98 results about "Shake-Flask Culture" 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 relates to the field of microbial genetic transformation, and discloses a preparation method of Ilyonectria fungus protoplast and a construction method of a genetic transformation system.The preparation method comprises the following steps that S11, Ilyonectria fungus hyphae are prepared, Ilyonectria fungi are cultured on a PDA solid culture medium for 14 d, then the Ilyonectria fungi are inoculated to a CMC liquid culture medium to be subjected to shake-flask culture for 4 d and filtered, filtrate is centrifuged for 10 min at the speed of 10000 rpm, and spores are collected; diluting the concentration of spores to 107 pieces / mL, inoculating the spores into a YEPD liquid culture medium, carrying out shake-flask culture for 2 days, and filtering and collecting hyphae; and S12, preparing the Ilyonectria fungus protoplast, namely adding the hypha prepared in the step S11 into the enzymatic hydrolysate, performing enzymolysis in a shaker at the temperature of 30 DEG C at the rotating speed of 150-200rpm for 3-5 hours, filtering, centrifuging filtrate at the temperature of 4 DEG C, discarding supernate, adding 0.7 M NaCl for centrifuging, and discarding the supernate, thereby obtaining the Ilyonectria fungus protoplast. The genetic transformation constructed by the invention can be used for exploring the specific action and mechanism of a specific pathogenic gene in the pathogenic process of Ilyonectria fungi through gene knockout or overexpression experiments.

The invention relates to engineering Escherichia coli for producing L-fucose and application of the engineering Escherichia coli, the production of the L-fucose is enhanced through a metabolic engineering strategy by utilizing a mode of combining genome integration and plasmid expression path genes, and the efficient production of the L-fucose is realized by optimizing a double-plasmid system and enhancing precursor supply. In addition, a hok / sok system and a cysC auxotroph system are introduced, and the plasmid-based engineering strain without antibiotic dependence is successfully constructed. The strain produces 7.99 g / L of L-fucose and 61.91 g / L of L-fucose through shake-flask culture and fed-batch culture under the condition that antibiotics are not added, the highest L-fucose yield reported so far is provided, and the use of antibiotics is completely not needed in the fermentation process.

The invention relates to recombinant escherichia coli and a method for synthesizing sialic acid lactose-N-tetrasaccharide c, and belongs to the technical field of microbial metabolism engineering. According to the research, engineering escherichia coli BAZ for producing LNTri II is taken as an initial strain, two key glycosyl transferases, namely beta 1, 4-galactosyl transferase and alpha 2, 6-sialyltransferase, and three CMP-Neu5Ac pathway enzymes, namely NeuC, NeuB and NeuA, are introduced, and related competitive genes are knocked out; under the conditions of shake-flask culture and fed batch culture, the LST c yield of 1.718 g / L and the LST c yield of 9.745 g / L are achieved through the engineering bacterium respectively, and the huge potential of a microbial cell factory in the aspect of efficient production of complex breast milk oligosaccharides is shown.

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 present invention provides a method for increasing the fermentation yield of L-lysine, and belongs to the field of L-amino acid fermentation. The method for increasing the fermentation yield of L-lysine is composed of the following steps: preparing a shake flask culture solution, preparing a seed solution, and fermenting and culturing. The method for increasing the fermentation yield of L-lysine of the present invention can effectively improve the tolerance of the bacterial body to high osmotic pressure, improve the acid production performance in the late fermentation period, and improve the single-tank yield and sugar-acid conversion rate of L-lysine; at the same time, further reduce the adverse effects of osmotic pressure regulators on the fermentation stability of L-lysine, and effectively avoid the problem of excessive addition of osmotic pressure regulators inhibiting bacterial metabolism, extending the fermentation production cycle, and reducing fermentation efficiency.

The invention discloses an expression system suitable for short-chain non-specific peroxidase and application of the expression system, and belongs to the technical field of enzyme engineering and genetic engineering. According to the expression system disclosed by the invention, a nucleotide sequence of short-chain non-specific peroxidase with SUMO and 8HIS tags is connected to a pET28a vector to obtain a recombinant vector, and escherichia coli BL21 (DE3) is transformed to obtain a genetically engineered bacterium; genetically engineered bacteria are subjected to two-stage temperature control culture in a ZYM5052 liquid culture medium, and meanwhile, 5ALA is added into the culture medium. According to the invention, an escherichia coli self-induction system is used, so that a shake-flask culture can grow at high density in a short time, the culture medium contains rich nutrient substances, and monitoring of cell density and adding of IPTG are not needed in the expression process. Meanwhile, a heme precursor 5ALA is added into an escherichia coli self-induction culture medium, and finally the high-activity short-chain non-specific peroxidase is obtained.

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

The invention relates to the technical field of mycelium culture, in particular to a culture method and application of selenium-enriched cordyceps militaris mycelium. The culture method comprises the following steps: carrying out PAD culture medium culture, shake-flask culture and fermentation tank aerated culture on a cordyceps militaris mother strain to obtain a cordyceps militaris liquid strain; inoculating the cordyceps militaris liquid strain into a liquid culture medium for aerated culture to obtain selenium-rich organic selenium cordyceps militaris liquid; the preparation method comprises the following steps: respectively crushing soybeans, wheat and corn, mixing to obtain mixed powder, soaking the mixed powder with water, and airing to obtain a solid culture medium; and inoculating the selenium-rich organic selenium cordyceps militaris bacterial liquid into a solid culture medium for aerated culture to obtain selenium-rich cordyceps militaris mycelia. The culture method is simple in step and easy to operate, the selenium content of the cultured selenium-enriched cordyceps militaris mycelium is high, and the culture method has wide application prospects in food, health care products, traditional Chinese medicinal materials and feed.

The invention discloses a method for preparing a pilose antler mushroom stick by using soybean hull residues and cattle feed, and the preparation method comprises the following steps: (1) pretreatment of soybean hulls: decomposing cellulose and hemicellulose in the soybean hulls by using an enzyme preparation to destroy the compact structure of the cellulose and hemicellulose; destroying bean skin cell membranes and fiber structures by using a high-voltage pulse electric field to form a porous structure; (2) activating a strain: performing slant inoculation on flammulina velutipes, and putting the flammulina velutipes into a liquid culture medium for shake-flask culture; (3) biotransformation of the pilose antler mushroom: putting the liquid strain in the liquid culture medium into the beancurd skin compound fungus bag, and obtaining a pilose antler mushroom stick after the bag is full; the beancurd skin compound bacteria comprise the following components in parts by weight: 45-55 parts of beancurd skin, 15-20 parts of corncob, 15-17 parts of bran, 13-15 parts of rice bran, 0.5-0.8 part of shell ash and 0.5-0.8 part of humic acid; the cattle feed is prepared from the following components in parts by weight: 180 to 220 parts of rapeseed meal, 120 to 160 parts of bran, 10 to 14 parts of shell ash, 0.4 to 0.8 part of table salt and 450 to 780 parts of needle mushroom sticks. The feed disclosed by the invention can effectively regulate the digestive ability and growth and development speed of cattle, and the growth and development speed of the fed cattle is averagely higher than that of cattle fed by conventional feed.

The invention relates to a construction method and application of recombinant escherichia coli for increasing the yield of lactoyl-N-tetrasaccharide, and belongs to the field of microbial genetic engineering. According to the invention, three genes tauB, nikD and livM screened by using a transcriptome technology are proved to be capable of promoting the generation of LNT and LNTri II. An engineering strain MG-LNT-09 is used as a starting strain, the biosynthesis efficiency of endogenous transporter genes tauB, nikD, livM, LNT and LNTri II derived from Escherichia coli K-12 derivative bacteria MG1655 is remarkably improved through single plasmid overexpression, the yield of the LNT in shake-flask culture is as high as 7.77 g / L, and the yield of the LNTri II is as high as 3.03 g / L. The optimized strain MG3WB of which the tauB gene is integrated to the hlyE site obtains the maximum LNT titer of 40.35 g / L in a 5L bioreactor without using any antibiotics or plasmids, has a good industrial prospect, and lays a foundation for further industrial production and synthesis of other complex breast milk oligosaccharides.

The invention discloses a Ustilaginoidea virens inoculant, a preparation method and application of the Ustilaginoidea virens inoculant in Ustilaginoidea virens inoculation, and belongs to the technical field of agricultural microorganisms. According to the method, two-step shake-flask culture is combined with the steps of filtering, centrifuging and spore germination, so that the ustilaginoidea virens inoculum taking germinated spores (GC) as the core is prepared, and the problems of hypha aging, low spore activity, asynchronous infection and the like in the existing inoculation method are effectively solved. The inoculant has a tender hypha structure and strong infection activity, and the inoculation efficiency and the pathogenesis consistency are remarkably improved. When being used for preparing transcriptome samples, metabolome samples and other omics samples, the strain avoids non-specific immune interference caused by PAMPs released by broken hyphae, ensures synchronous infection process, and is real and reliable in immune response. The invention provides a stable and efficient inoculation system with a clean background for the research on the ustilaginoidea virens-rice interaction mechanism, and has a good application prospect.

The application belongs to the technical field of microbial fermentation, and discloses soy sauce starter and a preparation method and application thereof. The preparation method of the soy sauce starter comprises the following steps: extracting a starter substrate, performing squeezing treatment, obtaining wet material and clear liquid; filtering the clear liquid to obtain filtered clear liquid; taking dry material and adding the wet material to obtain raw material; sterilizing the raw material to obtain starter material; inoculating spore powder of Aspergillus oryzae secondary strain into the starter material to obtain inoculated starter material; and culturing the inoculated starter material through starter culture including primary shake flask culture and secondary shake flask culture to obtain soy sauce starter. The preparation method innovatively constructs a synergistic regulation system of "physical structure improvement-nutrition precise supplement", solves the environmental protection treatment problem of the starter substrate, significantly improves the sensory quality and spore production effect of the soy sauce starter, and provides a sustainable development solution with economic and environmental protection benefits for the traditional fermentation industry.

This invention relates to the field of fungal strain cultivation technology, and particularly to a method for isolating and cultivating fungal strains from matsutake fruiting bodies. The method involves collecting matsutake mushrooms that are 70%–80% mature and free from insects, disinfecting them with alcohol, longitudinally splitting them in half, removing the flesh from the cap and stipe, and inoculating them onto a slant culture medium to obtain a primary strain. This primary strain is then inoculated into a culture medium and shaken to obtain a primary shake flask strain, which is then inoculated into a culture medium. The strain is then shaken and cultured, and the concentration of metabolites, dissolved oxygen, and pH in the culture medium are collected in real time to construct a strain growth time prediction model to predict the stage culture duration. The cell proliferation trend is determined based on the number of matsutake mycelial cells. Based on a comparison of the actual culture time during the initial adjustment period and the stage culture time, the adjustment method for the logarithmic growth phase of the current batch is determined. Based on a comparison of the shake flask culture time and the predicted culture time, the adjustment method for the initial culture conditions of the next batch is determined. This invention effectively optimizes shake flask culture conditions and improves strain quality.

The present invention discloses a laccase, its gene and application, belonging to the technical field of genetic engineering. The laccase gene of the present invention is obtained by codon optimization of the laccase gene of Cerrena unicolor, and the nucleotide sequence is as shown in SEQ ID NO.2. The laccase gene provided by the present invention is recombinantly expressed by transforming Aspergillus niger, and high-activity laccase can be mass-produced industrially. The highest enzyme activity in shake flask culture reaches 39 U / ml, and after culturing in a 7L fermenter for 125 h, the total laccase activity reaches 9234 U / ml. At the same time, the laccase provided by the present invention and the mediator methyl syringate have a significant destructive effect on zearalenone, which is beneficial to grain transportation and storage.

The invention relates to the field of scalp care, and particularly discloses a preparation method of a scalp and skin care preparation, which comprises the following steps: S1, a strain activation step: taking bacillus subtilis as a preparation strain, inoculating the strain in a preservation state into an LB agar culture medium with the pH value of 7, and performing 2-3 generations of culture rejuvenation to obtain vigor-rich strain seeds; s2, shake-flask culture: selecting the vigor-rich strain seeds, and inoculating the vigor-rich strain seeds into an LB liquid culture medium with the pH value of 7; culturing the inoculated LB liquid culture medium in a shaking table to obtain a shake flask bacterial liquid; s3, a fermentation culture step: firstly, preparing a fermentation culture medium, and adjusting the pH value of the fermentation culture medium to 7; then carrying out fermentation operation; according to the technical scheme, the product has high content of active components, the release efficiency of active substances of the bacillus subtilis preparation is high, and the final preparation has the safety, antibacterial and repairing capabilities.

The utility model provides a kind of continuous air-lift type yeast culture device, including enrichment tank, overflow module, air supplement module and pH adjustment module, the overflow module includes overflow collection tank, overflow pipe being connected between overflow collection tank and enrichment tank;The air supplement module includes gas distributor being arranged in enrichment tank and the gas inlet pipe, gas pump for gas distributor supply;The pH adjustment module includes lye tank etc.The utility model has the advantages that: by setting enrichment tank, overflow module, air supplement module and pH adjustment module, air supplement module is supplemented into gas in enrichment tank, for yeast growth use.pH electrode detects the pH value in enrichment tank, and controller controls the flow in enrichment tank according to pH value, and the pH value in enrichment tank is real-time regulated and controlled.Overflow module collects liquid overflowing from enrichment tank.Solve the problem of low mass transfer efficiency, pH regulation lag and insufficient scale in traditional shake flask culture.

The invention belongs to the field of microbial fermentation, and particularly relates to a culture method of lactic acid bacteria, which comprises culture of D-lactic acid bacteria and L-lactic acid bacteria, and is characterized in that the culture method of the D-lactic acid bacteria comprises five stages of strain screening, microbial inoculum preparation, shake-flask culture, seed culture and fermentation culture, according to the method, the five stages of D-lactic acid bacteria culture and L-lactic acid bacteria culture are divided, the process is standard, and operation is convenient. And by virtue of MRS culture medium enrichment and liquid chromatography detection, high-yield and high-purity strains are selected. Bacterial strain activity can be preserved for a long time through microbial inoculum preparation, and flask shaking and seed culture build a firm foundation for fermentation. According to the fermentation culture, parameters such as temperature, pH, dissolved oxygen and the like are accurately regulated and controlled, and batch feeding and an advanced monitoring system are adopted, so that the growth requirements of strains are met, high yield is maintained, and the yield and purity of lactic acid are improved. Components of the optimized MRS culture medium can be adjusted according to needs, the content and purity of lactic acid can be accurately measured through a detailed detection method, reliable data support is provided for culture, and it is guaranteed that the culture effect is stable and reliable.

The invention discloses a method for rapidly discriminating the culture stage of a yeast seed solution based on a Raman spectrum. The method specifically comprises the following steps: selecting a yeast single colony and culturing in a YPD test tube to prepare a primary seed solution; inoculating the primary seed solution into a shake flask containing 200 mL of a YPD liquid culture medium for shake culture; in the shake-flask culture process, a 785 nm Raman spectrometer is used for in-situ collection of a Raman spectrum, and sampling is carried out every 30 min to determine OD600; and establishing a yeast seed solution culture stage detection model based on the collected spectral data and OD600 data. According to the method, frequent sampling is not needed, the method can be used for rapidly distinguishing the hysteresis phase, the logarithmic growth phase and the stable phase of the yeast seed liquid, the optimal inoculation opportunity can be scientifically determined, and the consistency of the fermentation process and the product quality stability are improved.

The application discloses a method for improving cellulase activity by using a combination of an endogenous promoter and an associated 5' UTR of Trichoderma reesei and an engineering strain constructed by the method. TrAE The 5' UTR upstream of the TrAE gene TrAE Overexpressing a transcriptional regulator xyr1 The expression cassette of the gene is transformed into Trichoderma reesei Δ ku70 Rut‑C30 The application also discloses a high-yield cellulase Trichoderma reesei strain QEaeuX, and a method for producing high-activity cellulase by using the strain. ku70 Rut‑C30 The cellulase activity produced by the strain in a 96 h shake flask culture is 2.6 IU / mL, which is 101% higher than that of the original strain Δ The application has a good industrial development and application prospect.

The invention belongs to the technical field of microbial engineering and synthetic biology, and particularly relates to a schizochytrium limacinum mutant strain A1-11 and application thereof in synthesis of odd-carbon-chain fatty acid. According to the invention, ARTP plasma mutagenesis is carried out on wild-type Aurantiochytrium sp. Sdu080, a mutagenic strain A1-11 with genetic stability is successfully screened, and compared with an original strain, the total proportion of C15: 0 and C17: 0 of the mutagenic strain under a conventional shake-flask culture condition is increased from 7.17% to 33.13%. Furthermore, after 20 mM of L-valine is added into the fermentation liquid culture medium, the proportion of odd fatty acid of A1-11 can be further increased to 52.14%. The schizochytrium limacinum A1-11 provided by the invention not only remarkably breaks through the limitation of the odd fatty acid yield and proportion in the prior art, but also has the advantages of simple process, easiness in large-scale fermentation and the like.

The invention relates to the technical field of microbial fermentation, and discloses a fermentation method of a mixture of tetraacetyl phytosphingosine and triacetyl dihydrosphingosine, comprising the following steps: step (1), glycerol tube preservation; step (2), primary shake flask culture: taking a strain preservation glycerol tube, inoculating 1% (v / v) seed volume into a seed culture medium of a primary seed bottle, and culturing in a box shaker at 28°C and 240rpm for 72h; step (3), secondary shake flask culture: taking a primary seed bottle, inoculating 1% (v / v) inoculation volume into a secondary seed bottle, and culturing in a box shaker at 28°C and 240rpm for 48h; step (4), seed tank culture; step (5), transfer to a fermentation tank; step (6), fermentation. The invention adopts Wickham syringa to simultaneously ferment and produce TAPS and TriASa in the same fermentation system, and can obtain mixtures of TAPS and TriASa in different proportions by regulating dissolved oxygen, thereby greatly improving production efficiency and saving production costs.

The present application belongs to the technical field of oscillator, especially a constant-temperature oscillator for vaccine shake flask culture, aiming at the problem that the existing constant-temperature oscillator cannot clamp and fix the shake flask, and the corresponding water bath constant-temperature oscillator and air bath constant-temperature oscillator need to be used, thereby greatly increasing the culture cost, the present application proposes the following scheme, including a box body, a shaking plate is arranged in the box body, a plurality of clamping members for clamping the shake flask are arranged on the top of the shaking plate, U-shaped handles are fixed on the two sides of the top of the shaking plate, the shaking plate is lifted by the U-shaped handles, a temperature sensor is fixed on the inner wall of the bottom of the box body, a water tank and a heating tank are further included, the water tank is fixed on the bottom of the box body, the present application can perform air bath and water bath on the shake flask on the same equipment, the water bath and the air bath can be freely switched by the switching structure, the operation is simple, the cost of vaccine shake flask culture is greatly reduced, and the shake flask can be clamped in all directions by the cooperation of the clamping members, the first spring and the pressing plate during shaking.

The application discloses a kind of based on low salt culture and feeding optimization's high-efficiency fermentation method of schizochytrium, comprising: S1.low salt culture medium is used to carry out multiple rounds of screening and domestication to schizochytrium TKD-1, obtain the schizochytrium species of low salt high-yield DHA;S2.the schizochytrium species of good breeding is sequentially passed through shake flask culture, primary seed tank expansion culture and secondary seed tank expansion culture, obtain secondary seed liquid;S3.secondary seed liquid is connected into fermenter, carries out fermentation, obtains schizochytrium fermentation liquor;Wherein, in the fermentation process, the way of feeding is used to add glucose to fermenter, while detecting the glucose concentration in fermentation liquor on line to dynamically adjust the feeding rate of glucose;S4.after fermentation, enzyme hydrolysis method is used to break up bacteria, after centrifugation, extract DHA, obtain the oil rich in DHA.The method provided by the application can shorten the fermentation period, improve the biomass and DHA yield of schizochytrium, and reduce the production cost.

The invention discloses a high-temperature-resistant yeast tank type solid-state fermentation process, relates to the technical field of solid-state fermentation, and aims to solve the problem of poor yeast fermentation effect. Intelligent equipment such as a distributed temperature sensor and a mass flow meter is adopted, and real-time monitoring and accurate regulation and control of environmental parameters in the fermentation tank are achieved. The difficulty and errors of manual operation are reduced, the stability and controllability of the fermentation process are improved, growth and metabolism of strains can be optimized by adjusting the pH value or the dissolved oxygen concentration, so that the yield and quality of products are improved, abnormal changes in the fermentation process can be found in time through real-time monitoring, and the production cost is reduced. The comprehensive monitoring is beneficial to more accurate judgment of the fermentation state, so that more reasonable adjustment is made, the steps of first-stage shake-flask culture, second-stage high-temperature-resistant culture and third-stage seed amplification culture are clear, and the stability and activity of the strain in the amplification culture process are ensured.

The invention relates to the technical field of fermentation engineering, and discloses a method for producing cytidine by fermentation, which comprises the following steps: inoculating a cytidine-producing strain into an eggplant bottle culture medium for activation culture; inoculating the activated cytidine-producing strain into sterile water, and carrying out shake-flask culture to obtain a primary seed solution; inoculating the first-stage seed solution into a second-stage seed culture medium for expanding culture to obtain a second-stage seed solution; inoculating the secondary seed solution into a fermentation culture medium for fermentation, supplementing an IPTG (isopropyl-beta-d-thiogalactoside) inducer for three times in the fermentation process, and gradually decreasing the amount of the supplemented IPTG inducer; glucose is supplemented when the reducing sugar content in the fermentation liquor is 0.3-0.5%, the dissolved oxygen content is controlled to be 10-30% in the sugar supplementing process until supplementing is completed, and fermentation is stopped when the pH of the fermentation liquor rises again; and carrying out heating sterilization, filtration, concentration and cooling crystallization on the fermented fermentation liquor to obtain a cytidine finished product. Therefore, the yield and the purity of a cytidine finished product are improved, the production cost is reduced, and industrial production is facilitated.

The invention provides a high-yield tetraacetyl phytosphingosine strain and application thereof, and belongs to the technical field of biology. Wild Wickerhamomyces ciferrii RK01 which is separated from rotten wood in a mountain forest in Yunnan China for the first time is used as an original strain, ascospores are generated by the original strain, so that a haploid strain with better tetra-acetyl phytosphingosine (TAPS) production performance is obtained through separation, and a mutant strain W. ciferrii CG223 with high yield of TAPS is obtained by performing multiple rounds of ARTP iterative mutagenesis on the haploid strain. The strain can produce 4.02 g / LTAPS after being cultured for 72 hours under a batch shake-flask culture condition, which is 11.82 times that of a wild strain, and can produce 47.3 g / LTAPS after being cultured for 96 hours in a 20L fermentation tank, so that the yield of TAPS is remarkably increased, and large-scale production of TAPS is facilitated.

The invention discloses a construction method and application of escherichia coli for producing a heparin precursor by using cane sugar. The invention provides a method for constructing a recombinant strain, which comprises the following steps: overexpressing a heterologous sucrose-6-phosphohydrolase gene sacA and a sucrose phosphorylase gene spI in escherichia coli to obtain an engineering strain which can grow by using sucrose as a carbon source and can effectively synthesize a heparin precursor; a sucrose specific enzyme IIBC component gene sacP of a phosphotransferase system and a sucrose-6-phosphohydrolase gene sacA are co-expressed, and recombinant bacteria can more efficiently synthesize a heparin precursor in shake-flask culture, so that the yield of the heparin precursor is increased. The recombinant strain constructed by the invention has a good application prospect.

The invention relates to an alpha-1, 3-fucosyltransferase mutant and an application thereof in preparation of 3-fucosyllactose, the alpha-1, 3-fucosyltransferase mutant is based on SEQ ID NO.1, and any one or combination of more sites in 221, 225, 250 and 254 of the SEQ ID NO.1 is mutated. Compared with the prior art, the multiple alpha-1, 3-FucT mutants provided by the invention can effectively improve the 3-FL secretion capability of alpha-1, 3-FucT, and the 3-FL production level can reach 3.68 g / L after shake-flask culture, which is 11.15 times that of the original alpha-1, 3-FucT strain; the level of 3-FL produced by amplified fermentation culture can reach 25.28 g / L, and the strain has good industrial application potential.