295 results about "Shake flask" patented technology

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 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 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 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 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 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 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 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 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.

The invention discloses a lactococcus lactis strain with high nisin yield and application, and belongs to the technical field of industrial microorganisms. According to the invention, a nisin-tolerant nisin high-producing strain (Lactococcus lactis MN6) is successfully bred on the basis of compound mutation of ethyl methane sulfonate and heavy ion beam and in combination with nisin resistance screening, the strain can tolerate a fermentation environment with the nisin concentration of 6000 IU. ML <-1 >, the nisin yield through shake flask horizontal fermentation can reach 10776 IU. ML <-1 >, which is 9.76 times of the shake flask fermentation yield of original bacteria, the strain has good passage stability, and the strain can be used for producing a high-yield strain of nisin. The method is more suitable for industrial production.

The invention relates to bacillus subtilis for expressing bovine-derived beta-lactoglobulin and a construction method of the bacillus subtilis, and belongs to the technical field of microorganisms. According to the bacillus subtilis provided by the invention, bacillus subtilis BSF01 is used as a host, a constitutive promoter P566 is used for heterologous expression of a bovine-derived beta-lactoglobulin coding gene optimized by a codon, the yield of beta-lactoglobulin obtained by shake flask fermentation for 30 hours reaches 0.37 g / L, the yield of beta-lactoglobulin obtained by 3L fermentation tank fermentation for 44 hours reaches 3.06 g / L, the production intensity is 0.07 g / L / h, and the production cost is low. The bovine-derived beta-lactoglobulin is efficiently and insolubly expressed in a food safety level strain, the food industrial application of the beta-lactoglobulin is promoted, and the problems of food safety and excessive glycosylation possibly existing in heterologous expression of the beta-lactoglobulin in the prior art are solved.

The invention discloses a construction method of nicotinamide engineering bacteria for cosmetics, which comprises the following steps: amplifying target DNA, recovering the target DNA, carrying out seamless cloning or CPEC connection on the recovered DNA fragment and a vector, carrying out seamless cloning by using a seamless cloning kit of an all-type gold company, carrying out connection by using a sterile ultrapure water complementing system, and carrying out plasmid construction. The method comprises the following steps: inoculating a liquid culture medium with a strain which is verified to be correct, carrying out plasmid extraction on the obtained bacterial liquid by using an AxyPrep plasmid small extraction kit, carrying out shake flask small test on enzyme activity, thallus propagation and protein expression after chassis strain gene editing, adding a substrate 3-cyanopyridine solution into the crushed thallus for reaction, and carrying out HPLC (High Performance Liquid Chromatography) detection on nicotinamide. According to the method, the nitrile hydratase is expressed through metabolism of microorganisms, conversion from 3-cyanopyridine to nicotinamide is achieved through one-step reaction after cell disruption, the enzyme activity of the nitrile hydratase is improved, and meanwhile the generation amount of the by-product nicotinic acid is reduced.

The invention relates to the field of microorganisms, in particular to bacillus velezensis and application of the bacillus velezensis in preparation of surfactin, the preservation number is CCTCC NO: M 20251689, the bacillus velezensis is a wild type bacillus velezensis strain, and the surfactin yield of the bacillus velezensis is 2-3 times higher than that of a conventional wild strain. At the shake flask level, the yield of the surfactin reaches 10.4 g / L, when fed-batch fermentation is carried out in a 5L fermentation tank, the yield reaches 22.3 g / L, the yield and production intensity are higher than the currently known wild fungus fermentation level, and the method has huge industrial application potential.

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 relates to the technical field of microbial directional cultivation, in particular to a gene editing-based MICP functional bacteria directional cultivation method and system, and the method comprises the following steps: obtaining an original strain library with MICP activity, carrying out strain activation on the original strain library, carrying out high-throughput sequencing on the original strain library, and carrying out gene mining on an initial sequence; the method comprises the following steps: carrying out gene editing on strains in an initial strain library by using a gene editing tool, carrying out shake-flask activation on engineered candidate strains to obtain an activated recombinant bacterium solution, carrying out high-density culture on the activated recombinant bacterium solution by using a pre-constructed fermentation tank, screening out an MICP strain with the optimal function, carrying out continuous passage bacterium function detection on the optimal MICP strain, and carrying out high-density culture on the MICP strain with the optimal function. And based on the MICP functional bacteria, finishing directional cultivation of the MICP functional bacteria based on gene editing. According to the invention, directional cultivation of the MICP functional bacteria is efficiently realized by accurately targeting the key gene with the MICP function, and the MICP functional bacteria are corrected when the problems of function instability and the like exist, so that the more stable MICP functional bacteria are obtained.

The invention discloses a genetically engineered bacterium for high yield of L-alanine as well as a construction method and application of the genetically engineered bacterium, a dominant strain is obtained by knocking out an alanine racemase gene dadX and integrating coding sequences of alanine dehydrogenase from different sources, and the shake flask yield is 10.8 g / L; the activity of a corresponding mutant M18L / E75A / A78S / I266V is 4.3 times that of a wild type; 6 by-product related genes are knocked out, meanwhile, a multi-copy strain integrated with alanine dehydrogenase is constructed, and the yield of E.coli A-LB6 in a 5L fermentation tank is increased to 133.3 g / L. Through a dynamic regulation and control strategy, an oxygen-sensitive promoter Pomp * is adopted to replace a phosphoenolpyruvate carboxylase promoter, so that succinic acid byproducts are reduced by 85.8%, and the yield reaches 143.5 g / L after 48 hours of aerobic and anaerobic two-stage fermentation. And finally, by regulating and controlling the ATP synthetase gene cluster atp promoter, the yield of L-alanine in a 50 L fermentation tank reaches 151.3 g / L, and the saccharic acid conversion rate reaches 95.2%. The combination of polygene knockout and dynamic metabolism regulation effectively enhances the biosynthesis ability of alanine, and provides an efficient engineering strain for industrial production.

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 provides a saccharomyces cerevisiae strain with high phytosphingosine production. On the basis of the saccharomyces cerevisiae strain with high phytosphingosine production, the invention constructs an engineering saccharomyces cerevisiae strain for efficiently synthesizing phytosphingosine, reduces the expression of proteins encoded by LCB4, SHM2, CHA1, ORM2, ELO3 and SHM1 genes, and increases the expression of proteins encoded by TSC10, SYR2 and LCB2 genes by using a constitutive promoter, and the phytosphingosine shake flask amount reaches 9.52 mg / g dry weight, and then increases the expression of proteins encoded by the HAC1 gene by using a constitutive promoter, and the phytosphingosine shake flask amount is further increased by 4.34 times to 41.33 mg / g dry weight, and the phytosphingosine production reaches the highest production of shake flask fermentation reported so far. The production of phytosphingosine produced by fermentation in a 5L fermenter reaches 150.54 mg / g dry weight, and the titer is 2817 mg / L.

The invention discloses a series of recombinant bacteria for expressing a steroid compound taking a cyclopentane multi-hydrogen phenanthrene structure as a structural mother nucleus, such as recombinant strains for expressing cholesterol, 22-hydroxycholesterol, diosgenin or polyphyllin II, and construction and application of the strains. The invention provides a recombinant bacterium which can efficiently express a steroid compound taking cyclopentane multi-hydrogen phenanthrene as a structural mother nucleus, for example, the yield of diosgenin can be increased to the level of 40 mg / L or above, heterologous synthesis of the polyphyllin II is also realized to obtain a polyphyllin II synthetic strain, the yield of the polyphyllin II in a shake flask reaches 0.13 mg / L, and the yield of the polyphyllin II in the shake flask reaches 0.13 mg / L. The strain is the first strain for heterologous synthesis of the polyphyllin II at present.

The invention relates to the technical field of synthetic biology, in particular to a squalene high-yield saccharomyces cerevisiae engineering bacterium and a construction method and application thereof.Saccharomyces cerevisiae CEN.PK.2-1D is used as an original strain, pentose phosphate pathway genes ZWF1, GND1, TAL1 and TKL1 and IDP1 genes of TCA circulation are integrated in a genome of the original strain through homologous recombination, and the squalene high-yield saccharomyces cerevisiae engineering bacterium is obtained. Constructing a dual-channel cofactor supply system to improve the intracellular NADPH level and the NADPH / NADP + ratio; according to the saccharomyces cerevisiae genetic engineering strain Sc-Sq04 constructed by the method disclosed by the invention, the yield of squalene obtained by shake flask fermentation reaches 741.08 + / -28.9 mg / L by further integrating all genes of an MVA way, ERG20 and ERG9 genes, and a new thought is provided for transforming saccharomyces cerevisiae to synthesize terpene compounds and promoting industrial application.

The invention discloses a strain and method for increasing the yield of ergothioneine in saccharomyces cerevisiae and application, and belongs to the field of bioengineering. The invention relates to a method for improving the yield of ergothioneine in saccharomyces cerevisiae through a protein fusion technology, gene integration optimization and a culture medium strategy. A flexible linker FL1 is designed to fuse EGT1 and EGT2, the gene copy number and the SAM metabolic pathway are optimized, and a specific culture medium formula is combined, so that the shake flask fermentation yield reaches 122.5 mg / L, and the yield of a 10L fermentation tank reaches 2540 mg / L. The invention provides an efficient technical scheme for industrial production of ergothioneine.