210 results about "Shake flask" patented technology

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

The invention discloses a preparation method of a novel composite carbon source. The preparation method comprises the following steps: S1, preparing a solution; s2, preparing a culture medium; s3, extracting a genome; s4, extraction of plasmid DNA (deoxyribonucleic acid); s5, preparation of agarose gel electrophoresis; s6, constructing a linearized vector plasmid; s7, preparing an electrotransfection competent cell; and S8, electric conversion treatment. According to the preparation method of the novel composite carbon source, pfkA and pfkB can be respectively, independently and simultaneously knocked out when the carbon source is prepared by utilizing a gene editing technology, the consumption of a glucosamine precursor 6-fructose phosphate in an EMP pathway can be reduced, more glucose is used for synthesizing a target product, and by optimizing the preparation process of the composite carbon source, the yield of the target product is improved. The shake-flask fermentation yield of the glucosamine can be obviously improved, the conversion rate of the composite carbon source is also improved, and cell growth and synthesis of the target product glucosamine can be reasonably balanced, so that the yield of the glucosamine is improved.

The invention discloses recombinant poria cocos chitin endonuclease as well as a gene, a preparation method and application thereof. The nucleotide sequence of the gene is shown as SEQ ID NO.1 in a sequence table. According to the nucleotide sequence as shown in SEQ ID NO.1, high-level recombinant secretory expression of target protein in pichia pastoris host bacteria can be realized by taking pichia pastoris inducible expression plasmid pPICZ alpha A as a carrier. Under a shake flask fermentation condition, the average secretory expression quantity can reach 300.3 mg / L. The recombinant enzyme obtained by nickel ion affinity chromatography purification not only can effectively hydrolyze colloidal chitin to generate chitosan oligosaccharide (COS), but also can directly degrade chitin-containing raw materials such as shrimps, crabs and the like to generate COS. In addition, the enzyme also shows the activity of hydrolyzing polysaccharides such as cellulose, pectin, agar and the like, and has important potential values in the fields of functional chitosan oligosaccharide preparation, biological energy production, medicine development, feed, environmental protection, agricultural application and the like.

The invention discloses a stenotrophomonas sp. A322 strain and an application of the stenotrophomonas sp. A322 strain in synthesis of acarbose. The stenotrophomonas sp. A322 is preserved in the China Center for Type Culture Collection, and the preservation number of the stenotrophomonas sp. A322 is CCTCC (China Center for Type Culture Collection) NO: M 2025917. The stenotrophomonas sp. A322 can be used for synthesizing acarbose by using maltose and glucose as carbon sources, and the acarbose can be synthesized by using the stenotrophomonas sp. The stenotrophomonas sp. A322 is subjected to fermentation culture in a 100 mL shake flask fermentation system for 1 to 7 days, and the acarbose yield is 0.4 to 4 g / L. The high performance liquid chromatography retention time and the nuclear magnetic resonance carbon spectrum chemical shift of the acarbose obtained by fermentation, separation and purification of the Stenotrophomonas sp. A322 are consistent with those of a standard substance, and the high performance liquid chromatography retention time and the nuclear magnetic resonance carbon spectrum chemical shift of the acarbose obtained by fermentation, separation and purification of the Stenotrophomonas sp. A322 are consistent with those of the standard substance. Activity detection shows that the separated and purified acarbose sample has alpha-amylase inhibitory activity.

The invention provides a method for producing L-carnosine, a genetic engineering strain as well as a preparation method and application of the genetic engineering strain, and belongs to the field of genetic engineering. According to the genetic engineering strain provided by the invention, by knocking out a glucose-6-phosphate isomerase coding gene pgi, knocking out an L-threonine / L-homoserine transporter coding gene rhtA or knocking out a dipeptide transfer protein coding gene dppABCD, the yield of extracellular L-carnosine is increased, the shake flask yield of the finally constructed strain reaches 9.1 g / L, the 5L fermentation tank yield reaches 65 g / L, and the yield of the extracellular L-carnosine reaches 9.1 g / L; and the method has a good application prospect in L-carnosine biosynthesis.

The invention provides a beta-glucosidase mutant from Paenibacillus phyllosphaerae, and a method for efficiently preparing rare ginsenoside F2 by using an enzyme method and a whole-cell method, and particularly provides a beta-glucosidase mutant from Paenibacillus phyllosphaerae and a method for efficiently preparing rare ginsenoside F2 by using a beta-glucosidase mutant from Paenibacillus phyllosphaerae. The method comprises the following steps: engineering-modifying beta-glucosidase by utilizing computer-aided design, constructing a mutant library, carrying out primary screening and secondary screening to obtain two high-activity mutants Q7D and G189A, constructing a combined mutant Q7D / G189A, transforming the combined mutant Q7D / G189A into genetically engineered bacterium escherichia coli for expression, and producing F2 by utilizing a whole-cell method. Finally, the highest yield of 18.01 g / L is achieved after the reaction is carried out for 10 h in a 5 L fermentation tank and is 6.82 times that of a shake flask, and the purity of F2 reaches up to 82.20% after the reaction is carried out for 24 h finally. The invention not only provides an efficient solution for large-scale production of rare saponin F2, but also provides a transferable framework for designing and engineering diversified glycoside hydrolase in customized biological catalysis.

The invention discloses a pinene synthase mutant and application thereof in pinene production, and belongs to the technical field of bioengineering. Isoleucine at the 409th site of pinene synthase is mutated into valine, phenylalanine at the 443rd site of pinene synthase is mutated into alanine, overexpression is carried out in serratia marcescens (HBQA7), and an engineering strain ST17 is constructed. Under the condition of shake flask fermentation, the pinene yield of the engineering strain ST17 reaches 0.80 g / L; the yield is obviously increased to 43.2 g / L through amplification culture in a 30 L fermentation tank. The invention provides an efficient, economic and environment-friendly novel method for industrial biosynthesis of pinene.

1. Name of the product in this design: Packaging Bottle (Explosive Shake Bottle). 2. Purpose of this design: This product is a packaging bottle used to hold liquids. 3. The key design feature of this product is its shape. 4. The image or photograph that best illustrates the design's key points: a 3D model. 5. Other matters requiring explanation: Part A is made of transparent material. The white liquid in this design product is the contents and is not part of the design for which protection is sought.

Provided is an engineered bacterium for inhibiting the synthesis of dodecanedioic acid. The expression of a related redox gene is knocked out or down-regulated in the genome of the engineered bacterium. The engineered bacterium can effectively reduce the production of the by-product dodecanedioic acid and prevent the peroxidation of 12-hydroxydodecanoic acid during the synthesis of nylon 12 monomer. Further provided is a method for constructing an engineered bacterium for inhibiting the synthesis of dodecanedioic acid. The method involves synthesizing nylon 12 monomer in Escherichia coli by using a quorum-sensing system in Vibrio fischeri. The established Escherichia coli containing a self-induction system can produce the nylon 12 monomer from glucose, and the generation of the by-product dodecanedioic acid can be effectively reduced. After fermentation in a shake flask for 72 h, the yield of the nylon 12 monomer is more than 100 mg / L, and the yield of the by-product dodecanedioic acid is reduced to less than 200 mg / L (the proportion in the product is less than 20%).

This invention discloses a high-yield lactyl-3-4 ...5-4-4-4-4-4- N This invention relates to plasmid-free genetically engineered strains of tetrasaccharides and their applications, belonging to the field of microbial genetic engineering. The invention screened strains with higher activity. β -1,3-galactosyltransferase gene PmgalT Computer-aided enzyme function modification was used to achieve a shake-flask yield of 10.05 g / L for the constructed mutant. Subsequently, genome integration was performed... breast Multi-copy integration PmgalT * V27I / V39I / Y147F ‑galE‑galU A plasmid-free strain was constructed, enabling it to grow in shake flasks with lactoyl- N The yield of tetrasaccharides reached 14.4 g / L. Using glucose as the sole carbon source, the highest yield of 68.19 g / L was achieved at 64.5 h; using glycerol as the sole carbon source, the highest yield of 62.88 g / L was achieved at 60.5 h, providing a basis for its industrial production.

This invention discloses a nicotinamide phosphoribosyltransferase mutant, a recombinant strain, and a method for constructing the same. The mutants are I457V, G344A, Q378A, T283G, or F182A, obtained by single-point mutation of the wild-type nicotinamide phosphoribosyltransferase shown in SEQ ID NO:1. This invention utilizes a method derived from... Schizotequatrovirus By rationally designing and site-directed mutagenesis of KVP40 nicotinamide phosphoribosyltransferase, combined with metabolic engineering to block the NMN degradation pathway and enhance substrate synthesis and product transport, the yield of β-NMN by the recombinant strain during shake-flask fermentation reached 4.82 g / L. Through optimization of fed-batch fermentation conditions, the highest yield of β-NMN during fed-batch fermentation in shake flasks reached 15.60 g / L.

The present invention discloses a patchouli alcohol synthase mutant and a method for fermenting patchouli alcohol using recombinant Escherichia coli, belonging to the field of bioengineering. The present invention modifies patchouli alcohol synthase and constructs corresponding recombinant Escherichia coli to obtain a recombinant bacterium capable of efficiently fermenting and producing patchouli alcohol, and a fermentation method thereof. The recombinant strain can ferment and produce high-value-added patchouli alcohol using inexpensive glucose as a substrate. After 96 hours of shake flask fermentation, the patchouli alcohol yield reaches 448.1 mg / L, with a yield of 39.1 mg / g DCW and a volumetric production rate of 112 mg / L / day. In a 5L fermenter, the patchouli alcohol yield can reach 2128 mg / L, with a volumetric production rate of 304 mg / L / day.

The invention discloses an escherichia coli strain with high yield of riboflavin as well as a construction method and application of the escherichia coli strain. A series of Escherichia coli strains with high yield of riboflavin are constructed, the constructed Escherichia coli with high yield of riboflavin is safe and harmless, glucose is used as a substrate for fermentation under the aerobic condition of a shake flask, the yield of riboflavin is 1117.8 mg / L, 1330.1 mg / L, 1430.8 mg / L, 1508.7 mg / L and 1640.8 mg / L in sequence, and the yields are 112.5 mg / g of glucose, 134.1 mg / g of glucose, 146.3 mg / g of glucose, 158.8 mg / g of glucose and 168.1 mg / g of glucose in sequence; when the strain SN04M is subjected to fed-batch fermentation in a 1L fermentation tank, the yield of riboflavin reaches 8.52 g / L, and the total yield is 189.9 mg / g glucose. According to the invention, the fermentation yield of riboflavin and the substrate utilization rate of riboflavin to glucose can be obviously improved.

The present application relates to a kind of by knocking out Bacillus subtilis SacB gene constructed chassis strain and its application, belong to genetic engineering, enzyme engineering and food engineering technical field.The present application is by SacB gene on the genome of Bacillus subtilis Knocking out, obtain a kind of can efficiently express aminopeptidase, alkaline protease Bacillus subtilis expression system, lay the foundation for the efficient expression of protein, promote the efficient expression and industrial production of aminopeptidase, alkaline protease.The chassis strain described above, in the case of carrying aminopeptidase, alkaline protease expression cassette, respectively, 48H shake flask fermentation, fermentation broth aminopeptidase enzyme activity is as high as 1666.7U / mL, with the enzyme activity of starting strain as contrast, enzyme activity is increased by about 30%;Fermentation broth alkaline protease enzyme activity is as high as 6348U / mL, with the enzyme activity of starting strain as contrast, enzyme activity is increased by about 20%.

The invention relates to pichia pastoris with high yield of Brazilian sweet protein and application of the pichia pastoris, and belongs to the technical field of fermentation. The invention finds that two molecular chaperones ERV29 and CPR5 from saccharomyces cerevisiae jointly strengthen the expression of the Brazilian sweet protein so as to significantly improve the yield of the Brazilian sweet protein, the Pichia pastoris provided by the invention is subjected to fermentation culture, the yield of the Brazilian sweet protein in a shake flask reaches 1.8 g / L, the yield of the Brazilian sweet protein in a 5-L fermentation tank reaches 11.3 g / L, which is the highest yield of the Brazilian sweet protein in the existing strain, and the Pichia pastoris provided by the invention has a wide application prospect. The method has a wide application prospect in industrial production of Brazilian sweet protein.