337 results about "Pichia pastoris" patented technology

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 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 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 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 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.

The invention relates to pichia pastoris for producing brazitame protein as well as a construction method and application of the pichia pastoris, and belongs to the technical field of microorganisms. ExpL SP is used as a signal peptide in pichia pastoris, heterologous expression of Brazilian sweet protein is carried out through an AOX1 promoter, the ExpL SP and a Pro region of an alpha mating factor are further fused and transformed to obtain a new signal peptide to enhance the secretion efficiency, and the new signal peptide and a ribosomal protein synthesis factor Bcy1 are co-expressed to improve the protein synthesis capacity. The constructed pichia pastoris can be used for efficiently secreting the Brazilian sweet protein, the problems that in the prior art, when microorganisms are used for fermenting the Brazilian sweet protein, the Brazilian sweet protein is mistakenly folded, the sweet taste is reduced or lost are solved, the shake flask yield is stabilized at 450 mg / L, the fermentation tank yield reaches 5.215 g / L, and a high-cost-performance scheme is provided for industrial production of the Brazilian sweet protein.

The invention discloses recombinant laminin with anti-aging and repairing effects and a preparation method of the recombinant laminin, relates to the technical field of recombinant proteins, and aims to solve the technical problems that existing laminin is relatively low in yield and limited in application. According to the recombinant laminin with the anti-aging and repairing effects, the sequence of the recombinant laminin is shown as SEQ ID No.3, the total number is 363aa, and the predicted molecular weight is 40.4 kD. Compared with the prior art, the recombinant laminin is synthesized by embedding a laminin gene sequence into a pichia pastoris genome for heterologous expression. A cytological detection method for evaluating the efficacy of a skin care product is applied, and a research result shows that the recombinant laminin can remarkably promote cell adhesion and migration activity and can stimulate cells to generate collagen, so that the anti-aging and repairing functions of the recombinant laminin are shown.

The invention discloses a method for efficiently expressing FAD-dependent glucose dehydrogenase in pichia pastoris. The method comprises the following steps: (1) constructing a recombinant plasmid containing a promoter, a signal peptide, a glucose dehydrogenase gene and a purification tag; the nucleotide sequence of the signal peptide is as shown in SEQ ID No.2; (2) transforming the recombinant plasmids into host bacteria pichia pastoris to obtain recombinant pichia pastoris engineering bacteria capable of expressing FAD-dependent glucose dehydrogenase; and (3) carrying out fermentation culture, separation and purification on the recombinant pichia pastoris engineering bacteria to obtain the FAD-dependent glucose dehydrogenase. According to the invention, the expression level and the final yield of the FAD-GDH are obviously improved.

The invention relates to the technical field of ergothioneine, in particular to a process method for extracting ergothioneine from pichia pastoris fermentation liquor, which comprises the following steps: taking the pichia pastoris fermentation liquor containing ergothioneine, detecting isoelectric points of the fermentation liquor and charges carried by small molecular substances, then adding corresponding flocculants according to the charges carried, adjusting the pH value to be equal to the isoelectric points, and finally extracting the ergothioneine from the pichia pastoris fermentation liquor containing ergothioneine. (diluting the obtained first turbid solution with water, then adjusting the pH value to be equal to an isoelectric point, treating the second clear solution collected after filtration with an ultrafiltration membrane, adjusting the pH value of the collected ultrafiltration clear solution to be acidic, passing through a cationic resin column, eluting with an eluent, concentrating the collected eluent with a nanofiltration membrane, concentrating in vacuum, cooling, carrying out suction filtration, and washing to obtain the product. And drying to obtain the ergothioneine product with high yield and high purity.

The invention relates to a method for inducing pichia pastoris to express human alpha-lactalbumin by using ethanol, and belongs to the technical field of genetic engineering. Recombinant pichia pastoris using ethanol as an inducer for heterologous expression of human alpha-lactalbumin is constructed, three different expression vectors are used in the construction process, the first expression vector uses an ethanol inducible promoter to express fusion polypeptide of DNA binding protein and a transcription activator, and the second expression vector uses an ethanol inducible promoter to express fusion polypeptide of human alpha-lactalbumin. The second expression vector sequentially expresses a protein binding sequence, a methanol inducible promoter, an alpha signal peptide and human alpha-lactalbumin, the third expression vector overexpresses a signal recognition particle subunit participating in a protein targeting endoplasmic reticulum, and the yield of the constructed recombinant pichia pastoris shake flask fermentation human alpha-lactalbumin is 23.3 mg / L; the yield of the human alpha-lactalbumin reaches 1.1 g / L in a 3L fermentation tank, and methanol is not required to be added as an inducer in the fermentation process, so that the problems of toxicity and safety possibly caused by methanol are avoided, and efficient production of the human alpha-lactalbumin is realized.

The present invention belongs to the field of microbial metabolic engineering and synthetic biology technology applications, and particularly relates to a Pichia pastoris engineered strain that produces high sclareol production, as well as its construction method and application. A sclareol biosynthesis pathway is constructed in a host strain, and the intracellular mevalonate metabolic pathway and the central metabolic pathway are optimized, thereby obtaining an engineered bacterial strain A; the host strain is Pichia pastoris; or, metabolic regulatory factors are overexpressed or knocked out in the above-obtained engineered bacterial strain A, thereby obtaining an engineered bacterial strain B; or, in the above-obtained engineered bacterial strain B, a cell compartmentalization strategy is used to target the synthesis pathway to the peroxisome and optimize it, thereby obtaining an engineered bacterial strain C. The present invention provides a Pichia pastoris chassis cell for synthesizing the diterpenoid compound sclareol and an engineered strain for synthesizing sclareol; by introducing and optimizing the expression of the sclareol synthesis pathway, the sclareol yields in shake flask batch fermentation and bioreactor batch fed-batch fermentation reach 631.6 mg / L and 10.5 g / L, respectively.

The invention relates to the technical field of biological materials, in particular to an easy-to-produce recombinant humanized collagen purification process which comprises the following steps: performing centrifugal separation on a fermentation broth of a pichia pastoris expression strain to obtain a fermentation supernatant, and performing ultrafiltration concentration to obtain a fermentation supernatant concentrated solution; adding an ammonium sulfate solution into the fermentation supernatant concentrated solution, stirring, carrying out centrifugal separation, and collecting a precipitate I; dissolving the precipitate I with purified water, stirring and dissolving, repeating the step S2 again, and collecting a precipitate II; and dissolving the precipitate II with purified water, and then carrying out ultrafiltration, liquid change and concentration to obtain a purified recombinant humanized collagen solution. The recombinant humanized collagen purification process provided by the invention is simple in pretreatment process, easy to produce, relatively low in cost, small in protein loss and relatively high in protein recovery rate and purity; the prepared recombinant humanized collagen can be used as a raw material in the industries of medical beauty masks or biological dressings and the like, and can also be used for a subsequent refined chromatography process.

The invention relates to the technical field of biology, in particular to a pichia pastoris recombinant strain for expressing non-specific peroxygenase CciUPO and application of the pichia pastoris recombinant strain. The pichia pastoris recombinant strain is obtained by the following steps: S1, synthesizing a sequence of a CciUPO original gene sequence optimized based on a yeast codon; s2, carrying out recombination with the EV signal peptide after the directed evolution; s3, connecting the recombined target gene with an expression vector to construct a recombinant plasmid; and S4, transforming the recombinant plasmid into pichia pastoris competent cells, and screening out a recombinant strain containing the CciUPO recombinant gene. The expression quantity of the CciUPO obtained through the operation is increased by 10 times compared with that of a wild type CciUPO, the CciUPO has strict catalytic regioselectivity, can catalyze vitamin D3 to synthesize calcifediol, the selectivity is as high as 95%, the catalytic conversion rate of toluene is 95%, the selectivity is 78%, and the CciUPO has a relatively high application prospect in industrial production.

The invention specifically discloses a recombinant XVII type collagen and a preparation method thereof, and relates to the technical field of genetic engineering. The invention provides a recombinant XVII type collagen protein and a preparation method of the recombinant XVII type collagen protein. Compared with the prior art, the recombinant XVII type collagen pichia pastoris engineering strain provided by the invention is high in yield, the protein is not easy to degrade, and the recombinant XVII type collagen pichia pastoris engineering strain has a stable triple helix structure.

The invention relates to the technical field of gene engineering, in particular to a method for reducing acetic acid accumulation in the process of ethanol-induced expression of alpha-lactalbumin by regulating pichia pastoris. According to the recombinant yeast strain provided by the invention, the accumulation amount of acetic acid is greatly reduced in the process of inducing expression of target protein by adopting ethanol, acetic acid can be basically not accumulated, relatively high biomass and target protein yield can be achieved, and the highest yield of human alpha-lactalbumin up to now is achieved; and a strain resource and a foundation are provided for application of an ethanol-induced pichia pastoris expression system and development of bioengineering milk protein. The invention provides a method for solving the problem of acetic acid accumulation of pichia pastoris in the process of inducing expression of the target protein by using ethanol, and the method provides an effective strategy for popularization and application of an ethanol-induced expression system and improvement of the yield of the target protein induced and expressed by using the ethanol. The method has important significance on production of proteins such as alpha-lactalbumin and the like.

The invention discloses an optimized recombinant chicken double-cell factor as well as a preparation method and application thereof. The chicken recombinant double-cell factor coding gene provided by the invention contains complete ChIL-2 and ChIFN-alpha structures. The preparation process comprises the following steps: selecting a pichia pastoris expression system; optimizing the recombinant chicken double-cell factor according to the codon preference of pichia pastoris; inserting the optimized gene sequence into an expression vector, and introducing host bacteria to construct a recombinant yeast engineering strain; carrying out inoculated culture by using the obtained recombinant yeast, and screening strains containing target protein based on expression level to obtain required culture bacteria; and breeding and culturing the obtained high-expression-level culture bacteria. According to the invention, ChIL-2 and ChIFN-alpha are connected through a modified flexible linker, and a nucleotide sequence is replaced by preferred codons of pichia pastoris, so that a chicken recombinant cell factor can be secreted to a supernatant, the defect of ChIL-2 expression quantity is overcome, and the problem that proteins of ChIL-2 and ChIFN-alpha mainly exist in bacteria and inclusion bodies in prokaryotic expression is solved.