicing, the nucleotide sequence of the recombinant collagen is optimized through pichia pastoris codon preference, then a corresponding genetic engineering yeast strain is constructed and obtained, and the recombinant collagen is successfully prepared through fermentation. Experiments prove that the recombinant IV-type humanized collagen prepared by the invention is large in molecular weight and high in yield, and compared with commercially available recombinant collagen, the recombinant IV-type humanized collagen has better free radical scavenging performance and cell proliferation and migration promoting performance, and has good antioxidant and skin barrier repair functions, so that the recombinant IV-type humanized collagen has good practical application value.

The invention relates to a recombinant pichia pastoris strain for producing astaxanthin as well as a construction method and application of the recombinant pichia pastoris strain. The recombinant pichia pastoris strain is obtained by expressing dehydrogenase Zwf1, phosphate dehydrogenase Gnd2, NADH kinase Pos5 and fusion protein CrtZ-Linker-CrtW of beta-carotene ketolase CrtW and beta-carotene hydroxylase CrtZ in host bacteria. Wherein the host bacterium is a pichia pastoris gene modified strain PP-L. The fermentation conditions of the recombinant strain are optimized based on cofactor engineering, and the astaxanthin production capacity of the pichia pastoris is further improved. The construction method of the recombinant pichia pastoris is simple, synthesis of astaxanthin can be better promoted, the engineering strain can efficiently synthesize astaxanthin by using glucose and methanol through fermentation optimization, and industrial production is facilitated.

The invention relates to the technical field of biological fermentation, in particular to a methanol-induced fermentation process for expressing III type recombinant human collagen, which comprises the following steps: inoculating a pichia pastoris fermentation seed solution into a fermentation culture medium, performing fermentation culture to a glycerol culture stage, and entering a methanol-induced culture stage after glycerol feeding is finished and pH is rebounded. According to the methanol-induced fermentation process for expressing the III-type recombinant human collagen, provided by the invention, after the glycerol feeding is finished, the pH rebound is taken as an index, and methanol induction is started after the pH rebound, so that thallus growth is obviously accelerated, and the protein expression quantity is improved, and therefore, the III-type recombinant human collagen can be industrially produced; and greater economic values can be created for enterprises.

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 provides a micromolecular recombinant XVII type collagen as well as a preparation method and application thereof, and relates to the technical field of protein engineering. The novel recombinant human XVII type collagen is obtained by optimizing and screening a plurality of spiral region sequences such as a collagen structural domain, a C-terminal region and a middle region of the human XVII type collagen. The amino acid composition of the recombinant XVII type collagen is consistent with that of a natural collagen alpha1 chain, immunological rejection is not generated when the recombinant XVII type collagen is applied to a human body, the recombinant XVII type collagen can be widely applied to surgical medical treatment and medical beauty industries, the molecular weight of the collagen is small, efficient secretion soluble expression in eukaryotic host cells such as pichia pastoris can be achieved, and the recombinant XVII type collagen has a good application prospect. And industrial large-scale production is carried out. According to the present invention, the verification results show that the micromolecular recombinant XVII type collagen further has basement membrane anti-aging activity, wherein the basement membrane anti-aging activity specifically comprises the improvement of the expression levels of the IV type collagen, the VII type collagen and the XVII type collagen;

The invention relates to the technical field of microorganisms, in particular to recombinant pichia pastoris for expressing plastic degrading enzyme LCCICCG as well as a construction method and application of the recombinant pichia pastoris. According to the invention, pichia pastoris is taken as a host cell, the coding gene lccICCG of the plastic degrading enzyme is heterologously expressed, and single-copy or multi-copy recombinant pichia pastoris is constructed. By optimizing culture conditions, the expression quantity and biomass of the plastic degrading enzyme protein are remarkably improved; by optimizing a fermentation strategy, high-density fermentation production of the LCCICCG protein in a 5L fermentation tank is realized, and a technical basis is provided for realizing large-scale industrial production of the protein. The improvements not only improve the production efficiency and reduce the cost of industrial production, but also provide powerful support for commercial application of the plastic depolymerases.

The present invention provides a yeast recombinant type XVII humanized collagen and its preparation method, in particular, a recombinant humanized collagen expressing the 15th helical region of type XVII collagen, a recombinant engineering bacterium and its preparation method, belonging to the technical fields of genetic engineering and synthetic biology. The present invention provides the full-length sequence of the 15th helical region of type XVII collagen, and mutants are obtained by mutating amino acid residues based on this sequence; for the first time, the present invention expresses the full-length sequence of the 15th helical region of type XVII collagen in Pichia pastoris, establishes a complete set of fermentation processes and purification processes, improves the protein expression level and reduces degradation, and through experiments, it is verified that the obtained protein has biological activities such as cell adhesion activity and cell migration promotion activity that reach or exceed those of natural human collagen, can achieve the purpose of real product application, and can be widely applied in the fields of medicine, medical devices, biological materials, tissue engineering, cosmetics, etc.

The invention relates to a recombinant pichia pastoris genetically engineered bacterium for expressing recombinant full-chain-length humanized collagen as well as a preparation method and application of the recombinant pichia pastoris genetically engineered bacterium, and belongs to the technical field of genetic engineering. The recombinant pichia pastoris genetically engineered bacterium disclosed by the invention expresses recombinant full-chain-length humanized collagen, and a ku70 gene of the recombinant pichia pastoris genetically engineered bacterium is knocked out and knocked into a recombinant 4-proline hydroxylase encoding gene. Recombinant full-chain-length humanized collagen is optimized, a 4-proline hydroxylase gene and a collagen gene are co-expressed in pichia pastoris to obtain hydroxylation modified recombinant collagen, and a corresponding recombinant pichia pastoris genetically engineered bacterium is provided, has proteolytic enzyme A defect and hydroxylation modification capability, and can be used for preparing a recombinant collagen product. The stability of the collagen in the fermentation process is improved. The recombinant full-chain-length humanized collagen provided by the invention is 100% homologous with III-type collagen derived from a human body, so that the immune risk is extremely low, and the biological activity is more perfect.

The embodiment of the invention provides a recombinant pichia pastoris strain as well as a construction method and application thereof. The construction method comprises the following steps: by taking a pichia pastoris strain as a host strain, constructing a dual-synthesis path of phenylalanine ammonialyase and tyrosine ammonialyase in the host strain to obtain a first engineering strain; removing chorismic acid feedback inhibition of the first engineering strain based on metabolic engineering to obtain a second engineering strain; optimizing an aromatic amino acid synthesis route of the second engineering strain to obtain a third engineering strain; knocking out a branch metabolic pathway of the third engineering strain to obtain a fourth engineering strain; increasing precursor supply of the fourth engineering strain to obtain a recombinant pichia pastoris strain; according to the construction method provided by the embodiment of the invention, the synthesis path of p-coumaric acid can be systematically optimized, the recombinant pichia pastoris strain for efficiently producing p-coumaric acid by taking methanol as a carbon source is constructed, and compared with a host strain, the recombinant pichia pastoris strain has the advantage that the yield of p-coumaric acid is remarkably increased.

The invention relates to the technical field of microorganisms, and provides pichia pastoris CMRC 13Y with high protein yield and application of the pichia pastoris CMRC 13Y. The pichia pastoris CMRC 13Y is separated from naturally-fermented dried milk cakes, the classification name of the pichia pastoris CMRC 13Y is Pichia kudriavzevii, and the preservation number of the pichia pastoris CMRC 13Y is CGMCC No.34655. The pichia pastoris CMRC 13Y has high acid resistance, and the growth of the pichia pastoris CMRC 13Y is not obviously influenced along with the reduction of the pH value within the pH value range of 3.0-7.0; a yeast protein product prepared from the strain CMRC 13Y is subjected to sensory flavor determination, has no earthy smell in the aspect of smell, has fragrance, is fine and smooth in mouth feel, and has no unpleasant taste; the strain can achieve high-biomass growth and high-protein expression, has the advantages of being green, environmentally friendly, high in protein yield and the like, can be used for preparing microbial protein related products and is wide in application prospect.

The invention discloses a preparation method and application of recombinant humanized III-type collagen, and belongs to the technical field of genetic engineering. According to the invention, three segments with good hydrophilicity and high solubility are screened from a humanized III-type collagen sequence through a bioinformatics method, and coding genes of the three segments are integrated into a pichia pastoris genome in a multi-copy form after being subjected to pichia pastoris codon preference optimization, so that a recombinant strain of the humanized III-type collagen is obtained. The recombinant strain secretes and expresses collagen, the purity can reach 95% or above only through one-step purification, the downstream separation and purification difficulty is obviously reduced, and the production process is easy to amplify. The prepared three recombinant human-derived III-type collagen proteins all have the activity of promoting cell migration and the activity of promoting cell proliferation, and have good application value.

The invention provides a pichia pastoris strain for efficiently converting methanol to produce high-methionine-content single-cell protein and application of the pichia pastoris strain, and belongs to the technical field of biology. According to the invention, a strain of pichia pastoris (named as Pichiapastoris CN-SCP 00 with a preservation number of CCTCC (China Center For Type Culture Collection) NO: M2025657) is obtained through separation, and is subjected to multiple rounds of ARTP iterative mutation breeding, so that a strain Pichiapastoris CN-SCPYE421 (with a preservation number of CCTCC NO: M2025658) for efficiently converting methanol to produce single-cell protein containing high methionine is obtained. Compared with a commercial strain, the Pichiapastoris CN-SCPYE421 has higher methanol tolerance and utilization capacity, the fermentation time is remarkably shortened, and in addition, the Pichiapastoris CN-SCPYE421 can well grow at the temperature of 35 DEG C. The Pichiapastoris CN-SCPYE421 is subjected to fermentation culture in a 5L fermentation tank, the dry weight of thalli reaches 120-150g / L, the protein content reaches 65-75% of the dry weight of the thalli, the Pichiapastoris CN-SCPYE421 is rich in various essential amino acids, and the methionine content reaches 2.3%. The invention provides an excellent strain for industrial development of single-cell protein, and has great social significance and economic value for the feed protein industry.

The invention discloses a recombinant pichia pastoris strain with high yield of retinaldehyde as well as a construction method and application of the recombinant pichia pastoris strain. The recombinant pichia pastoris strain is obtained by expressing dehydrogenase Zwf1, phosphate dehydrogenase Gnd2, NADH kinase Pos5 and two copied beta-carotene-15, 15 '-monooxygenase BCMO in host bacteria. Wherein the host bacterium is a pichia pastoris gene modified strain PP-B (Propene Polymer-B). The retinaldehyde production performance of the recombinant strain is verified based on cofactor engineering and the copy number of key enzymes, and the retinaldehyde production capacity of the pichia pastoris is further improved. The recombinant pichia pastoris disclosed by the invention is simple in construction method, can better promote the synthesis of retinol, and is beneficial to industrial production as the engineering strain can efficiently synthesize retinol by using methanol as a unique carbon source through amplification fermentation in a 5L fermentation tank.

The present invention relates to an enzyme catalyst for preparing a dicarboxylic acid and a method for preparing a dicarboxylic acid using the same. The enzyme catalyst is prepared by a method comprising the following steps: (1) carrying out fermentation culture on enzyme-producing microorganisms in a culture medium to obtain a crude enzyme solution, and (2) immobilizing the crude enzyme solution on a molecular sieve to obtain the enzyme catalyst, wherein the enzyme producing microorganisms are selected from candida tropicalis, candida sake, pichia pastoris, saccharomyces cerevisiae, lactobacillus brevis, corynebacterium glutamicum, bacillus subtilis, bacillus cereus, escherichia coli and pseudomonas aeruginosa. The method comprises the following steps: in a PBS buffer solution, mixing triglyceride with the enzyme catalyst, and carrying out catalytic reaction under stirring at 20-50 DEG C; and then carrying out hydrolysis reaction at the temperature of 250-300 DEG C under the pressure of 30-100 bar. According to the method, the enzyme catalyst and the molecular sieve are immobilized, so that other oxidants or catalysts are not required to be added while the dicarboxylic acid with higher yield is obtained.

A recombinant collagen and a preparation method and use thereof, and in particular, a full-length collagen a1 chain produced through recombinant expression, and a preparation method and use thereof are provided. When a variant (which is denoted as a1 (I) M1) of a human type I collagen a1 chain (a1 (I) chain) and a variant (which is denoted as a1 (II) M6) of a human type II collagen a1 chain (a1 (II) chain) constructed in the present disclosure are produced through recombinant expression in Pichia pastoris, a main degradation product (a main degradation band) in substantially the same proportion as a target product (a target band) of a full-length a1 chain occurring during the recombinant expression of native full-length a1 (I) chain and a1 (II) chain is eliminated, and a yield of the target product is improved.

The invention discloses a method for separating and purifying bovine lactoferrin by using a ligand affinity chromatography technology, and belongs to the technical field of protein purification. The method comprises the following steps: introducing a bovine lactoferrin gene into pichia pastoris, fermenting to obtain a pichia pastoris fermentation solution, centrifuging, resuspending, homogenizing at high pressure, centrifuging and filtering the pichia pastoris fermentation solution to obtain a filtrate, separating and purifying bovine lactoferrin in the filtrate by using an affinity chromatography technology, immobilizing a triazine dye ligand in a filler of a chromatographic column, and purifying the triazine dye ligand to obtain the bovine lactoferrin. An eluent is prepared, the concentration of NaSCN in the eluent is 0.2-0.5 mol / L, the volume concentration of ethylene glycol in the eluent is 15%-20%, the concentration of NaCl in the eluent is 1-2 mol / L, and the bovine lactoferrin in the chromatographic column is eluted. According to the method, the composition of the eluent is optimized, the three components in the eluent are matched with one another and have a synergistic effect, the stability of the bovine lactoferrin is protected, the purity of the bovine lactoferrin is guaranteed, meanwhile, the elution efficiency of the bovine lactoferrin is improved, and the dosage of the eluent is reduced.

The invention relates to pichia pastoris engineering bacteria and a preparation method thereof, and a method for producing cellooligosaccharide / disaccharide by using corn straws or wheat straws, and belongs to the technical field of genetic engineering. A cellulose incision enzyme gene and a cellulose excision enzyme gene are transferred into pichia pastoris engineering bacteria. The preparation method comprises the following steps: respectively inserting a cellulose incision enzyme gene and a cellulose excision enzyme gene into plasmids to construct expression vectors; transferring the expression vector into a competent cell of pichia pastoris GS115; and screening and identifying to obtain successfully transformed pichia pastoris engineering bacteria. According to the present invention, the pichia pastoris heterologous expression cellulose incision enzyme and cellulose excision enzyme are adopted to construct the pichia pastoris engineering strain capable of degrading the plant straw into the cellooligosaccharide / disaccharide, such that the reliable chassis strain is provided for the production of the starch from the cellulose;

The invention relates to a composition for relieving, repairing, tightening and resisting wrinkles as well as a preparation method and application of the composition. The composition is composite fermented oil obtained by fermenting vegetable fat by adopting a combination of enterococcus monteilii and pichia pastoris. In the fermentation process, lemon leaves and pseudo-ginseng leaves are added at the same time, and microbial fermentation liquor with special effects is prepared; and then mixing the fermentation liquor with silybum marianum seed oil, green tea oil, jojoba oil, macadamia nut oil, hazelnut oil and rice germ oil to prepare the compound fermentation oil. According to the method, the glycolipid surfactant is creatively added in the fermentation process, so that full contact and fermentation of the microbial bacterial liquid and the vegetable oil are remarkably promoted, and the problem that the microbial bacterial liquid and oil are difficult to mix in the vegetable fat fermentation process is solved, so that the contact efficiency between microorganisms and the vegetable oil is improved; the biotransformation efficiency is improved; furthermore, the content of flavonoids and polyphenols in the fermented oil is increased, and the moisturizing, whitening, soothing, tightening and anti-wrinkle properties of the fermented oil are enhanced.

The invention belongs to the technical field of cosmetic production, and discloses fermented oil with moisturizing, repairing and anti-oxidation effects, the fermented oil with the moisturizing, repairing and anti-oxidation effects is prepared by fermenting plant raw materials through strains, and the plant raw materials comprise soybeans, mint and onions. The strain is selected from at least one of lactobacillus plantarum, lactobacillus rhamnosus and pichia pastoris; the mass ratio of the soybeans to the mint to the onions in the plant raw materials is 3: (1-2): (0.1-1); the mass ratio of the strains to the plant raw materials is (1-3): 50; the fermented oil with good free radical scavenging capacity and cell migration promoting capacity is obtained by fermenting plant raw materials through the strain, and in addition, the invention further discloses a preparation method and application of the fermented oil with the moisturizing, repairing and anti-oxidation effects and cosmetics.

The application provides a recombinant Pichia pastoris strain and a construction method and application thereof. The construction method comprises the following steps: taking a Pichia pastoris strain as a host strain, constructing a double synthesis pathway of phenylalanine ammonia lyase and tyrosine ammonia lyase in the host strain to obtain a first engineering strain; relieving the branch acid feedback inhibition of the first engineering strain based on metabolic engineering to obtain a second engineering strain; optimizing the aromatic amino acid synthesis pathway of the second engineering strain to obtain a third engineering strain; knocking out the branch metabolic pathway of the third engineering strain to obtain a fourth engineering strain; increasing the precursor supply of the fourth engineering strain to obtain the recombinant Pichia pastoris strain. The construction method can systematically optimize the synthesis path of p-coumaric acid, and the recombinant Pichia pastoris strain for efficiently producing p-coumaric acid with methanol as a carbon source is constructed. Compared with the host strain, the yield of p-coumaric acid of the recombinant Pichia pastoris strain is significantly improved.

The invention relates to a method for producing vanillin from natural lignocellulose biomass through co-culture of pichia pastoris, which comprises the following steps: respectively constructing a xylan hydrolysis recombinant strain (for releasing ferulic acid) and a vanillin synthesis recombinant strain (for converting ferulic acid) to realize conversion production from a natural xylan substrate to vanillin. In order to reduce the generation of by-products, 15 genes for coding aldehyde dehydrogenase (ALDRs), alcohol dehydrogenase (ADHs) and aldehyde ketoreductase (AKRs) are further knocked out from the vanillin synthesis recombinant strain, so that the vanillin can be further converted into the by-product vanillic acid, thereby improving the yield and purity of the vanillin.

The invention discloses a recombinant engineering bacterium for expressing fetuB and application of the recombinant engineering bacterium, and relates to the technical field of genetic engineering. According to the recombinant engineering bacteria, pichia pastoris serves as host bacteria, after fetuB genes are introduced, efficient expression of the fetuB in the pichia pastoris is achieved by optimizing codons and improving fermentation conditions, and the expression quantities of CBS7435 under the shake-flask culture condition are 320 + / -25 mg / L, SMD116 under the shake-flask culture condition is 280 + / -30 mg / L, and SuperMan5 under the shake-flask culture condition is 580 + / -45 mg / L. After fermentation conditions are optimized, the culture yield of a fermentation tank can reach 27.35 g / L. The fetuin B disclosed by the invention is good in biological activity, high in expression, simple in operation process and suitable for large-scale industrial production.

The invention discloses recombinant human chemical fibronectin as well as a coding gene and application thereof. The method comprises the following steps: performing codon optimization on an amino acid sequence of recombinant human chemical fibronectin according to codon preference of pichia pastoris to obtain a coding gene nucleotide sequence SEQ ID NO.2, cloning the coding gene nucleotide sequence SEQ ID NO.2 between EcoRI and NotI restriction enzyme cutting sites of a pichia pastoris expression vector pPIC9K to obtain a recombinant expression vector pPIC9K-FNc, and transferring the recombinant expression vector pPIC9K-FNc into a P.pastoris GS115 expression host cell to obtain the recombinant human chemical fibronectin. Recombinant pichia pastoris P.pastoris GS115 / pPIC9K-FNc is obtained through genetic mycin G418 screening, then recombinant bacteria are subjected to fermentation culture and are subjected to induced expression by using methanol, and then the recombinant human chemical fibronectin is obtained after purification. The fibronectin produced by the method is large in molecular weight and has better biological activities such as wrinkle resistance and barrier repair.

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 relates to ester-producing aroma-producing yeast for making hard liquor as well as a preparation method and application thereof, and belongs to the technical field of liquor making. According to the invention, two microorganisms with high ester yield, namely saccharomyces cerevisiae and pichia pastoris, are screened, and the saccharomyces cerevisiae and the pichia pastoris which are compounded in different proportions are subjected to compound fermentation, so that the yields of ethyl acetate produced by the saccharomyces cerevisiae and the pichia pastoris which are compounded in different proportions are different. The yeast for making hard liquor is subjected to enhanced fermentation according to the optimal inoculation proportion, the content of ethyl acetate, ethyl hexanoate and ethyl lactate of the prepared yeast for making hard liquor is greatly increased, and the saccharifying power, the liquefying power, the fermenting power and the esterifying power of the yeast for making hard liquor are also remarkably improved. In addition, the invention also finds that the pichia pastoris and the saccharomyces cerevisiae of the strengthened yeast are always maintained at a relatively high level in the fermentation process, so that the quality of the yeast can be remarkably improved by compounding the pichia pastoris and the saccharomyces cerevisiae, and the strong aroma is given to the liquor in the fermentation of the Luzhou-flavor liquor.

The invention relates to a recombinant III-type human collagen expression strain with a whole chain length and a triple helix structure as well as a preparation method and application thereof, and belongs to the technical field of genetic engineering bacteria. The recombinant pichia pastoris genetically engineered bacterium expresses recombinant III-type human collagen with a full chain length and a triple helix structure, and a ku70 gene of the recombinant pichia pastoris genetically engineered bacterium is knocked out and knocked in a recombinant 4-proline hydroxylase coding gene and an FKBP22 coding gene. The recombinant pichia pastoris genetically engineered bacterium provided by the invention can be used for obtaining the recombinant full-chain-length triple-helix recombinant III-type human collagen with a higher expression level through high-density fermentation, does not contain heterologous sequences, has higher hydroxyproline content and thermal stability, is close to natural human collagen, has a stable triple-helix structure, is high in thermal stability, and can be used for preparing the recombinant III-type human collagen. The application in the fields of medical health and medical beauty such as medical beauty filling and tissue repair can be met.

The invention discloses pichia kudriavzevii for high yield of L-malic acid as well as a construction method and application thereof, and belongs to the technical field of biology, the pichia kudriavzevii is named as YM-023 and classified as Pichia kudriavzevii, the preservation number is CGMCC No. 35487, the preservation time is July 31, 2025, and the preservation number is CGMCC No. 35487. The preservation unit is China General Microbiological Culture Collection Center (CGMCC). The method comprises the following steps: starting from an acid-resistant yeast Kudriavzevii pichia pastoris strain YL-000 (the preservation number is CGMCC No.35680), carrying out metabolic engineering transformation to obtain a transformed strain, and ensuring that the transformed strain can realize efficient production of L-malic acid in a fermentation mode of adding a small amount of calcium carbonate neutralizer.

The invention relates to recombinant pichia pastoris capable of efficiently expressing III-type human-like collagen and application of the recombinant pichia pastoris, and belongs to the technical field of microorganisms. According to the invention, an III-type human-like collagen expression cassette is constructed in pichia pastoris GS115 and co-expressed with a molecular chaperone, a transcription factor and a translation factor, so that the expression efficiency of the III-type human-like collagen is improved, and the shake-flask fermentation yield reaches 0.93 g / L. And then fermentation conditions, material supplementing conditions, methanol feeding induction conditions and the like are optimized, so that the yield of the III-type human-like collagen in a 5L fermentation tank by the constructed recombinant pichia pastoris reaches 10.3 g / L, the efficient production of target protein is realized, a favorable tool is provided for the application of the III-type human-like collagen in the fields of food, cosmetics, medicines and the like, and the engineering application prospect is broad. Good industrial application prospects are realized.