379 results about "Shake flask" 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 present invention relates to a recombinant yeast with high squalene production, a construction method and a use thereof, wherein the recombinant yeast is a saccharomyces cerevisiae that expresses or overexpresses HMGR gene, ADA gene, LEU2 gene, MVA pathway gene and squalene synthase gene ERG9. The present invention integrates acetaldehyde dehydrogenase gene ADA and HMG-CoA reductase gene tHMGR in multiple copies in saccharomyces cerevisiae, integrates the screened high-activity phosphoketolase gene PK and phosphotransacetylase gene PTA, weakens epoxysqualene synthesis, strengthens the mevalonate pathway, overexpresses yeast endogenous squalene synthase gene, etc. to obtain a recombinant yeast with high squalene production, so that the production in the shake flask stage of the production process exceeds 5g / L, and the production in the fed batch fermentation stage exceeds 50g / L, breaking through the current bottleneck of microbial synthesis of squalene production, and is suitable for practical promotion and application.

The invention discloses a porcine muscle stem cell line capable of being cultured in vitro for a long time and application of the porcine muscle stem cell line, and belongs to the technical field of stem cells and cell culture meat. The porcine muscle stem cell line is prepared by the following steps: performing CDKN2A gene knockout on porcine muscle stem cells by using a CRISPR / Cas9 gene editing technology, performing monoclonal screening and genotype identification to obtain a cell strain with successful CDKN2A knockout, and then performing carrier-free and serum-free suspension culture domestication on the cell strain. And a carrier-free and serum-free porcine muscle stem cell line capable of being subjected to in-vitro long-term suspension culture is obtained. Verification shows that the CDKN2A knockout porcine muscle stem cell line subjected to carrier-free and serum-free suspension culture domestication not only can be subjected to carrier-free and serum-free suspension culture in a shake flask for a long time, but also can maintain a good differentiation characteristic. The porcine muscle stem cell line which can be cultured on a large scale, is derived from edible animals and can be cultured in vitro for a long time is created, and excellent cell resources are provided for commercial production of cultured meat.

The invention relates to a saccharomyces cerevisiae strain for efficiently synthesizing cordycepin as well as a construction method and application of the saccharomyces cerevisiae strain, and belongs to the technical field of microbial genetic engineering. The construction method provided by the invention comprises the following steps: carrying out multi-copy genome integrated expression on heterologous codon optimized 2 '-carbonyl-3'-deoxyadenosine reductase genes and 3 '-adenosine monophosphate phosphohydrolase genes, so as to increase the dosage of key enzymes; and supplementing the defective gene of the engineering strain to obtain the saccharomyces cerevisiae strain B5U3 with high yield of cordycepin. According to the obtained saccharomyces cerevisiae strain for efficiently synthesizing the cordycepin, the cordycepin yield reaches 2.92 g / L after shake flask fermentation, the cordycepin yield reaches 7.5 g / L after fermentation in a fermentation tank, and a foundation is laid for safer and more reliable industrial production of the cordycepin.

The invention belongs to the technical field of biology, and discloses a Tibetan pig source bacillus subtilis, the classification name is bacillus subtilis Yirun2024, the Latin name is bacillus subtilis Yirun2024, the preservation number is CCTCC NO: M 20241752, the preservation date is August 7, 2024, the preservation unit is China Center for Type Culture Collection, and the preservation address is Wuhan University, Wuhan, China. When the bacillus subtilis is fermented for 12 hours in a shake flask, the endo-cellulase activity, the exo-cellulase activity and the beta-glucosidase activity of the bacillus subtilis are respectively up to 2902.72 U / mL, 11026.33 U / mL and 2983.04 U / mL, which are far better than those of similar engineering bacteria. Meanwhile, the invention further provides application of the bacillus subtilis, the bacillus subtilis has important significance in the field of promotion of application of fiber resources, and meanwhile, the bacillus subtilis can keep a high survival rate in a high-temperature environment.

The invention relates to the technical field of genetic engineering, and particularly discloses a genetic engineering bacterium for producing melanin as well as a construction method and application of the genetic engineering bacterium. According to the application, the yield of the melanin can be effectively increased by modifying a metabolic pathway of the genetically engineered bacterium, and the yield of the melanin reaches 6.2 g / L and 18.5 g / L respectively through shake-flask culture and 5L fermentation tank culture. Compared with the prior art, the yield of melanin produced by the genetically engineered bacterium is improved by 30% at the shake flask fermentation level, the fermentation yield of a fermentation tank is improved by 4.3 times, in addition, expensive tyrosine is changed into glucose as a fermentation precursor substance, the production cost is greatly reduced, and an efficient and feasible solution is provided for large-scale production of melanin.

The invention provides a recombinant strain for controllably synthesizing high-proportion 4-hydroxybutyric acid (4HB) polyhydroxyalkanoate (PHA) as well as a construction method and application of the recombinant strain. The recombinant strain disclosed by the invention can be used for efficiently producing high-proportion poly (3-hydroxybutyrate-4-hydroxybutyric acid) (P34HB for short) by using glucose as a unique carbon source or a 4HB structure related or non-related carbon source in amplification production of a shake flask, a 7L fermentation tank, a 100L fermentation tank and a 5000L fermentation tank.

The present invention relates to a genetically engineered strain of Saccharomyces cerevisiae for the synthesis of 7-DHC exocytosis, and a construction method and application thereof. The present invention constructs a recombinant S.cerevisiae strain sc13 that can efficiently transport 7-DHC to the extracellular space. When it is used for 500mL shake flask biphasic fermentation, the total production of 7-DHC reaches 28.189mg / g (secretion amount 11.701mg / g), compared with the control sc1 strain, the total production of 7-DHC increases by 14.54 times, and the total extracellular secretion increases by 13.77 times, wherein the secretion production of extracellular 7-DHC accounts for 41.51%. The recombinant strain constructed by the present invention has stronger exocrine ability and higher yield, provides a guiding idea for the synthesis of 7-DHC and the simplification of the separation and extraction of 7-DHC, and has broad application prospects.

The invention relates to the technical field of microorganisms and particularly discloses bacillus velezensis for producing tetramethylpyrazine and application of the bacillus velezensis, the preservation number of the strain is CCTCC NO: M20232058, and the bacillus velezensis is obtained by separating and purifying from high-temperature Daqu of Maotai-flavor liquor in the liquor industry of Jin-brand Maotai Town in Guizhou. The bacillus velezensis disclosed by the invention can produce 1875.4 mg / L of tetramethylpyrazine by performing shake flask fermentation on a liquid culture medium for 7 days, and the tetramethylpyrazine can reach 4810.8 mg / L by optimizing a culture medium formula and culture conditions. The bacillus velezensis is applied to small-scale brewing of Maotai-flavor liquor, the content of tetramethylpyrazine in the Maotai-flavor liquor reaches 34.5 + / -1.4 mg / L and is increased by 325.93% compared with a contrast, and the bacillus velezensis can be used for increasing the content of tetramethylpyrazine in the Maotai-flavor liquor.

The invention discloses an oligosaccharide enzyme fermentation process control system and method based on pH and dissolved oxygen electrode feedback, relates to the technical field of oligosaccharide enzyme fermentation, and aims to improve the yield and activity of oligosaccharide enzyme. The system comprises a pH electrode, a dissolved oxygen electrode, a stirrer, a ventilation system and a temperature control system, and accurate control is realized through a data acquisition and processing unit, a logic control unit and a human-computer interface. The control method comprises the steps of seed culture, shake flask fermentation, fermentation tank culture, monitoring and evaluation as well as optimization and adjustment. Through a segmented oxygen control strategy and dissolved oxygen and stirring linkage control, the dissolved oxygen level is accurately controlled, the yield and activity of the oligosaccharide enzyme are remarkably improved, the fermentation process conditions are optimized, and the monitoring and evaluation capability is enhanced. The method is suitable for industrial production of the oligosaccharide enzyme, and is beneficial for improving the product quality and the economic benefit.

The invention discloses a kitchen waste oil-based sophorolipid high-yield candida utilis strain and an application thereof. Belongs to the technical field of directional domestication breeding. According to the method, wild candida bombicola is used as an original strain, and is sequentially domesticated in a culture medium containing kitchen waste oil with the concentration gradient of 2%, 4%, 6%, 8% and 10% to obtain the S.bombicola x10. The invention further discloses an application of the domesticated strain in fermentation production of sophorolipid, after the strain is subjected to shake flask fermentation, the sophorolipid yield reaches 60.19 g / L and is improved by 16.22% compared with that of a wild strain, and the acid type sophorolipid yield is improved by 32.11%. When the concentration of the lactone sophorolipid generated by fermentation is 1.25 mg / mL, the growth of the bacillus subtilis is completely inhibited. The invention provides an excellent production strain for industrial production of sophorolipid, and has good theoretical research significance and practical application value.

The invention discloses an aspergillus niger recombinant bacterium AnCT-XynB-K-agdA and application thereof, and belongs to the technical field of microbiology and bioengineering. According to the invention, Aspergillus niger AnCat is taken as an expression host, firstly, an acid protease regulatory factor prtT is knocked out to obtain a defective strain, and then different chaperonins are subjected to fusion expression by optimizing integration sites of a laccase lcc9 expression cassette, so that the high-expression laccase chaperonin is obtained. According to the method, an expression vector pC3-5 'agdA-PcitA-XynB-L-lcc9-hph-3' agdA is constructed, a recombinant bacterium AnCT-XynB-K-agdA is further obtained, the enzyme activity of the extracellular laccase subjected to shake flask fermentation reaches 1821.2 U / L, and finally, the recombinant efficient expression of the laccase Lcc9 in aspergillus niger is realized.

The invention discloses a double-function flavin adenine dinucleotide synthetase mutant and application, the double-function flavin adenine dinucleotide synthetase is called RFK / FADS for short, and the amino acid sequence of the double-function flavin adenine dinucleotide synthetase is shown as SEQ ID NO.1; and the RFK / FADS mutant is RFK / FADSF55A, RFK / FADSG43Q, RFK / FADSG43Q-F55H, RFK / FADSG43Q-F55N or RFK / FADSG43Q-F55N-F129Y, and the RFK / FADSG43Q-F55N and the FADSG43Q-F55N-F129Y can be used for preparing the According to the genetically engineered bacterium disclosed by the invention, flavine adenine dinucleotide is produced through a simple fermentation process by taking glucose as a substrate under a shake flask aerobic condition, and the yields of the strains ZF02, ZF03, ZF04, ZF05 and ZF06 are respectively increased by 120%, 130%, 166%, 246% and 286% in sequence compared with those of ZF01.

The present invention discloses a yeast engineering bacterium containing a (-)-α-bisabolol synthase mutant and its application. By designing (-)-α-bisabolol synthase, the present invention obtains a mutant with high expression level, co-expresses the mutant with ERG20, highly expresses the key rate-limiting enzymes in the metabolic pathway, deletes the promoter to reduce the side reaction pathway, and adopts a multi-copy strategy to improve the metabolic flux in the (-)-α-bisabolol biosynthesis pathway. The shake-flask fermentation yield of the yeast engineering bacterium constructed by the present invention is close to 70 mg / L, which is about 35 times higher than that of the starting strain, and has broad application prospects in the biological fermentation production of (-)-α-bisabolol.

The invention discloses mutant protein of dihydroxyacetone phosphate phosphatase and application of the mutant protein. The invention provides a phosphodihydroxyacetone phosphatase mutant protein, which is characterized in that residues of phosphodihydroxyacetone phosphatase corresponding to 21st, 36th, 42nd, 49th, 64th, 82nd, 150th and 244th sites of an amino acid sequence shown in a sequence 1 are mutated to obtain a protein with phosphodihydroxyacetone phosphatase activity; and the synthesis capability of the 1, 3-dihydroxyacetone is improved. The invention also provides related biological materials and derivatives of the protein, and application of the protein in preparation of 1, 3-dihydroxyacetone. The yield of 1, 3-dihydroxy acetone of the protein is greatly increased, compared with starting wild type dihydroxy acetone phosphate phosphatase, the yield of 1, 3-dihydroxy acetone obtained through shake flask fermentation of the dihydroxy acetone phosphate phosphatase mutant SF6-11 is increased by 1.65 times, and the yield of 1, 3-dihydroxy acetone is increased to 4.54 + / -0.09 g / L from 2.75 + / -0.07 g / L.

The invention discloses coprinus cinereus sourced laccase as well as a recombinant expression strain and a high-efficiency expression method thereof, and belongs to the technical field of microbiology and bioengineering. According to the invention, aspergillus niger MA70.15 is used as an experimental strain, and a random integration strategy is adopted, so that a pC3-PcitA-SPGlaA-lcc9-TtrpC-pyrG expression vector is integrated in an upstream region of a promoter of host bacteria citrate synthase in a form of nine continuous copies. In order to explore the influence of particles and osmotic regulators in a fermentation culture medium on laccase production through fermentation of recombinant aspergillus niger, the particles and osmotic regulators of different types and concentrations are added, so that the enzyme activity of the laccase fermented in a shake flask of the recombinant strain AnRcgL1 reaches 1566.7 U / L, the enzyme activity fermented in a 1L fermentation tank reaches 1961 U / L, and the recombinant high-efficiency expression of the laccase Lcc9 in the aspergillus niger is successfully realized.

The invention belongs to the technical field of gene engineering, and particularly relates to a strain for efficiently expressing beta-lactoglobulin as well as a construction method and application of the strain. By utilizing the beta-lactoglobulin expression vector disclosed by the invention, beta-lactoglobulin is guided to be secreted out of a host cell by an exogenous signal peptide, and efficient secretory expression of the beta-lactoglobulin in the host cell is realized by integrating a multi-copy expression cassette through a genome and co-expressing ATP enzyme Ssa4 participating in protein folding and coping with pressure; the amino terminal and the carboxyl terminal of the secreted and expressed beta-lactoglobulin do not contain redundant amino acids, and do not contain animal-derived components. Meanwhile, the invention provides a construction method and a fermentation culture method of the engineering strain, compared with a single-copy genetically engineered strain, the expression quantity of the genetically engineered strain in shake-flask fermentation is increased by 2.47 times, and the genetically engineered strain has a good industrial application prospect.

The utility model provides a shake flask fixing support based on a shaking table, which belongs to the technical field of cell culture equipment, the shaking table is provided with a tray, the shake flask fixing support is connected with the shaking table, the shake flask fixing support comprises a plurality of supporting rods and a fixing frame, and the fixing frame and the tray are parallel to each other and are horizontally arranged. The supporting rods are perpendicular to the fixing frame and the tray and are connected with the fixing frame and the tray respectively. A first fixing net is arranged on the tray, a second fixing net is arranged on the fixing frame, the first fixing net is formed by staggering first connecting ropes, and the second fixing net is formed by staggering second connecting ropes; the first connecting rope is detachably connected with the tray, and the second connecting rope is detachably connected with the fixing frame. The fixing support can be matched with a tray to fix the lower portion of the shake flask and the bottleneck portion of the shake flask, so that the anti-overturning effect is achieved, the fixing support is suitable for shake flasks of different specifications, cost is saved, and efficiency is higher.

The invention belongs to the technical field of microorganism mutation breeding, and particularly relates to a nisin high-yield mutant strain with enhanced production performance. The mutant strain is specifically lactococcus lactis 2686, and the preservation number of the mutant strain is CCTCC (China Center for Type Culture Collection) NO: M 2023219. The strain has relatively high osmotic pressure tolerance, can still normally grow and produce nisin when the osmotic pressure is higher than 0.66 osM / kg H2O, meanwhile, the strain has excellent nisin production capacity, the titer reaches 9834 IU / mL when the strain is fermented in a shake flask for 21 hours, and the titer reaches 21000 IU / mL when the strain is fermented in a fermentation tank for 22 hours. The method is high in yield, good in stability, low in production cost and suitable for further industrial application.

The invention discloses a high-yield delta-tocotrienol genetically engineered bacterium and application thereof, and belongs to the technical field of gene recombination. The invention discloses a genetically engineered bacterium for high yield of delta-tocotrienols, which is characterized in that yarrowia lipolytica ATCC MYA-2613 is used as an original strain, and a ku70 gene is knocked out; the gene is used for expressing and coding 4-hydroxyphenylpyruvate dioxygenase, the gene is used for coding uromelanic acid phytate transferase and the gene is used for coding tocopherol cyclase; intensifying a shikimic acid pathway; a mevalonic acid pathway is strengthened; an isopentenol path is introduced; carrying out protein fusion on the HPD and the HPT; truncating a signal peptide of a gene encoding a tocopherol cyclase and back-filling a leucine defect. The genetically engineered bacterium constructed by the invention has industrial stability, can be amplified by more than 10 times in a 2L fermentation tank level compared with a shake flask level, can produce 616.45 mg / L of delta-tocotrienol after being cultured for 240 hours, and has industrial application prospects.

The invention provides a genetically engineered bacterium for high yield of 9 alpha-hydroxyandrostane-4-ene-3, 17-diketone as well as a construction method and application of the genetically engineered bacterium for high yield of 9 alpha-hydroxyandrostane-4-ene-3, 17-diketone. According to the invention, the production of by-products 4-HBC, 4-AD and 9-OH-3-OPCM is eliminated by blocking the expression of Opccr, SalA and TeB in mycobacterium neoaurum and overexpressing KshA1, the production limitation of 9-OHAD is liberated by overexpressing rate-limiting enzyme ChsH1-2, and finally, under the condition that the concentration of phytosterol is 20g / L, the purposes that the yield of 9-OHAD is 13.4 g / L, the molar yield is 91.1% and almost no by-product exists are achieved. When amplification is carried out at the concentration of 45g / L of phytosterol in a 5L bioreactor, soybean oil and (2-hydroxypropyl)-beta-cyclodextrin are used as auxiliaries, and a 9-OHAD conversion result at the concentration of 20g / L in a shake flask is basically reproduced.

The invention discloses a yarrowia lipolytica engineering strain with high yield of resveratrol as well as construction and application of the yarrowia lipolytica engineering strain, and belongs to the technical field of synthetic biology and metabolic engineering. The invention provides a yarrowia lipolytica engineering strain. A 4-coumaric acid-CoA ligase gene, a multi-copy resveratrol synthase gene, a transketolase 1 gene, a fusion gene of a histidine phosphate aminotransferase gene and a tyrosine ammonia lyase gene, a glycolytic pathway hexokinase gene, a 6-phosphofructokinase-1 gene and a phosphoglycerate kinase gene are integrated, so that the recombinant protein is obtained. According to the present invention, by using glucose as the substrate, the high yield of resveratrol can be achieved by using glucose as the substrate, the resveratrol yield during shake-flask culture fermentation can achieve 3.157 g / L, the resveratrol yield can be increased to 30.7 g / L during 5L-scale fed-batch fermentation, and the resveratrol yield can be increased to 30.7 g / L when the resveratrol yield is increased to 30.7 g / L when the resveratrol yield is increased to 30.7 g / L when the resveratrol yield is increased to 30.7 g / L; the maximum value is reported by the yarrowia lipolytica system at present.

The invention belongs to the technical field of fermentation, and particularly relates to a saccharomyces cerevisiae culture with banana fragrance as well as a preparation method and application of the saccharomyces cerevisiae culture. The preparation method of the yeast culture comprises the following steps: S1, preparing a single colony plate: performing activation culture on saccharomyces cerevisiae to obtain a saccharomyces cerevisiae single colony plate; s2, shake-flask seed culture: inoculating the saccharomyces cerevisiae single colony plate into a YPD liquid culture medium for amplification culture to obtain a shake-flask seed culture solution; s3, aerobic fermentation: inoculating the shake flask seed culture solution into a fermentation tank, and performing aerobic fermentation by controlling dissolved oxygen in stages to obtain a saccharomyces cerevisiae culture; the separated saccharomyces cerevisiae L9 strain is adopted as a fermentation strain for fermentation, aerobic fermentation is performed in a staged dissolved oxygen control mode, and the prepared fermentation product is rich in banana fragrance and has the skin care effects of promoting cell proliferation and migration, promoting repair, reducing inflammatory factors and skin pigmentation and improving skin moisture content.

The invention discloses a 4-hydroxyphenylacetic acid 3-hydroxylase mutant and application thereof, recombinant expression plasmids and corresponding genetically engineered bacteria M214V, R290N and E294I are constructed by using genes for coding the mutants (EcHpaBM214V, EcHpaBR290N and EcHpaBE294I) and nucleotide sequences for coding EcHpaC genes respectively, and after the genetically engineered bacteria disclosed by the invention are subjected to shake flask fermentation in a 30mL M9Y culture medium containing 50mu g / mL chloramphenicol for 24 hours, the yield of the mutants is increased, and the yield of the mutants is increased. The yield of the tanshinol is 748.04 mg / L, 895.55 mg / L and 793.36 mg / L. The method breaks through the limitation that the substrate generated by the 4-hydroxyphenylacetic acid 3-hydroxylase through catalysis is poor in specificity and low in catalysis efficiency, and the yield of the tanshinol is high. The method has the advantages of being high in catalytic efficiency, simple in process and environmentally friendly.

The invention provides a recombinant Aspergillus niger strain with high yield of trans-aconitic acid, and belongs to the technical field of gene engineering. The recombinant aspergillus niger strain can simultaneously express aconitase and aconitic acid isomerase. According to the genetic engineering aspergillus niger strain provided by the invention, the trans-aconitic acid fermentation yield is generally higher than that of an original strain, and the strain with the highest yield can reach 51g / L on a shake flask level, so that the genetic engineering aspergillus niger strain has very high application value.

The invention discloses a method for obtaining and culturing ED pathway regulated and controlled engineering bacteria for fermentation production of vitamin B12, a mutant strain is based on ensifer adhesion, an ED pathway key gene edd gene in the ensifer adhesion of a vitamin B12 production strain is overexpressed by a homologous recombination single exchange method, an edd gene overexpressed mutant strain is constructed, and the edd gene overexpressed mutant strain is used for producing the vitamin B12. The purpose of disturbing the ED pathway to realize the metabolic migration of central carbon is achieved, and the method is applied to the production of vitamin B12. A shake flask fermentation experiment proves that overexpression of the edd gene can remarkably promote synthesis of a VB12 product, and the edd gene has good commercial and scientific research values.

The invention discloses a bifunctional flavin adenine dinucleotide synthetase mutant and an application of the bifunctional flavin adenine dinucleotide synthetase mutant in FAD synthesis. The mutant disclosed by the invention is RFK / FADSC18R or RFK / FADSC18R-R46H, and the structural formula of the mutant is as shown in the specification. The amino acid sequence of the RFK / FADSC18R is as shown in SEQ ID NO. 15, and the amino acid sequence of the RFK / FADSC18R-R46H is as shown in SEQ ID NO. 16. A genetically engineered bacterium carrying the bifunctional flavin adenine dinucleotide synthetase mutant gene plasmid is used for producing flavin adenine dinucleotide by using glucose as a substrate through a simple fermentation process under a shake flask aerobic condition, and the FAD yields of strains XF02 and XF03 containing mutant genes are 79.31 mg / l and 167.56 mg / l in sequence; and compared with a strain XF01 containing a non-mutated gene, the mutant gene is improved by 58.62% and 235.12% respectively. A microbial'cell factory 'is constructed through a metabolic engineering strategy, and the modified microorganisms can generate required natural products in microbial cells by utilizing low-price raw materials. The progress not only reduces the production cost of the FAD, but also improves the environmental sustainability of the production process of the FAD.

The invention relates to the field of microbial genetic transformation, and discloses a preparation method of Ilyonectria robus fungus protoplast and a construction method of a genetic transformation system.The preparation method comprises the following steps that S11, I.robus hyphae are prepared, after I.robus is cultured on a PDA solid culture medium for 14 days, the I.robus hyphae are inoculated to a CMC liquid culture medium to be subjected to shake-flask culture, and spores are filtered and collected; diluting the spore concentration, inoculating into a YEPD liquid culture medium, carrying out shake-flask culture, filtering and collecting hyphae; and S12, preparing enzymatic hydrolysate, namely adding the hyphae prepared in the step S11 into the enzymatic hydrolysate prepared in the step S12, carrying out shake flask enzymolysis, filtering, centrifuging filtrate, discarding supernate, adding a 0.7 M NaCl solution, centrifuging, and discarding the supernate, so as to obtain the I.robus fungus protoplast. The protoplasts with proper quantity and good quality can be efficiently obtained, and the transformation efficiency of a subsequent genetic transformation system is improved; and the constructed genetic transformation system can be used for construction of a mutant library of the I.robusta strain, interaction of a host plant and the I.robusta strain and research on the pathogenesis of the I.robusta strain.

The invention discloses a germacene A synthase mutant and application thereof, and belongs to the technical field of enzyme engineering and microbial engineering. Aiming at the bottleneck of the catalytic efficiency of the germacene A synthase, the optimal germacene A synthase mutant DlGASS32H-I97V-Y315F is obtained through semi-rational design based on sequence conservative analysis by taking the germacene A synthase from fungi with a separation wheel layer carbon shell as an object, and the optimal germacene A synthase mutant DlGASS32H-I97V-Y315F is verified through shake flask fermentation in engineering bacteria saccharomyces cerevisiae, so that the yield of beta-elemene is increased by 24%. The combined mutant obtained through a key gene conserved sequence mutation prediction strategy shows remarkably improved catalytic performance, the effectiveness of guiding an enzyme directed evolution strategy based on multi-species sequence conservative analysis is verified, and the yield of germacene A or beta-elemene is remarkably improved. Meanwhile, the method has the advantages of simplicity and convenience in operation, environmental friendliness, mild reaction conditions and the like, and has a very good application prospect in the field of production of germacene A.

The invention discloses a bumblebee candida for efficiently producing sophorolipid as well as a construction method and application of the bumblebee candida. According to the invention, metabolic transformation is carried out on a wild type strain of candida bumblebee, a peroxisome membrane transporter gene PXA1 is knocked out, so that a beta-oxidation pathway of fatty acid competing with a sophorolipid synthesis pathway is moderately inhibited, and on the basis, an endogenous potent promoter pENO is utilized to enhance expression of CYP52M1; therefore, the bumblebee candida engineering bacteria for efficiently producing sophorolipid are constructed. Compared with an original strain, the recombinant candida bumblebee has the advantages that efficient conversion of a fermentation substrate and high yield of sophorolipid can be realized, and the sophorolipid yield of shake flask fermentation reaches 88.50 g / L. The strain is fermented in a 5L fermentation tank and fermented for 7 days after rapeseed oil is added, 232.27 g / L of crude sophorolipid is produced, and high productivity is kept.