WO2017139959A1 - 一种高产cAMP的酵母菌株及其应用 - Google Patents

一种高产cAMP的酵母菌株及其应用 Download PDF

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WO2017139959A1
WO2017139959A1 PCT/CN2016/074067 CN2016074067W WO2017139959A1 WO 2017139959 A1 WO2017139959 A1 WO 2017139959A1 CN 2016074067 W CN2016074067 W CN 2016074067W WO 2017139959 A1 WO2017139959 A1 WO 2017139959A1
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yeast
camp
gene
ura3
genetic modification
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邹少兰
高文萱
胡芸
杨顺华
马平生
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Tianjin University
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Definitions

  • the invention belongs to the technical field of genetic engineering and microbial fermentation, and particularly relates to a yeast strain capable of excessively synthesizing extracellular cAMP, a construction method thereof, a fermentation process and application in the fields of medicine, animal husbandry, food or chemical industry.
  • Cyclic adenosine monophosphate is a physiologically active substance widely present in human body. It acts as a second messenger in cells, affecting glucose metabolism, fat metabolism, nucleic acid synthesis, protein synthesis, etc. Important adjustments. At present, cAMP is widely used in medicine: clinically, cAMP is used for the treatment of angina pectoris, myocardial infarction, myocarditis and cardiogenic shock; it has certain effects on improving symptoms such as palpitations, shortness of breath and chest tightness in rheumatic heart disease; Combined with the efficacy of chemotherapy, it can also be used for the induction of acute leukemia; in addition, it has a certain effect on chronic bronchitis, various hepatitis and psoriasis in the elderly.
  • cAMP Cyclic adenosine monophosphate
  • cAMP can also be used as a pharmaceutical intermediate to prepare dibutyryl adenosine monophosphate and cyclophosphamide adenosine to improve fat solubility and thereby exert more effective physiological and pharmacological effects.
  • cAMP can also be used as a food additive for livestock and poultry, simulating the action of growth hormone under ex vivo conditions, promoting the growth of livestock and poultry, and increasing the yield of high quality poultry products.
  • the production methods of cAMP include chemical synthesis method, enzymatic conversion method and fermentation method.
  • all domestic and international industrial production uses chemical synthesis method using adenosine-5'-monophosphate (AMP) as raw material, which uses high-efficiency separation column for intermediate separation, which is complicated in operation and expensive in reagents involved.
  • AMP adenosine-5'-monophosphate
  • the use of large amounts of pyridine as a solvent also causes serious environmental pollution.
  • the enzymatic conversion method uses adenylate cyclase (EC4.6.1.1, Cyr1p) as an enzyme source to catalyze the production of cAMP in ATP raw materials.
  • this method has high substrate, low content of adenylate cyclase, and difficult purification.
  • cAMP by bacterial fermentation methods such as Brevibacterium liquefaciens, Microbacterium, Corynebacterium, Arthrobacter, Escherichia coli has been studied, but it faces problems such as poor repeatability, unstable yield, high cost, and difficulty in industrialization.
  • Yeast especially Saccharomyces cerevisiae, is one of the most important industrial microorganisms. It not only has the advantages of mature industrial application technology, strong stress resistance, and food safety GRAS microorganisms, but also the earliest microbial bacteria for whole genome sequencing. It is one of the most important model organisms in molecular biology and genetics research. The basic research related to cAMP signaling pathway and its regulation is deep, and the genetic manipulation techniques are perfect.
  • the object of the present invention is to overcome the deficiencies of the prior art mentioned above, and to provide a yeast strain which is genetically modified and capable of excessively synthesizing extracellular cAMP, which is easy to culture, is suitable for large-scale fermentation production, and is easy to industrialize. Recombination methods and applications. specifically,
  • the present invention provides a yeast strain which has undergone first and second genetic modification, wherein the first gene is protein kinase A (PKA) catalytic subunit coding gene TPK1, TPK2 And TPK3, by modifying the first gene to completely inhibit PKA activity or expression, thereby eliminating feedback inhibition of cAMP, but at the same time inhibiting the growth of yeast, the second genetic modification eliminates the growth caused by the first genetic modification Inhibition, thereby allowing normal growth of the yeast, the cAMP production of the yeast is increased, wherein the increase in cAMP production is relative to the cAMP production of the unmodified yeast.
  • PKA protein kinase A
  • the second gene includes, but is not limited to, a protein kinase Rim15 encoding gene RIM15, a transcription factor Msn1/Msn2 encoding gene MSN1/MSN2, a protein kinase Yak1 encoding gene YAK1 or a protein kinase Sch9 encoding gene SCH9, preferably YAK1.
  • the second genetic modification causes the activity or expression of the enzyme encoded by the modified gene to be completely inhibited, or the activity of the encoded enzyme is increased or Da.
  • the second genetic modification completely inhibits the activity or expression of the protein kinase Rim15 encoding gene RIM15, the transcription factor Msn1/Msn2 encoding gene MSN1/MSN2, the protein kinase Yak1 encoding gene YAK1 encoding enzyme, or the protein kinase Sch9 encoding gene
  • the activity of the SCH9 encoding enzyme is increased or overexpressed.
  • the above yeast further includes a third genetic modification to reduce the degradation of cAMP, thereby increasing cAMP production.
  • the third gene is the cAMP phosphodiesterase encoding gene PDE1 and/or PDE2, preferably PDE1. More preferably, the third genetic modification results in complete inhibition of the activity or expression of the enzyme encoded by the modified gene.
  • the above genetic modification method such that the activity or expression of the encoded enzyme is completely inhibited includes point mutation, deletion, insertion, antisense polynucleotide, siRNA, microRNA, CRISPR, more preferably deletion and point mutation; thereby increasing the activity of encoding the enzyme or Over-expressed genetic modification methods include point mutations, ligation of strong promoters, ligation of enhancers, and increased copy number
  • the above yeast further includes a fourth genetic modification to increase the positive regulation of the synthesis of cAMP precursors in the purine synthesis pathway, thereby increasing the synthesis of cAMP precursors, thereby increasing cAMP production.
  • the fourth gene includes, but is not limited to, the transcription factors Bas1, Bas2 encoding genes. More preferably, the genetic modification increases the expression of the Bas1/Bas2 complex.
  • the modification manner of the above fourth gene includes, but is not limited to, point mutation, ligation of a strong promoter, ligation enhancer, increase of copy number, fusion co-expression, and more preferably fusion co-expression.
  • the manner in which the gene is modified comprises the use of a selectable marker gene.
  • the selectable marker gene includes, but is not limited to, URA3, LEU2, HIS3, TRP1, LYS2. More preferably, the URA3 selectable marker gene is used. Still more preferably, the gene is modified in a manner that recycles the selectable marker gene without retaining the selectable marker gene.
  • the yeast strain described in the present invention is more yeast than the yeast modified without the first and second genes, or the yeast without the first, second and third genetic modifications, or the first, second, third and third Four genetically modified yeast strains have higher extracellular cAMP production.
  • the yeast strain described herein has an extracellular cAMP yield of up to 9721.6 [mu]mol/L.
  • the yeast strain described in the present invention is any of Saccharomyces cerevisiae, Saccharomyces pastorianus, Pichia stipitis, Saccharomyces Bayanus and Candida shehatae.
  • Saccharomyces cerevisiae is preferred.
  • the first genetic modification was introduced into the yeast to eliminate the feedback inhibition of cAMP, but at the same time, the growth of the yeast was inhibited.
  • the second genetic modification eliminated the growth inhibition caused by the first genetic modification, thereby allowing the yeast to grow normally. .
  • the method further comprises performing a third genetic modification on the yeast to reduce the degradation of cAMP, thereby further increasing cAMP production.
  • the method further comprises performing a fourth genetic modification on the yeast to increase the positive regulation of the synthesis of the cAMP precursor in the purine synthesis pathway, increase the synthesis of the cAMP precursor, and increase the cAMP production.
  • the fermentation method includes a fermentation medium component such as a carbon source, a nitrogen source, and a trace element. Also includes temperature and speed.
  • the fermentation medium has a glucose concentration of 20-150 g/L, a yeast extract of 10-20 g/L, a peptone of 20-40 g/L, and an adenine addition amount of 0-1.25 g/L. .
  • the present invention also provides products in the fields of medicine, animal husbandry, food or chemical industry, such as drugs, feeds, foods and the like, comprising the yeast strain or fermentation broth of any of the above.
  • the invention provides a genetically modified yeast strain capable of excessively synthesizing extracellular cAMP, a recombinant method for constructing the same, and an application thereof. More specifically,
  • the yeast of the present invention includes a first genetic modification and a second genetic modification. Further, the yeast further includes third and fourth genetic modifications. Among them, the first genetic modification eliminates the feedback inhibition of cAMP, but at the same time, the growth of yeast is inhibited; the second genetic modification eliminates the growth inhibition caused by the first genetic modification, thereby enabling the yeast to grow normally; Genetic modification reduces the degradation of cAMP, and the fourth genetic modification increases the positive regulation of the synthesis of cAMP precursors in the purine synthesis pathway, increasing the synthesis of cAMP precursors and increasing cAMP production.
  • the first genetic modification can completely inhibit the activity or expression of PKA by modifying the catalytic subunits of protein kinase A (PKA), TPK1, TPK2 and TPK3, and the feedback inhibition of cAMP by cKA is Elimination, but at the same time, the growth of yeast is inhibited; the second genetic modification eliminates the growth inhibition caused by the first genetic modification, so that the yeast can grow normally; the second genetic modification can make the activity or expression of the protein kinase Yak1, etc.
  • PKA protein kinase A
  • the third genetic modification completely inhibits the phosphodiesterase Pde activity or expression of hydrolyzed cAMP;
  • Four genetic modifications increase the positive regulation of the synthesis of the cAMP precursor AMP, such as the transcription factors Bas1, Bas2, or the expression of the AMP synthesis, thereby increasing AMP synthesis and ultimately increasing cAMP production.
  • the present invention describes the elimination of feedback inhibition of cAMP by PKA by first and second genetic modification of yeast strains to increase synthetic extracellular cAMP production.
  • PKA is currently the only known cAMP target in yeast. It is generally believed that fluctuations in cAMP concentration exert effects through the PKA pathway; in other words, the intracellular cAMP signaling pathway is regulated by the strict negative feedback of PKA.
  • Yeast PKA is a tetrameric protein composed of two catalytic subunits and a dimeric regulatory subunit. PKA is present as a passivation complex when intracellular lack of cAMP or cAMP levels is low.
  • cAMP When intracellular cAMP levels rise, cAMP binds to the regulatory subunit of PKA, and the catalytic subunit of PKA dissociates from the regulatory subunit, activating a series of protein phosphorylation cascades, resulting in changes in the activity of these proteins.
  • the level of intracellular cAMP is regulated at different levels; the primary effector enzyme is adenylate cyclase Cyr1, and Cyr1 catalyzes the synthesis of cAMP by ATP.
  • the regulatory subunit of PKA is encoded by the gene BCY1, and the catalytic subunit is encoded by the genes TPK1, TPK2 and TPK3.
  • Protein kinase Sch9 shares homology with the catalytic subunit of PKA. Under normal physiological conditions, Sch9 and PKA have different substrates; however, increasing the activity of either kinase compensates for growth defects caused by deletion of another kinase. .
  • the first genetic modification in the present invention eliminates the feedback inhibition of cAMP by PKA, and the PKA activity or expression is completely inhibited, and the second genetic modification eliminates the growth inhibition of the yeast strain due to the first genetic modification, thereby being able to be normal.
  • the first genetic modification in the present invention eliminates the feedback inhibition of cAMP by PKA, and the PKA activity or expression is completely inhibited
  • the second genetic modification eliminates the growth inhibition of the yeast strain due to the first genetic modification, thereby being able to be normal.
  • the nucleic acid sequence encoding the catalytic subunits Tpk1, Tpk2 and Tpk3 can be carried out.
  • the nucleic acid sequence can be a regulatory region or a coding region.
  • Tpk1 The nucleic acid sequence of Tpk1 can be found, for example, in GenBank Accession No. NC_001142.9.
  • Tpk2 The nucleic acid sequence of Tpk2 can be found, for example, in GenBank Accession No. NC_001148.4.
  • Tpk3 The nucleic acid sequence of Tpk3 can be found, for example, in GenBank Accession No. NC_001143.9.
  • Strategies for maintaining normal growth of strains in which PKA activity is completely inhibited include, but are not limited to, genetic modification of the protein kinase Rim15 encoding gene RIM15, transcription factor Msn1/Msn2 encoding gene MSN1/MSN2 or protein kinase Yak1 encoding gene YAK1, and the result of the modification The corresponding enzyme activity or expression is completely inhibited, and the strain is thus allowed to grow normally.
  • Strategies for maintaining the normal growth of strains in which PKA activity is completely inhibited include, but are not limited to, genetic modification of the protein kinase Sch9-encoding gene SCH9, the result of which results in the corresponding enzyme activity or The expression is increased and the strain is thus allowed to grow normally.
  • the nucleic acid sequence of Yak1 can be found, for example, in GenBank Accession No. NC_001142.9.
  • Rim15 The nucleic acid sequence of Rim15 can be found, for example, in GenBank Accession No. NC_001138.5.
  • the nucleic acid sequence of Msn1/Msn2 can be found, for example, in GenBank Accession No. NC_001147.6, NC_001145.3.
  • nucleic acid sequence of Sch9 can be found, for example, in GenBank Accession No. NC_001140.6.
  • the first and second genetic modifications are performed to completely inhibit the activity or expression of the enzyme, or to increase the activity or expression of the enzyme
  • four genes of TPK1, TPK2, TPK3 and the second gene are present in the yeast strain.
  • the modification step except that the combination of TPK1, TPK2, and TPK3 is simultaneously inactivated in the haploid yeast cell (the combination cannot survive due to growth defects), the above four genes may be modified in any order or in any combination.
  • yeast strains described above that have been modified by the first and second genes described above produce more extracellular cAMP relative to yeast strains that have not been modified with the first and second genes.
  • the nucleic acid sequence of Pde1 can be found, for example, in GenBank Accession No. NC_001139.9.
  • the nucleic acid sequence of Pde2 can be found, for example, in GenBank Accession No. NC_001147.6.
  • a genetic modification method that allows the activity or expression of the encoded enzyme to be completely inhibited may be a point mutation, a deletion or insertion of one or more nucleotides of the nucleic acid sequence of the gene, and a point mutation such as a single nucleotide conversion ( ⁇ to ⁇ or Pyrimidine to pyrimidine) or transversion (purine to pyrimidine or pyrimidine to purine). Mutations in a nucleic acid can result in the replacement of one or more conserved or non-conservative amino acids in the polypeptide it expresses.
  • This substitution may result in a change in the conformation of the polypeptide, or may lose some or all of its function, and a frameshift mutation may occur. This will result in the entire polypeptide chain starting to encode a completely different polypeptide from this point, or it may form a stop codon in advance to make the polypeptide chain defective or even silence the gene.
  • the nucleic acid sequence for deletion or insertion can be obtained by PCR or chemical synthesis, or can be obtained by cell replication amplification.
  • a genetic modification that completely inhibits gene enzymatic activity or expression can also be achieved by providing or expressing antisense oligonucleotides, siRNA, microRNA or other nucleic acid that can prevent the translation of the mRNA of the gene to be modified into a protein.
  • Transcriptional activator-like effector nuclease (TALEN) and CRISPR technology - Clustered Regularly Interspaced Short Palindromic Repeats can also be used to inactivate in recent years. gene.
  • a genetic modification referred to in the present invention that completely inhibits the activity or expression of a gene refers to a reduction in the activity of the enzyme/polypeptide (compared to unmodified yeast or wild-type yeast) by at least 95% (eg At least 96%, 97%, 98%, 99%, or 100%).
  • Genetic modifications that result in increased or overexpressed activity of the encoded enzyme include, but are not limited to, point mutations, ligation of strong promoters, ligation of enhancers, and increased copy number.
  • a point mutation increases the activity of the polypeptide; increases the copy number of the nucleic acid sequence; links the nucleic acid sequence to an expressible strong promoter or enhancer by genetic modification; changes the promoter of the nucleic acid sequence or other regulatory factors to increase its expression level ( For example, increasing the binding strength of a promoter sequence to a transcription initiation factor; increasing the half-life of mRNA transcribed by the nucleic acid sequence; inhibiting degradation of the mRNA or polypeptide chain.
  • the genetic modification mentioned in the present invention for increasing the activity or expression of a gene enzyme means that the activity of the enzyme/polypeptide encoded by the genetically modified nucleic acid sequence or the efficiency of the action on the substrate is increased by at least 20% compared with the wild type. (eg at least 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or higher).
  • the yeast strain modified by the above first, second and third genes described in the present invention produces more extracellular cAMP than the yeast strain not modified by the first, second and third genes . Even more extracellular cAMP is produced relative to the first and second genetically modified yeast strains.
  • Increasing the extracellular cAMP level of yeast cells and modifying the genes of cAMP-PKA signaling pathway can also be achieved by regulating the purine synthesis pathway and increasing the synthesis amount of cAMP synthesis precursor AMP.
  • the expression of the ADE de novo pathway gene, ADE gene is positively regulated by the synergy between the two transcriptional regulators Bas1 and Bas2.
  • Bas1 is a Myb transcriptional regulator
  • Bas2 also known as Pho2
  • Pho2 is a homeodomain transcriptional regulator that binds to a specific promoter region in a complex form and synergistically activates the expression of 10 ADE genes.
  • Ade4p PRPP amide transferase, abbreviated as PRPPAT
  • Bas2p can sense the concentration of SAICAR. Change, when this concentration drops to a certain extent, Bas2p no longer forms a complex with Bas1p.
  • adenine enters the cell, and AMP is formed by the salvage pathway, and AMP is further converted into ADP and ATP, which also leads to inhibition of the synthesis of the entire de novo synthesis pathway.
  • the fourth genetic modification of the present invention increases AMP synthesis and increases cAMP production by increasing the positive regulation of AMP synthesis in the purine synthesis pathway.
  • the core of positive regulation of increasing AMP synthesis is to promote the formation of Bas1/Bas2 complex, increase the positive regulatory activity of the complex, and inhibit the feedback inhibition of adenine, ADP and ATP.
  • Bas1/Bas2 complex There are various strategies for promoting the formation of Bas1/Bas2 complex and improving the positive regulatory activity of the complex, which can increase the activity or expression of a single positive regulatory transcription factor Bas1 or Bas2, or directly express co-expressing Bas1 and Bas2, or in fusion.
  • the activity or expression level was further increased based on the expression of Bas1 and Bas2.
  • a point mutation increases the activity of the polypeptide; increases the copy number of the nucleic acid sequence; links the nucleic acid sequence to an expressible strong promoter or enhancer by genetic modification; changes the promoter of the nucleic acid sequence or other regulatory factors to increase its expression level ( For example, increasing the binding strength of a promoter sequence to a transcription initiation factor; increasing the half-life of mRNA transcribed by the nucleic acid sequence; inhibiting degradation of the mRNA or polypeptide chain.
  • an increase in the activity of a polypeptide encoded by a genetically modified nucleic acid sequence requires an increase in the efficiency of its action on the substrate by at least 20% compared to the wild type (eg, at least 25%, 30%, 40%, 50%, 60%, 70). %, 80%, 90%, 95%, 99%, 100% or higher).
  • Overexpression of a single copy or multiple copies of a nucleic acid sequence can be achieved by ligation into a vector or by integration into the genome of the yeast.
  • Vectors suitable for overexpression of nucleic acid sequences have been commercialized or can be obtained by genetic recombination techniques commonly used in the art. There are several methods in the art for integrating nucleic acid sequences in the yeast genome.
  • the vector containing the nucleic acid sequence can operably link some elements necessary for expression to the nucleic acid sequence of interest: a nucleic acid sequence (eg, an auxotrophic selectable marker gene, an antibiotic resistance gene) expressing a selectable marker; A gene (eg, 6xHis tag) that can be used to purify the polypeptide; it can also carry one or more origins of replication.
  • Elements necessary for expression include nucleic acid sequences, such as promoter sequences, that are capable of directing or regulating the nucleic acid sequence of interest.
  • Representative promoters include, without limitation, the PGK promoter, the TPI1 promoter, and the ADH1 promoter. Elements necessary for expression also include an enhancer sequence, a response element, or an inducing factor that regulates expression of the coding sequence.
  • the elements necessary for expression may be from bacteria, yeast, insects, plants, mammals, fungi or viruses, and the expression vector may carry elements from different sources.
  • elements necessary for expression see In Goeddel, 1990, Gene Expression Technology: Methods in Enzymology, 185, Academic Press, San Diego, CA.
  • the nucleic acid sequence that is optimal for a particular codon or the element necessary for expression can be selected for best expression in yeast. See Bennetzen & Hall, 1982, J. Biol. Chem., 257: 3026-31.
  • the nucleic acid sequence of Bas1 can be found, for example, in GenBank Accession No. NC_001143.9.
  • the nucleic acid sequence of Bas2 can be found, for example, in GenBank Accession No. NC_001136.10.
  • Yeast strains modified by the above first, second, third and fourth genes described above are compared to yeast strains not modified by the first, second, third and fourth genes Produce more extracellular cAMP. Even more extracellular cAMP is produced relative to the first and second genetically modified yeast strains or to the first, second and third genetically modified yeast strains.
  • a nucleic acid sequence e.g., an expression vector
  • methods include, without limitation, electroporation, calcium phosphate precipitation, heat shock, lipofection, microinjection, lithium chloride, lithium acetate, mercaptoethanol, and virus-mediated genes. Transfer method.
  • a "host cell” can be any cell that can be used in standard molecular biological manipulations to produce nucleic acids and polypeptides. These include, but are not limited to, bacterial cells (such as E. coli), insect cells, plant cells, and mammalian cells (such as CHO or COS cells). "Yeast cells” include the recombinant yeast cells described herein.
  • a "host cell” as referred to herein includes not only a mother cell to which nucleic acid is transduced, but also a daughter cell thereof.
  • selectable marker genes for use in screening for transfer to yeast cells include, but are not limited to, any auxotrophic genes available for yeast, such as URA3, LEU2, HIS3, TRP1, LYS2.
  • auxotrophic genes available for yeast
  • a URA3 gene which can be conveniently and repeatedly used is preferably used. Still more preferably, the URA3 gene is recycled without remaining in the genetically modified strain.
  • yeast strains capable of industrial fermentation include Saccharomyces pastorianus, Pichia stipitis, Saccharomyces Bayanus, and Candida shehatae.
  • suitable strains include Saccharomyces pastorianus, Pichia stipitis, Saccharomyces Bayanus, and Candida shehatae.
  • the pathways or gene names may be slightly different in these strains, but the same strategies and techniques can be applied to modify the corresponding pathways and homologous genes.
  • the genetically engineered yeast strains described herein are well known to those skilled in the art.
  • the present invention also describes a fermentation method for producing cAMP by the above genetically modified yeast strain.
  • the fermentation medium has a glucose concentration of 20-150 g/L, a yeast extract of 10-20 g/L, a peptone of 20-40 g/L, and an adenine addition amount of 0-1.25 g/L. .
  • the invention also describes the use of the above genetically modified yeast strains which can be used to ferment metabolic carbohydrates to produce extracellular cAMP. Further, the above-mentioned genetically modified yeast strain cells, and/or fermented fermentation broth containing extracellular cAMP, can be directly used as a substitute for cAMP. And as cAMP is generally applied to various fields, such as medicine, animal husbandry, food or chemical industry. Still further, the application includes applications in the preparation of products in the above fields, such as pharmaceuticals, foods, feeds and the like.
  • the present invention also provides a product comprising the above-mentioned genetically modified yeast strain cells, and/or fermented fermentation liquid containing extracellular cAMP, in the fields of medicine, animal husbandry, food or chemical industry, such as medicines, foods, Feed and so on.
  • the recombinant yeast strain obtained by the invention has low nutrient requirement, is easy to culture, is suitable for large-scale fermentation production and is easy to realize industrialization performance; and related genetic modification by genetic engineering means, while eliminating feedback inhibition of cAMP pathway and reducing cAMP degradation, increasing
  • the metabolic flow to the cAMP precursor AMP enables the resulting recombinant yeast strain to produce extracellular cAMP stably, continuously, and efficiently; by increasing the carbon source concentration, optimizing the carbon/nitrogen ratio, and adding precursors, the metabolic flux is further improved. Effectively flow to the target product to obtain an optimized, high-yield extracellular cAMP fermentation process with a cAMP concentration of 9721.6 ⁇ mol/L.
  • the invention overcomes the defects of the traditional chemical synthesis method, the raw materials are expensive, the by-products are many, the environmental pollution is serious, and the enzymatic conversion method is expensive, the stability of the adenylate cyclase is poor, and the process is simple.
  • the product is easy to separate and extract, low in raw material cost, safe and environmentally friendly, and can be industrialized. It opens up a new way for microbial fermentation to produce cAMP, which will promote green and clean production of high-value medicines and biochemical products, and can be widely used in medicine and animal husbandry. Industry, food or chemical industry has important economic and social significance.
  • Figure 1 is a schematic diagram of the plasmid of Example 1, wherein Figure 1A is the plasmid pUC18-URA3, and Figure 1B is the plasmid pUC18-TPK2p-TPK2t-URA3-TPK2t.
  • Figure 2 is a preliminary evaluation of cAMP production of five gene deletion strains of Example 5.
  • Figure 3 is a schematic representation of the plasmid pUC18-H1 YNRC ⁇ 9 -BAS1-BAS2-H2 YNRC ⁇ 9 -URA3-H2 YNRC ⁇ of Example 6.
  • uracil CMG CMG -URA flat short flat screened.
  • the components are as follows: amino acid base mixture, 0.83 g/L; yeast amino nitrogen source Yeast Nitrogen Base, abbreviated as YNB, 6.7 g/L; glucose, 20 g/L; agar powder, 15 g/L.
  • the amino acid base mixture therein is shown in Table 1.
  • the selection of the medium can be made by eliminating specific amino acid components.
  • the pH was adjusted to 5.6, the glucose was sterilized at 110 ° C for 15 min, and the other components were sterilized at 121 ° C for 21 min, and mixed before use.
  • the preparation method is as follows:
  • YCplac33 is available from Invitrogen.
  • pUC18 is available from companies such as Invitrogen and Promega.
  • Example 1 Construction of a yeast strain lacking the TPK2 gene
  • the plasmid pUC18-TPK2p-TPK2t-URA3-TPK2t was first constructed.
  • the plasmid was linearized by restriction enzyme digestion and transformed into yeast competent cells, and URA3 was used as a selection marker gene.
  • the yeast strain tpk2 ⁇ ::URA3 obtained by double-exchange of TPK2p downstream of TPK2p and URA3 as the left and right homologous arms was screened; and the strain ppk2 was detected by 5'-FOA plate: URA3 was left and right on the URA3 chromosome.
  • the yeast target strain tpk2 ⁇ which recombines between TPK2t and ejects the URA3 gene.
  • the primers for construction and identification are shown in Table 2, and the schematic diagram is shown in Figure 1.
  • PCR amplification of the URA3 gene using plasmid YCplac33 as a template using the sequences shown in SEQ ID No. 1 and SEQ ID No. 2 in Table 2 as the upstream and downstream primers P1 and P2, and the Fast Pfu polymerase produced by the full-scale gold company. Annealed at 50 ° C for 1 min and extended at 72 ° C for 1.5 min for a total of 32 cycles. A 1055 bp PCR fragment was obtained, and the fragment was digested with restriction endonuclease BamHI and SalI. The vector was ligated with the vector plasmid pUC18. The T4 DNA ligase was ligated and transformed into E. coli Top10 competent cells, and the transformant plasmid was extracted for restriction enzyme digestion to obtain a plasmid.
  • pUC18-BamHI-URA3-SalI (referred to as pUC18-URA3, see Figure 1A).
  • the Saccharomyces cerevisiae strain W303-1A chromosome was extracted, and the W303-1A chromosome was used as a template for PCR, and the terminator region of the TPK2 gene (abbreviated as TPK2t) was amplified, using SEQ ID No. 3 and SEQ ID No. 4 in Table 2.
  • TPK2t the terminator region of the TPK2 gene
  • SEQ ID No. 3 and SEQ ID No. 4 in Table 2.
  • the sequence shown is the upstream and downstream primers P3 and P4, and the Fast Pfu polymerase produced by Quanjin Company was annealed at 50 ° C for 1 min and extended at 72 ° C for 0.5 min for a total of 32 cycles.
  • a 526 bp PCR fragment was obtained, and the fragment and the vector plasmid pUC18-URA3 were digested with restriction endonucleases KpnI and BamHI.
  • the T4 DNA ligase was ligated and transformed into E. coli Top10 competent cells, and the transformant plasmid was extracted for restriction enzyme digestion.
  • the plasmid pUC18-TPK2t-URA3 was obtained.
  • the W303-1A chromosome was extracted, and the W303-1A chromosome was used as a template for PCR, and the promoter region of TPK2 gene (abbreviated as TPK2p) was amplified.
  • TPK2p the promoter region of TPK2 gene
  • the sequences shown in SEQ ID No. 5 and SEQ ID No. 6 in Table 2 were used.
  • the upstream and downstream primers P5 and P6, the Fast Pfu polymerase produced by Quanjin Company were annealed at 50 ° C for 1 min and extended at 72 ° C for 0.5 min for a total of 32 cycles.
  • a 532 bp PCR fragment was obtained, and the fragment was digested with restriction endonucleases Sac I and Kpn I to digest the fragment and the vector plasmid pUC18-TPK2t-URA3.
  • the T4 DNA ligase was ligated and transformed into E. coli Top10 competent cells, and the transformant plasmid was extracted.
  • the plasmid pUC18-TPK2p-TPK2t-URA3 was obtained by restriction enzyme digestion.
  • the PCR product containing the TPK2t sequence amplified in the above (2) was digested with Sal I and Pst I, and ligated with the same double-digested pUC18-TPK2p-TPK2t-URA3 large fragment to obtain plasmid pUC18-TPK2p- TPK2t-URA3-TPK2t (see Figure 1B).
  • the plasmid pUC18-TPK2p-TPK2t-URA3-TPK2t was digested with Sac I and Pst I to obtain the linearized DNA fragment TPK2p-TPK2t-URA3-TPK2t; then the lithium acetate method was used to transform the competent cells of Saccharomyces cerevisiae W303-1A strain.
  • the uracil auxotrophic selection marker (URA) was used for screening, that is, screening was performed using CMG- URA plates, and the obtained transformant strain was subjected to YPAD liquid culture, and chromosomal DNA was extracted, and the PCR method was used as a template to verify the primer pair.
  • the strain W303-1A (tpk2 ⁇ :: URA3) plate growth cells were further coated on 5'-FOA plates, thereby screening out the homologous recombination between two identical sequences (TPK2t) integrated on the chromosome. Colonies of the URA3 fragment. Using the same primer pair P7 and P8, the PCR product of the target strain that successfully popped URA3 was 1053 bp long, and the PCR fragment of the host strain W303-1A (tpk2 ⁇ :: URA3) without URA3 was 2592 bp long.
  • Example 2 Construction of a yeast strain lacking the TPK1 or TPK3 gene
  • plasmid pUC18-TPK1p-TPK1t-URA3-TPK1t for TPK1 gene deletion The plasmid pUC18-TPK1p-TPK1t-URA3-TPK1t was constructed based on the (1) pUC18-URA3 plasmid of Example 1, and the process was carried out in the same manner as in Example 1. The steps of (2), (3) and (4) are the same as the procedure for finally obtaining pUC18-TPK2p-TPK2t-URA3-TPK2t, and the primer pair used: amplification TPK1t is SEQ ID No. 9 and SEQ ID No. in Table 2.
  • the sequence shown by 10 is the upstream and downstream primers P9 and P10, and the product is 516 bp;
  • the amplified TPK1p is the sequence shown by SEQ ID No. 11 and SEQ ID No. 12 in Table 2, the upstream and downstream primers P11 and P12, and the product 572 bp.
  • the plasmid pUC18-TPK1p-TPK1t-URA3-TPK1t was digested with Sac I and Pst I to obtain a linearized DNA fragment TPK1p-TPK1t-URA3-TPK1t; then the S. cerevisiae W303-1A strain competent cells were transformed with uracil. (URA) auxotrophic selection markers were screened, that is, screened using CMG- URA plates, and the obtained transformant strains were further verified by PCR, and the primer pairs were SEQ ID No. 13 and SEQ ID No. 14 in Table 2.
  • the indicated sequences were the upstream and downstream primers P13 and P14, and the successfully integrated transformant PCR product was 2631 bp, and the control strain PCR product was 2476 bp.
  • the positive transformant strain was named W303-1A (tpk1 ⁇ ::URA3).
  • the strain W303-1A (tpk1 ⁇ :: URA3) plate growth cells were further coated on 5'-FOA (5'-orotic acid) plates to screen out two identical sequences (TPK1t) integrated on the chromosome. A colony that sheds URA3 fragments by homologous recombination.
  • the PCR product of the target strain that successfully popped URA3 was 1102 bp long, and the control PCR fragment without URA3 was 2631 bp long.
  • the 1102 bp PCR product was sequenced and the sequencing results confirmed that the expected changes occurred: 70 bases before the start codon of the TPK1 gene, the entire ORF region, and 116 bases after the stop codon were completely deleted.
  • a positive transformant strain W303-1A (tpk1 ⁇ ) in which the TPK1 gene was deleted and the URA3 gene was ejected was finally obtained.
  • the plasmid pUC18-TPK3p-TPK3t-URA3-TPK3t was digested with Sac I and Pst I to obtain the linearized DNA fragment TPK3p-TPK3t-URA3-TPK3t; then the S. cerevisiae W303-1A strain competent cells were transformed, the same example
  • the "W303-1A (tpk2 ⁇ ) strain construction" method was used to screen and identify W303-1A (tpk3 ⁇ ::URA3) strain and W303-1A (tpk3 ⁇ ) strain.
  • the primers used in the PCR method verification were the sequences shown in SEQ ID No. 19 and SEQ ID No.
  • the PCR product containing the URA3 gene was 2695 bp, and the integration was successful.
  • the transformant PCR product of the URA3 gene was 1116 bp, and the PCR product of the control strain was 2400 bp.
  • the 1116 bp PCR product was sequenced and the sequencing results confirmed that the expected change occurred: the entire ORF region of the TPK3 gene and 93 bases after the stop codon were completely deleted.
  • a positive transformant strain W303-1A (tpk3?) in which the TPK3 gene was deleted and the URA3 gene was ejected was finally obtained.
  • Example 3 Construction of a yeast strain lacking the YAK1 gene
  • YAK1 gene deletion strain The plasmid pUC18–YAK1p-YAK1t-URA3-YAK1t was digested with Sac I and Pst I to obtain a linearized DNA fragment YAK1p-YAK1t-URA3-YAK1t; then the S. cerevisiae W303-1A strain was transformed. Competent cells were screened and identified as "W303-1A (tpk2?) strain construction" in Example 1 to obtain W303-1A (yak1 ⁇ ::URA3) strain and W303-1A (yak1 ⁇ ) strain.
  • the PCR primers used in the PCR method were the sequences shown in SEQ ID No. 25 and SEQ ID No.
  • the primers were P25 and P26.
  • the successful integration and the PCR product containing the URA3 gene were 2529 bp, and the integration was successful.
  • the transformant PCR product of the URA3 gene was 1010 bp, and the PCR product of the control strain was 3614 bp.
  • the 1010 bp PCR product was sequenced and the sequencing results confirmed that the expected changes occurred: 40 bases before the start codon of the YAK1 gene, 146 bases after the entire ORF region and the stop codon were completely deleted.
  • a positive transformant strain W303-1A (yak1 ⁇ ) in which the YAK1 gene was deleted and the URA3 gene was ejected was finally obtained.
  • Example 4 Construction of a yeast strain that simultaneously lacks the TPK1, TPK2, TPK3, and YAK1 genes
  • the W303-1A (yak1 ⁇ ) strain was constructed in the same manner as in Example 3.
  • the deletion plasmid pUC18-TPK1p-TPK1t-URA3-TPK1t constructed in Example 2 was digested with Sac I and Pst I to obtain a linearized DNA fragment TPK1p-TPK1t-URA3-TPK1t; then, W303 in the first step was transformed.
  • -1A (yak1 ⁇ ) strain competent cells were screened using uracil (URA) auxotrophic selection markers, ie, screened using CMG- URA plates, and the obtained transformant strains were identified by the same method as in Example 2.
  • the TPK1 deletion strain W303-1A (yak1 ⁇ ) (tpk1 ⁇ :: URA3) was obtained, and further, the W303-1A (yak1 ⁇ ) (tpk1 ⁇ ) strain was obtained.
  • the W303-1B strain (MAT ⁇ leu2-3, 112ura3-1trp1-92his3-11, 15ade2-1can1-100) identical to the W303-1A strain except for the mating type genotype was obtained from W303-1A by mating type transformation, and W303-1B was obtained.
  • the two genes TPK2 and TPK3 were sequentially knocked out.
  • the plasmid pUC18-TPK2p-TPK2t-URA3-TPK2t constructed in Example 1 was digested with Sac I and Pst I to obtain a linearized DNA fragment TPK2p-TPK2t-URA3-TPK2t; then the S. cerevisiae W303-1B strain was transformed.
  • the cells were screened and identified in the same manner as in the "W303-1A (tpk2?) strain construction" method of Example 1 to obtain W303-1B (tpk2?::URA3) strain and W303-1B (tpk2?) strain.
  • the plasmid pUC18-TPK3p-TPK3t-URA3-TPK3t constructed in Example 2 was digested with Sac I and Pst I to obtain a linearized DNA fragment TPK3p-TPK3t-URA3-TPK3t; and then transformed into Saccharomyces cerevisiae W303-1B (tpk2) ⁇ ) strain competent cells, and the method of "W303-1A (tpk2 ⁇ ) strain construction" in Example 1 was screened and identified to obtain W303-1B (tpk2 ⁇ tpk3 ⁇ ::URA3) strain and W303-1B (tpk2 ⁇ tpk3 ⁇ ) ) strain.
  • the W303-1A (tpk1 ⁇ yak1 ⁇ ) strain was crossed with W303-1B (tpk2 ⁇ tpk3 ⁇ ) strain to obtain diploid strain, and then alleles were isolated by sporulation and sporulation, and W303- was obtained by screening and identification.
  • Identification primers for the identification of four gene knockouts are the same as before.
  • the sequencing results of the identified PCR products confirmed that the four gene nucleic acid sequences on the chromosomes of both strains were expected to change, and all of the ORF regions were deleted.
  • Seed culture pick up the colonies grown on the YPAD solid medium plate, insert into a test tube containing 5 mL of YPAD culture solution, culture at 30 ° C, 220 rpm overnight, and if necessary, carry out secondary expansion culture;
  • Fermentation medium yeast extract 10g/L, peptone 20g/L, glucose 20g/L, natural pH value; inoculate fresh seed liquid into 100ml volumetric flask containing 25ml fermentation medium, control initial The OD 600 value is about 0.1, and the fermentation is carried out at 30 ° C and 220 rpm;
  • OD 600 measured fermentation broth After fermentation samples were diluted appropriately measured OD 600, detected growth;
  • HPLC analysis of the concentration of cAMP in the fermentation supernatant, ie, extracellular cAMP concentration The method for HPLC analysis of cAMP was adjusted according to the method of the Pharmacopoeia of the People's Republic of China: 2010 Edition (edited by the National Pharmacopoeia Commission) on page 419.
  • the standard sample is filtered with a 0.2 ⁇ m filter and analyzed by HPLC to peak.
  • the area is subjected to a standard curve for cAMP concentration; 3) the concentration of cAMP in the fermentation broth sample is calculated by external standard quantitative method using a standard curve.
  • the maximum extracellular cAMP concentration of the two deletion strains was 144.0 and 155.1 times the maximum extracellular cAMP concentration of the control strain W303-1A, respectively. It should be noted that the cAMP concentration of the two deletion strains increased rapidly within 0-24 h, and the cAMP concentration decreased slightly after reaching the highest value at 24 h, but remained above 180 ⁇ mol/L until 96 h.
  • Example 5 Construction of a yeast strain that simultaneously lacks the TPK1, TPK2, TPK3, YAK1, PDE1 genes
  • PCR was carried out using the W303-1A chromosome as a template to amplify the terminator region of the PDE1 gene (abbreviated as PDE1t), and the sequences shown in SEQ ID No. 27 and SEQ ID No. 28 in Table 2 were used as the upstream and downstream primers P27.
  • P28 Fast Pfu polymerase produced by Quanjin Company, annealed at 50 ° C for 1 min, and extended at 72 ° C for 0.5 min for a total of 32 cycles.
  • a 500 bp PCR fragment was obtained, and the fragment and the vector plasmid pUC18-URA3 were digested with restriction endonucleases KpnI and BamHI.
  • the T4 DNA ligase was ligated and transformed into E. coli Top10 competent cells, and the transformant plasmid was extracted for restriction enzyme digestion.
  • the plasmid pUC18-PDE1t-URA3 was obtained.
  • PCR was carried out using the W303-1A chromosome as a template to amplify the promoter region of PDE1 gene (abbreviated as PDE1p), and the sequences shown in SEQ ID No. 29 and SEQ ID No. 30 in Table 2 were used as the upstream and downstream primers P29.
  • P30 Fast Pfu polymerase produced by Quanjin Company, annealed at 50 ° C for 1 min, and extended at 72 ° C for 0.5 min for a total of 32 cycles.
  • a 544 bp PCR fragment was obtained, and the fragment was digested with restriction endonucleases Sac I and Kpn I to digest the fragment and the vector plasmid pUC18-PDE1t-URA3, and the T4 DNA ligase was ligated to transform into E. coli Top10 competent cells, and the transformant plasmid was extracted.
  • the plasmid pUC18-PDE1p-PDE1t-URA3 was obtained by restriction enzyme digestion.
  • the amplified PCR product containing the PDE1t sequence was digested with Sal I and Pst I, and ligated with the same double-digested pUC18-PDE1p-PDE1t-URA3 large fragment to obtain plasmid pUC18-PDE1p-PDE1t-URA3- PDE 1t.
  • the plasmid pUC18-PDE1p-PDE1t-URA3-PDE 1t was digested with Sac I and Pst I, and the same method as in "W303-1A (tpk2 ⁇ ) strain construction in Example 1) was used in Example 4
  • the constructed W303-1A (tpk1 ⁇ tpk2 ⁇ tpk3 ⁇ yak1 ⁇ ) was constructed as the host, and W303-1A (tpk1 ⁇ tpk2 ⁇ tpk3 ⁇ yak1 ⁇ pde1 ⁇ ::URA3) and W303-1A(tpk1 ⁇ tpk2 ⁇ tpk3 ⁇ yak1 were constructed.
  • ⁇ pde1 ⁇ The constructed W303-1A (tpk1 ⁇ tpk2 ⁇ tpk3 ⁇ yak1 ⁇ was constructed as the host, and W303-1A (tpk1 ⁇ tpk2 ⁇ tpk3 ⁇ yak1 ⁇ pde1 ⁇ ::URA3) and W
  • the PCR primers used in the PCR method were the primers shown in SEQ ID No. 31 and SEQ ID No. 32 in Table 2 as the upstream and downstream primers P31 and P32.
  • the successful integration and the transformant PCR product containing the URA3 gene were 2958 bp, and the integration was successful.
  • the transformant PCR product of the URA3 gene was 1445 bp, and the PCR product of the control strain was 2635 bp.
  • the 1445 bp PCR product was sequenced and the sequencing results confirmed the expected change: the 40 bases before the start codon of the PDE1 gene, the entire ORF region and the 46 bases after the stop codon were completely deleted.
  • a positive transformant strain in which the PDE1 gene was deleted and the URA3 gene was ejected was finally obtained.
  • Seed culture is the same as in Example 4; 2. Fermentation: 1) Fermentation medium: yeast extract 10 g/L, peptone 20 g/L, glucose 20 g/L, natural pH; 2) Fermentation conditions: fresh seeds
  • Fermentation medium yeast extract 10 g/L, peptone 20 g/L, glucose 20 g/L, natural pH
  • Fermentation conditions fresh seeds
  • the solution was inoculated into a 100 ml volumetric flask containing 25 ml of fermentation medium, and the initial OD 600 value was controlled to be about 0.1, and fermentation was carried out at 30 ° C and 220 rpm.
  • the fermentation broth was analyzed in the same manner as the HPLC analysis method in Example 4. The results of extracellular cAMP production within 120 h are shown in Figure 2.
  • the results in Figure 2 show that: 1.
  • the maximum extracellular cAMP concentrations in the two deletion strains were 252.0 and 953.8 ⁇ mol/L, respectively, which were 173.8 and 657.8, respectively, of the maximum extracellular cAMP concentration (1.45 ⁇ mol/L) of the control strain W303-1A.
  • the ratio of the maximum extracellular cAMP concentration of W303-1B (tpk1 ⁇ tpk2 ⁇ tpk3 ⁇ yak1 ⁇ ) strain in Example 4 was 1.17 and 4.42 times; 2.
  • W303-1A (tpk1 ⁇ tpk2 ⁇ tpk3 ⁇ yak1 ⁇ pde1 ⁇ :: The cAMP concentration of URA3) was basically stable from 48h, while the cAMP concentration of W303-1A (tpk1 ⁇ tpk2 ⁇ tpk3 ⁇ yak1 ⁇ pde1 ⁇ ) increased continuously. 3. The retention of the selection marker URA3 could significantly increase the cAMP yield.
  • Seed culture was the same as in Example 4; 2. Fermentation: 1) Fermentation medium: yeast extract 10 g/L, peptone 20g / L, glucose 20 ⁇ 150g / L, natural pH; 2) fermentation conditions: fresh seed solution inoculated into a 100ml volumetric flask containing 25ml fermentation medium, control initial OD 600 value of about 1, 30 ° C, Fermentation at 220 rpm.
  • the fermentation broth was analyzed in the same manner as the HPLC analysis method in Example 4. The results of evaluation of extracellular cAMP for growth and fermentation production within 120 h are shown in Table 4.
  • the BAS1 and BAS2 genes are fused and co-expressed and integrated into the chromosome of the yeast cell. To this end, it is first necessary to construct a fusion co-expression integration vector with left and right homology arms.
  • the integration site selected here is reported in the literature (Bai Flagfeldt D, Siewers V, Huang L, et al. Characterization of chromosomal integration sites for heterologous gene expression in Saccharomyces cerevisiae. Yeast, 2009, 26(10): 545-551).
  • YNRC ⁇ 9 which has a high gene expression efficiency, is located on chromosome XIV.
  • a partial sequence of YNRC ⁇ 9 was amplified by PCR using the chromosomal DNA of W303-1A as a template as an integrated left and right homology arm.
  • the sequences shown in SEQ ID No. 33 and SEQ ID No. 34 in Table 2 were used as the upstream and downstream primers P33 and P34, and the Fast Pfu polymerase produced by Quanjin Company was annealed at 50 ° C for 1 min and extended at 72 ° C for 45 sec. In 32 cycles, a 521 bp PCR1 fragment was obtained (corresponding sequence used as the integrated left homology arm H1 YNRC ⁇ 9 ).
  • the 448 bp PCR2 fragment was amplified by the same method (the corresponding sequence was used as the integrated right homology arm H2 YNRC ⁇ 9). ).
  • the PCR1 and PCR2 products were used as templates, and P33 and P36 were used as primer pairs.
  • the 949 bp PCR12 fragment was amplified by cross-overlap extension PCR reaction. The 5' end of this fragment was added with the enzyme cleavage site EcoRI, and the 3' end was added with the enzyme cleavage site BamHI, and the enzyme cleavage sites SacI and Sal I were added in the middle.
  • the PCR product was digested with EcoRI and BamHI, and ligated with the same double-digested pUC18 large fragment, transformed into E. coli Top10 competent cells, and the transformant plasmid was extracted for restriction enzyme digestion to obtain plasmid pUC18-H1 YNRC ⁇ 9- SacI- SalI-H2 YNRC ⁇ 9 . Sequencing demonstrated that the cloned fragment did not have a mutation.
  • PCR amplification of the URA3 gene using YCplac33 as a template was used as a selectable marker gene for integration.
  • the sequences shown in SEQ ID No. 37 and SEQ ID No. 38 in Table 2 were used as the upstream and downstream primers P37 and P38, and the Fast Pfu polymerase produced by Quanjin Company was annealed at 50 ° C for 1 min and extended at 72 ° C for 1 min. For 32 cycles, a 1060 bp PCR1 fragment was obtained.
  • the H2 YNRC ⁇ 9 fragment was still amplified by PCR using the chromosomal DNA of W303-1A as a template, but the upstream and downstream primer pairs were replaced with the sequences shown in SEQ ID No.
  • the 2907 bp PCR1 fragment was obtained, and the 5' end of the fragment was ligated with SacI, including the 762 bp sequence upstream of the ATG of the BAS1 gene start codon and the 2112 bp ORF sequence including ATG, and deleted the 3' end of the ORF by 324 bp (including Stop codon) sequence.
  • PCR1 and PCR2 were used as primer pairs, and a 4578 bp PCR12 fragment was amplified by cross-overlap extension PCR reaction.
  • the PCR fragment was subjected to A-tail reaction, separated by agarose gel electrophoresis, and the gel was recovered.
  • the pGEM-T easy vector purchased from Promega was ligated according to the instructions, and the E. coli Top10 competent cells were transformed to extract transformants.
  • the plasmid was subjected to restriction enzyme digestion to obtain plasmid pGEM-T easy-BAS1-BAS2. Sequencing demonstrated that the cloned fragment did not have a mutation.
  • the plasmid pGEM-T easy-BAS1-BAS2 was digested with SacI and Sal I, and the large fragment SacI-BAS1-BAS2-Sal I was recovered.
  • the same double enzyme was digested with pUC18-H1 YNRC ⁇ 9 -SacI-SalI-H2 YNRC ⁇ 9 -
  • the large fragment of URA3-H2 YNRC ⁇ 9 was ligated to obtain the integration plasmid pUC18-H1 YNRC ⁇ 9 -BAS1-BAS2-H2 YNRC ⁇ 9 -URA3-H2 YNRC ⁇ 9 .
  • the plasmid pUC18-H1 YNRC ⁇ 9 -BAS1-BAS2-H2 YNRC ⁇ 9 -URA3-H2 YNRC ⁇ 9 was digested with Pvu II to obtain a linearized DNA fragment H1 YNRC ⁇ 9 -BAS1-BAS2-H2 YNRC ⁇ 9 -URA3-H2 YNRC ⁇ 9 ; then W303-1A was transformed ( Tpk1 ⁇ tpk2 ⁇ tpk3 ⁇ yak1 ⁇ pde1 ⁇ ) strain competent cells, and the method of "W303-1A(tpk2 ⁇ ) strain construction" in Example 1 was screened and identified to obtain W303-1A (tpk1 ⁇ tpk2 ⁇ tpk3 ⁇ yak1 ⁇ pde1 ⁇ BAS1BAS2-URA3) strain and W303-1A (tpk1 ⁇ tpk2 ⁇ tpk3 ⁇ yak1 ⁇ pde1 ⁇ BAS1BAS
  • the PCR primers used in the PCR method were SEQ ID No. 45 and SEQ ID No. 46 in Table 2.
  • the sequences were SEQ ID No. 45 and SEQ ID No. 46 were the upstream and downstream primers P45 and P46.
  • the integration of the transformant PCR product containing the URA3 gene was 7389 bp, and the integration was successful.
  • the transformant PCR product of the URA3 gene was 5929 bp, and the PCR product of the control strain was 1687 bp.
  • Example 5 has demonstrated that glucose concentration significantly affects the production of cAMP.
  • the molecular formula is C 10 H l2 N 5 O 6 P
  • the content of nitrogen and phosphorus, especially nitrogen may be the primary limiting factor for cAMP synthesis while increasing the glucose concentration.
  • the condition of extracellular adenine also greatly affects the expression level of each gene in the purine synthesis pathway, which in turn affects cAMP production. Therefore, the above-mentioned strains were examined for the production of cAMP under the two yeast extract/peptone levels (1*YP, 2*YP) and adenine addition. The specific operation is as follows: 1.
  • the seed liquid culture is the same as in the example 4; 2. Fermentation: 1) Fermentation medium: yeast extract 10 g/L, peptone 20 g/L, abbreviated as 1*YP, glucose 150 g/L, natural pH value; yeast extract 20g/L, peptone 40g/L, abbreviated as 2*YP, glucose 150g/L, natural pH value; adenine addition amount is 0.625, 1.25g/L, abbreviated as A0.625, A1.25; 2) Fermentation conditions: fresh seed solution was inoculated into a 100 ml volumetric flask containing 25 ml of fermentation medium, and the initial OD 600 value was controlled at about 1, 30 ° C, 220 rpm fermentation; 3) fermentation broth analysis: same HPLC analysis method in Example 4, simultaneous HPLC analysis of adenine: a, sample processing and HPLC analysis are the same as sample processing and HPLC analysis of cAMP analysis; b, adenine standard curve preparation: using adenine standard The
  • the fermentation results are shown in Table 5.
  • the results in Table 5 indicate that: 1. Under the fermentation conditions of 1*YP and 15% glucose, the maximum extracellular cAMP concentration of the fusion co-expressing BAS1-BAS2 strain is higher than that of W303-1A in Example 5 (tpk1 ⁇ tpk2 ⁇ tpk3 ⁇ yak1 The maximum extracellular cAMP concentration (3925.6 ⁇ mol/L) of ⁇ pde1 ⁇ ) strain was improved, the former was 1.108 times of the latter; 2. The effect of increasing yeast powder and peptone concentration was extremely significant; 3. Adding adenine could further increase extracellular cAMP production.

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Abstract

一种高产环磷酸腺苷（cAMP）的酵母菌株及其构建方法，所述方法为构建同时缺失蛋白激酶A催化亚基编码基因TPK1、TPK2、TPK3和蛋白激酶编码基因YAK1的酿酒酵母；还可以在缺失上述4种基因的基础上使所述酵母菌株再缺失磷酸二酯酶编码基因PDE1及转录因子Bas1和Bas2的编码基因；所述重组酵母菌株相对于未经基因修饰的酵母菌株而言，能更高效地生产胞外cAMP。

Description

一种高产cAMP的酵母菌株及其应用 技术领域
本发明属于基因工程和微生物发酵技术领域，具体涉及一种能过量合成胞外cAMP的酵母菌株及其构建方法、发酵工艺与在医药、畜牧业、食品或化工领域中的应用。
背景技术
环磷酸腺苷(cyclic adenosine monophosphate，简称cAMP)是人体内广泛存在的一种具有生理活性的重要物质，其作为细胞内的第二信使，对糖代谢、脂肪代谢、核酸合成、蛋白质合成等起着重要的调节作用。目前cAMP在医药方面用途广泛：临床上cAMP用于治疗心绞痛、心肌梗死、心肌炎及心源性休克等；对改善风湿性心脏病的心悸、气急、胸闷等症状有一定的作用；可提高急性白血病结合化疗的疗效，亦可用于急性白血病的诱导缓解；此外，对老年慢性支气管炎、各种肝炎和银屑病也有一定疗效。cAMP也可作为药物中间体制备二丁酰环磷腺苷和环磷腺苷葡甲胺，提高脂溶性，从而发挥更有效的生理及药理作用。cAMP还可用作畜禽食品添加剂，在离体条件下模拟生长激素的作用，促进畜禽生长，增加优质禽产品产量。
cAMP的生产方法有化学合成法、酶促转化法和发酵法三种。目前国内外产业化生产全部采用以腺苷一磷酸(Adenosine-5’-monophosphate,简称AMP)为原料的化学合成法，其采用高效分离柱进行中间体分离，操作复杂，且所涉及的试剂昂贵，采用大量的吡啶作为溶剂也造成严重的环境污染。酶促转化法即以腺苷酸环化酶(E.C.4.6.1.1，Cyr1p)为酶源催化ATP原料一步生产cAMP，但此法存在底物昂贵、腺苷酸环化酶含量低、纯化困难、稳定性差等限制，故目前还局限于实验室阶段，离产业化还有相当的距离。相比之下，微生物发酵法生产cAMP具有如下的一些优点：条件温和、工艺简单、能利用廉价的碳源，副产物少、环境污染小，可实现持续生产等等，是有潜力的、值得大力开发的工艺。
目前利用液化短杆菌、微杆菌、棒状杆菌、节杆菌、大肠杆菌等细菌发酵法生产cAMP已有一定的研究，但面临重复性差、产量不稳定、成本高、难以工业化等问题。而酵母尤其是酿酒酵母(Saccharomyces cerevisiae)作为最重要的工业微生物之一，不仅具有工业化应用技术成熟、抗逆性强、为食品安全性GRAS微生物的优点，同时作为最早实现全基因组测序的微生物菌种，又是分子生物学和遗传学研究的最重要模式生物之一，cAMP信号通路及其调控相关的基础研究深入，遗传操作技术手段完善。遗憾的是目前国内外的研究都集中于胞内cAMP的信号调控功能，还没有将其作为一个产品从而关注胞外生产情况的专门研究。若研究酵母胞外cAMP产生规律，并通过基因工程手段改造实现以过量生产胞外cAMP酵母为菌种的发酵法生产，将极大地降低成本、简化工艺，从而为微生物发酵生产cAMP开辟新的途径，推动高值医药和生化产品的绿色清洁生产，具有重要的经济价值和社会意义。
发明内容
3.1发明概述Brief Summary of the Invention
本发明的目的是克服上述现有技术存在的不足，提供一种容易培养、适合大规模发酵生产并易于实现产业化的、经过遗传改造而能过量合成胞外cAMP的酵母菌株，构建此菌株的重组方法及应用。具体地，
本发明提供一种酵母菌株，此酵母菌株经过了第一种和第二种基因修饰，其中，所述第一种基因是蛋白激酶A(protein kinase A，PKA)催化亚基编码基因TPK1，TPK2和TPK3，通过修饰第一种基因使PKA活性或表达被完全抑制，从而消除对cAMP的反馈抑制，但同时使得酵母的生长被抑制，第二种基因修饰消除了第一种基因修饰导致的生长抑制，从而使得酵母能够正常生长，所述酵母的cAMP产量上升，其中，所述cAMP产量上升，是相对于未经基因修饰的酵母的cAMP产量而言的。
优选的，所述第二种基因包括但不仅限于蛋白激酶Rim15编码基因RIM15、转录因子Msn1/Msn2编码基因MSN1/MSN2、蛋白激酶Yak1编码基因YAK1或蛋白激酶Sch9编码基因SCH9，优选YAK1。更优选的，所述第二种基因修饰使得所修饰基因编码酶的活性或表达被完全抑制，或者编码酶的活性提高或者过表 达。
优选的，第二种基因修饰使得蛋白激酶Rim15编码基因RIM15、转录因子Msn1/Msn2编码基因MSN1/MSN2、蛋白激酶Yak1编码基因YAK1编码酶的活性或表达被完全抑制；或者使得蛋白激酶Sch9编码基因SCH9编码酶的活性提高或者过表达。
进一步的，上述酵母还包括第三种基因修饰，以减少cAMP的降解，从而提高cAMP产量。
优选的，所述第三种基因为cAMP磷酸二酯酶编码基因PDE1和/或PDE2，优选PDE1。更优选的，所述第三种基因修饰使得所修饰基因编码酶的活性或表达被完全抑制。
优选的，上述使得编码酶的活性或表达被完全抑制的基因修饰方式包括点突变、缺失、插入、反义polynucleotides、siRNA、microRNA、CRISPR，更优选缺失和点突变；使得编码酶的活性提高或者过表达的基因修饰方式包括点突变、连接强启动子、连接增强子、提高拷贝数
进一步，上述酵母还包括第四种基因修饰，以增加嘌呤合成途径中cAMP前体物的合成的正调控，使cAMP前体物的合成上升，从而提高cAMP产量。
优选的，所述第四种基因包括但不仅限于转录因子Bas1、Bas2编码基因。更优选的，所述基因修饰使Bas1/Bas2复合物表达上升。
优选的，上述第四种基因的修饰方式包括但不仅限于点突变、连接强启动子、连接增强子、提高拷贝数、融合共表达，更优选融合共表达。
进一步的，所述基因的修饰方式包含使用选择标记基因。优选的，所述选择标记基因包括但不仅限于URA3、LEU2、HIS3、TRP1、LYS2。更优选使用URA3选择标记基因。更进一步优选的，所述基因的修饰方式是循环使用选择标记基因而不保留选择标记基因。
本发明所描述的酵母菌株，较未经第一和第二种基因修饰的酵母，或未经第一，第二和第三种基因修饰的酵母，或第一，第二，第三和第四种基因修饰的酵母菌株有更高的胞外cAMP产量。例如，本文描述的酵母菌株的胞外cAMP产量高达9721.6μmol/L。
本发明所描述的酵母菌株，为酿酒酵母(Saccharomyces cerevisiae)、巴斯德酵母(Saccharomyces pastorianus),树干毕赤酵母(Pichia stipitis),Saccharomyces Bayanus和休哈塔假丝酵母(Candida shehatae)中的任意一种。优选酿酒酵母(Saccharomyces cerevisiae)。
本发明的一个目的是提供一种构建胞外cAMP产量大幅提高的上述酵母菌株的方法。
首先在酵母中引入第一种基因修饰，消除对cAMP的反馈抑制，但同时使得酵母的生长被抑制，第二种基因修饰消除了第一种基因修饰导致的生长抑制、从而使得酵母能够正常生长。
进一步的，所述方法中还包括对酵母进行第三种基因修饰以减少cAMP的降解，从而进一步提高cAMP产量。
更进一步，所述方法中还包括对酵母进行第四种基因修饰以增加嘌呤合成途径中cAMP前体物的合成的正调控，使cAMP前体物的合成上升，提高cAMP产量。
本发明的一个目的是提供一种使用上述的酵母菌株生产cAMP的发酵方法。
进一步，所述发酵方法包括发酵培养基组分，如碳源、氮源、微量元素。还包括温度、转速。
优选的，所述发酵方法中，发酵用培养基的葡萄糖浓度为20-150g/L，酵母提取物10-20g/L，蛋白胨20-40g/L，腺嘌呤添加量为0-1.25g/L。
本发明的一个目的是提供一种利用上述的酵母发酵生产的发酵液。本发明的一个目的是提供上述任一的酵母菌株或者发酵液在生产cAMP中的用途。
本发明的一个目的是提供上述任一的酵母菌株或者发酵液在医药、畜牧业、食品或化工领域的应用。
本发明的一个目的是提供上述任一的酵母菌株或者发酵液在制备医药、畜牧业、食品或化工等领域的产品中的应用。
相应的，本发明还提供包含上述任一的酵母菌株或者发酵液的医药、畜牧业、食品或化工领域的产品，例如药物，饲料，食品等等。
若非特殊说明，本文涉及的所有专业术语、技术概念都是本领域技术人员所熟知的。尽管有多种 类似或等效的方法可以用来构建和测定本发明中涉及的酵母菌株，下文中将描述一种合适的方法和材料。这些材料、方法和例子只是作为举证，它不在任何程度上限制本发明。本文中提到的所有文献、专利和其他参考资料均尊重其完整性。本发明涉及的方法和材料将在下文中与详细说明和图表一同给出。
3.2发明详述detailed description
本发明提供的是一种经过遗传改造的、能过量合成胞外cAMP的酵母菌株、构建此菌株的重组方法及应用。更具体地，
如上所述，本发明的酵母包括第一种基因修饰和第二种基因修饰。进一步的，所述酵母还包括第三种和第四种基因修饰。其中，第一种基因修饰消除对cAMP的反馈抑制，但同时使得酵母的生长被抑制；第二种基因修饰消除了第一种基因修饰导致的生长抑制，从而使得酵母能够正常生长；第三种基因修饰减少cAMP的降解，第四种基因修饰增加嘌呤合成途径中cAMP前体物的合成的正调控，使cAMP前体物的合成上升，提高cAMP产量。
更具体地，第一种基因修饰可以通过修饰蛋白激酶A(protein kinase A，PKA)的催化亚基编码基因TPK1、TPK2和TPK3，使PKA活性或表达被完全抑制，PKA对cAMP的反馈抑制被消除，但同时使得酵母的生长被抑制；第二种基因修饰消除了第一种基因修饰导致的生长抑制，从而使得酵母能够正常生长；第二种基因修饰可以使蛋白激酶Yak1等的活性或表达被完全抑制，从而消除了因PKA活性或表达被完全抑制而导致的抑制细胞生长，因此菌株能够正常生长；第三种基因修饰使水解cAMP的磷酸二酯酶Pde活性或表达被完全抑制；第四种基因修饰使经嘌呤合成途径中合成cAMP前体物AMP的正调控例如转录因子Bas1、Bas2活性或表达上升，使AMP合成上升，最终提高cAMP产量。
本发明描述了通过酵母菌株的第一种和第二种基因修饰消除PKA对cAMP的反馈抑制来提高合成胞外cAMP产量。PKA是酵母中目前唯一已知的cAMP靶标，一般认为cAMP浓度上的波动通过PKA途径来施加效应；换言之，酵母细胞内cAMP信号通路受PKA的严格负反馈调控。酵母PKA是由两个催化亚基和一个二聚体的调节亚基组成的四聚体蛋白，在胞内缺乏cAMP或cAMP水平较低时，PKA以钝化复合体的形式存在。胞内cAMP水平上升时，cAMP与PKA的调节亚基结合，PKA的催化亚基从调节亚基上解离下来，激活一系列蛋白磷酸化级联反应，导致这些蛋白的活性发生变化，进而在不同水平上调控胞内cAMP的水平；其中的首要效应酶是腺苷酸环化酶Cyr1，Cyr1催化ATP合成cAMP。PKA的调节亚基由基因BCY1编码，催化亚基由基因TPK1、TPK2和TPK3编码。
当三个催化亚基Tpk1、Tpk2和Tpk3活性或表达同时被完全抑制时，PKA活性也就被完全抑制，PKA对cAMP的反馈抑制被消除。另一方面，当PKA活性被完全抑制时，蛋白激酶Rim15停留在细胞核内，激活转录因子Msn1/Msn2，Msn1/Msn2进而激活对细胞生长起负调控作用的蛋白激酶Yak1，使细胞停留在G1期而无法继续生长，因此PKA活性被完全抑制的酵母菌株需要进一步的基因修饰来确保菌株正常生长。
蛋白激酶Sch9与PKA的催化亚基具有同源性，在正常的生理条件下，Sch9与PKA具有不同的底物；但增加任一种激酶的活性都会补偿由于缺失另一个激酶而引起的生长缺陷。
本发明中的第一种基因修饰消除了PKA对cAMP的反馈抑制，使PKA活性或表达被完全抑制，第二种基因修饰消除了酵母菌株因第一种基因修饰导致的生长抑制、从而能够正常生长。消除PKA对cAMP的反馈抑制作用以及保持PKA活性被完全抑制情况下的菌株正常生长的策略有多种。使PKA活性或表达被完全抑制、消除PKA对cAMP的反馈抑制作用，可以在编码催化亚基Tpk1、Tpk2和Tpk3的核酸序列中进行，此核酸序列可以是调控区、编码区。
Tpk1的核酸序列可以在例如GenBank Accession No.NC_001142.9中找到。
Tpk2的核酸序列可以在例如GenBank Accession No.NC_001148.4中找到。
Tpk3的核酸序列可以在例如GenBank Accession No.NC_001143.9中找到。
保持PKA活性被完全抑制情况下的菌株正常生长的策略包括但不限于对蛋白激酶Rim15编码基因RIM15、转录因子Msn1/Msn2编码基因MSN1/MSN2或蛋白激酶Yak1编码基因YAK1进行基因修饰，修饰的结果使相应酶活性或表达被完全抑制，菌株从而得以正常生长。保持PKA活性被完全抑制情况下的菌株正常生长的策略还包括但不限于对蛋白激酶Sch9编码基因SCH9进行基因修饰，修饰的结果使相应酶活性或 表达被提高，菌株从而得以正常生长。
Yak1的核酸序列可以在例如GenBank Accession No.NC_001142.9中找到。
Rim15的核酸序列可以在例如GenBank Accession No.NC_001138.5中找到。
Msn1/Msn2的核酸序列可以在例如GenBank Accession No.NC_001147.6，NC_001145.3中找到。
Sch9的核酸序列可以在例如GenBank Accession No.NC_001140.6中找到。
不论以何种方式进行第一种和第二种基因修饰使酶活性或表达被完全抑制，或使酶活性或表达被提高，酵母菌株中TPK1、TPK2、TPK3和第二种基因共四个基因的修饰步骤，除了在单倍体酵母细胞中同时失活TPK1、TPK2、TPK3的组合(此组合因生长缺陷而不能存活)以外，上述四个基因可以不分先后、任一组合的被修饰。
本发明描述的经过上述第一种和第二种基因修饰的酵母菌株相对于未经第一种和第二种基因修饰的酵母菌株产生更多的胞外cAMP。
另一方面，cAMP一旦产生，唯一使它失活的方式是在环核苷酸磷酸二酯酶Pde的催化下将其水解为5’-AMP。因此，Pde也影响着胞内cAMP的水平。因此，本发明通过第三种基因修饰使Pde活性或表达被完全抑制，从而减少cAMP的降解、提高cAMP含量水平。酿酒酵母细胞本身包含两个cAMP磷酸二酯酶：Pde1编码一个低亲和力(Km＝20-250μM)的、特异性较低的酶，主要介导快速的cAMP信号传导；Pde2编码一个高亲和力(Km＝170nM)的、特异性很强的酶，主要控制cAMP的基础水平。
Pde1的核酸序列可以在例如GenBank Accession No.NC_001139.9中找到。
Pde2的核酸序列可以在例如GenBank Accession No.NC_001147.6中找到。
本发明所描述的第一种，第二种和第三种基因修饰可以通过本领域内已知的任意多种方法来实现。使得编码酶的活性或表达被完全抑制的基因修饰方式可以是对基因的核酸序列进行点突变、缺失或插入一个或者多个核苷酸，点突变如单核苷酸的转换(嘌呤到嘌呤或嘧啶到嘧啶)或者颠换(嘌呤到嘧啶或嘧啶到嘌呤)。核酸的突变可以导致在其表达的多肽中一个或者多个保守的或者非保守的氨基酸的置换，这种置换可能会导致多肽的构象发生变化，也可能失去部分或者全部功能，可能发生移码突变将导致整个多肽链从该点处开始编码一个完全不同的多肽，也可能提前形成一个终止密码子使得多肽链残缺，甚至使基因沉默。
缺失或插入用核酸序列可以用PCR或者化学合成的方法得到，也可以利用细胞复制扩增得到。使基因酶活性或表达被完全抑制的基因修饰还可以通过提供或表达反义寡核苷酸antisense polynucleotides、siRNA、microRNA或其它可以阻止待修饰基因的mRNA翻译为蛋白质的核酸的方法来实现。近年来发展的类转录激活因子效应物核酸酶(transcription activator-like effector nuclease，TALEN)和CRISPR技术——规律成簇的间隔短回文重复(Clustered Regularly Interspaced Short Palindromic Repeats)也可以用来失活基因。
本发明中提到的使基因酶活性或表达被完全抑制的基因修饰指的是能够使这种酶/多肽的活性降低(与未经基因修饰的酵母或野生型酵母比较)至少95％(例如至少96％，97％，98％，99％，或者100％)。
使得编码酶的活性提高或者过表达的基因修饰方式包括但不限于点突变、连接强启动子、连接增强子、提高拷贝数。例如，点突变提高多肽活性；提高核酸序列的拷贝数；通过基因修饰使核酸序列与一个可表达的强启动子或增强子连接；改变核酸序列的启动子或者其他调节因子从而提高其表达水平(例如提高启动子序列与转录起始因子的结合强度)；提高该核酸序列转录的mRNA的半衰期；抑制对mRNA或者多肽链的降解。
本发明中提到的使基因酶活性或表达被提高的基因修饰指的是一个经过基因修饰的核酸序列编码的酶/多肽的活性或对底物的作用效率与野生型相比提高至少20％(例如至少25％,30％,40％,50％,60％,70％,80％,90％,95％,99％,100％或者更高)。
本发明描述的经过上述第一种、第二种和第三种基因修饰的酵母菌株，相对于未经第一种、第二种和第三种基因修饰的酵母菌株产生更多的胞外cAMP。甚至相对于第一种和第二种基因修饰的酵母菌株产生更多的胞外cAMP。
提高酵母细胞胞外cAMP水平，对cAMP-PKA信号通路途径的基因进行修饰外，也可以通过调控嘌呤合成途径、增加cAMP合成前体物AMP的合成量来实现。酿酒酵母的嘌呤合成途径中，AMP从头合成途径基因即ADE基因的表达受2个转录调控因子Bas1和Bas2间的协同作用正调控。Bas1是Myb族转录调控因子，Bas2又名Pho2，是同源域(Homeodomain)转录调控因子，二者以复合物形式与特定启动子区域结合，协同作用激活10个ADE基因的表达。当胞内ADP、ATP浓度过高时，ADP、ATP反馈抑制从头合成途径第一个酶Ade4p(PRPP酰胺转移酶，简称PRPPAT)的活性，使得中间产物SAICAR的合成下降，Bas2p能感知SAICAR的浓度变化，当此浓度降至一定程度时，Bas2p不再与Bas1p形成复合物。当培养基中有相当量的腺嘌呤存在时，腺嘌呤进入胞内，通过补救途径形成AMP，AMP进一步转化为ADP和ATP，也导致整个从头合成途径合成受到抑制。
本发明的第四种基因修饰通过增加嘌呤合成途径中AMP合成的正调控，使AMP合成上升，提高cAMP产量。增加AMP合成的正调控的核心在于促进Bas1/Bas2复合物形成、提高复合物的正调控活性，抑制腺嘌呤、ADP和ATP的反馈抑制。而促进Bas1/Bas2复合物形成、提高复合物正调控活性的策略有多种，可以提高单个正调控转录因子Bas1或Bas2的活性或表达，也可以直接融合共表达Bas1和Bas2，或在融合共表达Bas1和Bas2的基础上进一步提高活性或表达量。有很多种方法能够使核酸编码的多肽活性提高。例如，点突变提高多肽活性；提高核酸序列的拷贝数；通过基因修饰使核酸序列与一个可表达的强启动子或增强子连接；改变核酸序列的启动子或者其他调节因子从而提高其表达水平(例如提高启动子序列与转录起始因子的结合强度)；提高该核酸序列转录的mRNA的半衰期；抑制对mRNA或者多肽链的降解。认定一个经过基因修饰的核酸序列编码的多肽活性的提高需要其对底物的作用效率与野生型相比提高至少20％(例如至少25％,30％,40％,50％,60％,70％,80％,90％,95％,99％,100％或者更高)。单拷贝或者多拷贝的核酸序列的过表达可以通过连接到载体中实现，也可以通过整合到酵母的基因组中实现。适用于核酸序列过表达的载体已经商业化或者可以通过本领域中常用的基因重组技术得到。在本领域中已经有一些在酵母基因组中整合核酸序列的方法。例如，Methods in Enzymology:Guide to Yeast Genetics and Molecular and cell Biology,Vol.194,2004,Abelson et al.,eds.,Academic Press。含有核酸序列的载体可以把一些对表达有必要的元件与目的核酸序列进行可操作的连接：可以带上表达选择标记的核酸序列(如营养缺陷型选择标记基因、抗生素抗性基因)；或者带上可用来纯化多肽的基因(例如6xHis tag)；还可以带有一个或多个复制起点。对表达有必要的元件包括能够指导或者调控目的核酸序列的核酸序列，例如启动子序列。代表性的启动子包括(不局限于此)PGK启动子，TPI1启动子，ADH1启动子。对表达有必要的元件还包括增强子序列，响应元件，或者调控编码序列表达的诱导因子。
对表达有必要的元件可以来自细菌，酵母，昆虫，植物，哺乳动物，真菌或者病毒，表达载体可以带有不同来源的元件。对表达有必要的元件可以参见in Goeddel,1990,Gene Expression Technology:Methods in Enzymology,185,Academic Press,San Diego,CA。在某些情况下，可以选择对特定密码子最适宜的核酸序列或对表达有必要的元件以便在酵母中得到最好的表达。参见Bennetzen&Hall,1982,J.Biol.Chem.,257:3026-31。
Bas1的核酸序列可以在例如GenBank Accession No.NC_001143.9中找到。
Bas2的核酸序列可以在例如GenBank Accession No.NC_001136.10中找到。
本发明描述的经过上述第一种、第二种、第三种和第四种基因修饰的酵母菌株相对于未经第一种、第二种、第三种和第四种基因修饰的酵母菌株产生更多的胞外cAMP。甚至相对于第一种和第二种基因修饰的酵母菌株或者相对于第一种、第二种和第三种基因修饰的酵母菌株产生更多的胞外cAMP。
在本发明的基因修饰过程中，核酸序列(如表达载体)可以通过多种方法转入酵母细胞或者其他宿主细胞中。这些方法包括(不局限于此)电穿孔法，磷酸钙沉淀法，热激法，脂质转染法，显微注射法，氯化锂法，醋酸锂法，巯基乙醇法和病毒介导基因转移法。
除了酵母细胞，“宿主细胞”还可以是任何可以用于标准分子生物学操作并产生核酸和多肽的细胞。包括但不仅限于细菌细胞(如E.coli)，昆虫细胞，植物细胞和哺乳动物细胞(如CHO或COS细胞)。 “酵母细胞”包括本文中描述的重组酵母细胞。本文所指的“宿主细胞”不仅包括对其进行核酸转导的母细胞，还包括其子细胞。
在本发明的基因修饰过程中，供转入酵母细胞筛选使用的选择标记基因包括但不仅限于酵母可用的任何营养缺陷型基因，如URA3、LEU2、HIS3、TRP1、LYS2。为了能进行尽可能多的基因修饰，同时避免营养缺陷型基因表达本身带来的可能干扰，优选能方便地被反复、循环使用的URA3基因。更进一步优选的，URA3基因被循环使用而不保留在被基因修饰的菌株中。
虽然本发明所应用的策略针对S.cerevisiae菌株的基因和多肽，但是相同的策略同样适用于其它可发酵的酵母，优选能够进行工业发酵的酵母菌株。除了S.cerevisiae菌株外，合适的菌株还包括巴斯德酵母(Saccharomyces pastorianus)、树干毕赤酵母(Pichia stipitis)、Saccharomyces Bayanus和休哈塔假丝酵母(Candida shehatae)。在这些菌株中通路或者基因名称或许稍有不同，但是可以应用同样的策略和技术来修饰相应的通路和同源基因。此处描述的基因工程酵母菌株是本领域的研究人员所熟知的。
本发明还描述了上述经过基因修饰的酵母菌株生产cAMP的发酵方法。
优选的，所述发酵方法中，发酵用培养基的葡萄糖浓度为20-150g/L，酵母提取物10-20g/L，蛋白胨20-40g/L，腺嘌呤添加量为0-1.25g/L。
本发明还描述了上述经过基因修饰的酵母菌株的用途，其可用于发酵代谢碳水化合物产生胞外cAMP。进一步，上述经过基因修饰的酵母菌株细胞，和/或经发酵得到的包含胞外cAMP的发酵液，可直接作为cAMP的替代品。并如同cAMP一般应用到各个领域，例如，医药、畜牧业、食品或化工领域等等。更进一步，所述应用包括在制备上述领域的产品中的应用，例如药物、食品和饲料等。相应的，本发明还提供包含上述经过基因修饰的酵母菌株细胞，和/或经发酵得到的包含胞外cAMP的发酵液的医药、畜牧业、食品或化工等领域的产品，例如药物、食品、饲料等。
3.3发明的优点和益处
本发明所得重组酵母菌株具有低营养需求、容易培养、适合大规模发酵生产并易于实现产业化的性能；通过基因工程手段进行相关基因修饰，在消除cAMP通路反馈抑制、减少cAMP降解的同时，增加通向cAMP前体物AMP的代谢流，使所得重组酵母菌株能稳定、持续、高效生产胞外cAMP；通过提高碳源浓度、优化碳/氮比、添加前体物等措施，使代谢流更有效地流向目的产物，得到优化的、高产胞外cAMP的发酵工艺，cAMP浓度可达到9721.6μmol/L。本发明克服了传统化学合成法工艺复杂、原料昂贵、副产物多、环境污染严重的缺点，也克服了酶促转化法底物昂贵、腺苷酸环化酶稳定性差等不足，具有工艺操作简单、产物容易分离提取、原料成本低、安全环保、可工业化生产的优点，为微生物发酵生产cAMP开辟了新的途径，将推动高值医药和生化产品的绿色清洁生产，能广泛应用于医药、畜牧业、食品或化工领域，具有重要的经济价值和社会意义。
附图说明
图1为实施例1质粒示意图，其中图1A为质粒pUC18-URA3，图1B为质粒pUC18-TPK2p-TPK2t-URA3-TPK2t。
图2为实施例5五个基因缺失菌株cAMP生产初步评价。
图3为实施例6质粒pUC18-H1YNRCΔ9-BAS1-BAS2-H2YNRCΔ9-URA3-H2YNRCΔ示意图。
具体实施方式
下面结合具体实施例对本发明所述技术方案作进一步的说明。可以理解的是，在此描述的特定实施方式通过举例的方式来表示，其并不作为对本发明的限制。若非特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段。在不偏离于本发明范围的情况下，本发明的主要特征可以用于各种实施方式。本领域的技术人员将会意识到或能够确认，仅仅使用常规实验，许多等同物都能应用于本文所描述的特定步骤中。这些等同物被认为处在本发明的范围之内，并且被权利要求所覆盖。
实施例中所涉及的几种培养基如下：
1)酵母菌株活化及种子液培养用培养基YPAD
酵母提取物(Yeast extracts)10g/L，蛋白胨(Peptone)20g/L，葡萄糖(Glucose)20g/L，腺嘌呤(Adenine)0.05g/L，自然pH值；固体培养时另加琼脂粉15g/L。
2)使用尿嘧啶(URA)营养缺陷型选择标记时的转化筛选培养基CMG-URA
使用缺省了尿嘧啶的基本培养基CMG平板简称CMG-URA平板进行筛选。组分如下：氨基酸碱基混合物，0.83g/L；不含氨基酸的酵母氮源Yeast Nitrogen Base，简称YNB，6.7g/L；葡萄糖，20g/L；琼脂粉，15g/L。其中的氨基酸碱基混合物见表1。
表1.氨基酸碱基混合物成分
腺嘌呤50mg/L 亮氨酸100mg/L 精氨酸20mg/L 赖氨酸30mg/L
天冬氨酸100mg/L 甲硫氨酸20mg/L 谷氨酸100mg/L 苯丙氨酸50mg/L
组氨酸100mg/L 丝氨酸150mg/L 异亮氨酸30mg/L 苏氨酸150mg/L
色氨酸l00mg/L 酪氨酸30mg/L 尿嘧啶50mg/L 缬氨酸150mg/L
注：省却特定的氨基酸成分，即可做成选择培养基。调pH值5.6，葡萄糖110℃灭菌15min，其它成分121℃灭菌21min，使用前混合。
3)5’-FOA平板(5’-乳清酸平板)
制备方法如下：
a、制备100ml 5’-FOA溶液，包括：
Figure PCTCN2016074067-appb-000001
用磁力搅拌器于45℃下不停搅拌，使其各个组分全部溶解，之后过滤除菌；
b、制备100ml琼脂粉Agar溶液，使其最终浓度达到3.0％，121℃灭菌15min后，将其冷却至45℃；
c、将100ml 5’-FOA与100ml Agar溶液混匀，防止出现气泡，倒入无菌培养皿中，冷却成型。
4)大肠杆菌培养用LB培养基
酵母提取物(Yeast extracts)5g/L，蛋白胨(Peptone)10g/L，氯化钠(NaCl)10g/L，调pH值至7.0；固体培养时另加琼脂粉15g/L。需要时添加抗生素，氨苄青霉素的添加终浓度100μg/mL。
实施例中所涉及的引物序列见表2。
表2实施例中用到的引物序列
Figure PCTCN2016074067-appb-000002
Figure PCTCN2016074067-appb-000003
Figure PCTCN2016074067-appb-000004
实施例中，酿酒酵母菌株W303-1A(＝ATCC208352)可以从美国ATCC购得。YCplac33可从Invitrogen公司买到。pUC18可从Invitrogen公司、Promega公司等多家公司买到。
实施例1：构建缺失TPK2基因的酵母菌株
利用同源重组双交换机制缺失酵母染色体上的TPK2基因，首先构建缺失用质粒pUC18-TPK2p-TPK2t-URA3-TPK2t，此质粒经酶切线性化后转化酵母感受态细胞，以URA3为筛选标记基因筛选得到以TPK2p和URA3下游的TPK2t为左、右同源臂发生双交换得到的酵母菌株tpk2△::URA3；再以5’-FOA平板筛选得到菌株tpk2△::URA3染色体上URA3左、右两侧TPK2t之间发生重组而弹出URA3基因的酵母目的菌株tpk2△。
构建和鉴定用引物见表2，示意图见图1。
一、pUC18-TPK2p-TPK2t-URA3-TPK2t构建
此质粒的构建涉及四个连接：
(1)pUC18-URA3构建
以质粒YCplac33为模板进行PCR扩增URA3基因，使用表2中的SEQ ID No.1、SEQ ID No.2所示序列为上、下游引物P1、P2，全式金公司生产的Fast Pfu聚合酶，50℃退火1min，72℃延伸1.5min，共32个循环。得到1055bp的PCR片段，使用限制性内切酶BamHI和SalI双酶酶切此片段和载体质粒pUC18，T4DNA连接酶连接后转化大肠杆菌Top10感受态细胞，提取转化子质粒进行酶切鉴定，得到质粒pUC18-BamHI-URA3-SalI(简称pUC18-URA3，见图1A)。
(2)pUC18-TPK2t-URA3构建
提取酿酒酵母菌株W303-1A染色体，以W303-1A染色体为模板进行PCR，扩增TPK2基因的终止子区(简称为TPK2t)，使用表2中的SEQ ID No.3、SEQ ID No.4所示序列为上、下游引物P3、P4，全式金公司生产的Fast Pfu聚合酶，50℃退火1min，72℃延伸0.5min，共32个循环。得到526bp的PCR片段，使用限制性内切酶KpnI和BamHI双酶酶切此片段和载体质粒pUC18-URA3，T4DNA连接酶连接后转化大肠杆菌Top10感受态细胞，提取转化子质粒进行酶切鉴定，得到质粒pUC18-TPK2t-URA3。
(3)pUC18-TPK2p-TPK2t-URA3构建
提取W303-1A染色体，以W303-1A染色体为模板进行PCR，扩增TPK2基因的启动子区(简称为TPK2p)，使用表2中的SEQ ID No.5、SEQ ID No.6所示序列为上、下游引物P5、P6，全式金公司生产的Fast Pfu聚合酶，50℃退火1min，72℃延伸0.5min，共32个循环。得到532bp的PCR片段，使用限制性内切酶Sac I和Kpn I双酶酶切此片段和载体质粒pUC18-TPK2t-URA3，T4DNA连接酶连接后转化大肠杆菌Top10感受态细胞，提取转化子质粒进行酶切鉴定，得到质粒pUC18-TPK2p–TPK2t-URA3。
(4)pUC18-TPK2p-TPK2t-URA3-TPK2t构建
将上述(2)中扩增的含TPK2t序列的PCR产物用Sal I和Pst I双酶酶切，与同样双酶酶切的pUC18-TPK2p-TPK2t-URA3大片段连接，得到质粒pUC18-TPK2p-TPK2t-URA3-TPK2t(见图1B)。
二、W303-1A(tpk2△)菌株构建
将质粒pUC18-TPK2p-TPK2t-URA3-TPK2t用Sac I和Pst I双酶酶切，得到线性化DNA片段TPK2p-TPK2t-URA3-TPK2t；然后利用醋酸锂法转化酿酒酵母W303-1A菌株感受态细胞，使用尿嘧啶营养缺陷型选择标记(URA)进行筛选，即使用CMG-URA平板进行筛选，对得到的转化子菌株进行YPAD液体培 养，提取染色体DNA，以其为模板进行PCR法验证，引物对为表2中的SEQ ID No.7、SEQ ID No.8所示序列为上、下游引物P7、P8，整合成功、含有URA基因的转化子PCR产物为2592bp，对照菌株PCR产物为2299bp。阳性转化子菌株命名为W303-1A(tpk2△::URA3)。
进一步将菌株W303-1A(tpk2△::URA3)平板生长菌体涂布于5’-FOA平板上，从而筛选出通过整合在染色体上的两个相同序列(TPK2t)之间同源重组而弹出URA3片段的菌落。用同上的引物对P7、P8验证，成功弹出URA3的目的菌株的PCR产物长1053bp，没有弹出URA3的宿主菌株W303-1A(tpk2△::URA3)的PCR片段长2592bp。对1053bp PCR产物进行了测序，测序结果证明发生了预期的变化：TPK2基因的起始密码子前的2个碱基、整个ORF区和终止密码子之后的107个碱基被完全缺失。这样，最终得到TPK2基因缺失、URA3基因弹出的阳性转化子菌株W303-1A(tpk2△)。
实施例2：构建缺失TPK1或TPK3基因的酵母菌株
一、W303-1A(tpk1△)构建
TPK1基因缺失用质粒pUC18-TPK1p-TPK1t-URA3-TPK1t的构建：以实施例1中(1)pUC18-URA3质粒为基础构建质粒pUC18-TPK1p-TPK1t-URA3-TPK1t，过程与实施例1中经过(2)、(3)和(4)步骤最终得到pUC18-TPK2p-TPK2t-URA3-TPK2t的过程一样，使用的引物对：扩增TPK1t为表2中的SEQ ID No.9、SEQ ID No.10所示序列为上、下游引物P9、P10，产物516bp；扩增TPK1p为表2中的SEQ ID No.11、SEQ ID No.12所示序列为上、下游引物P11、P12，产物572bp。
将质粒pUC18–TPK1p-TPK1t-URA3-TPK1t用Sac I和Pst I双酶酶切，得到线性化DNA片段TPK1p-TPK1t-URA3-TPK1t；然后转化酿酒酵母W303-1A菌株感受态细胞，使用尿嘧啶(URA)营养缺陷型选择标记进行筛选，即使用CMG-URA平板进行筛选，对得到的转化子菌株进一步用PCR法验证，引物对为表2中的SEQ ID No.13、SEQ ID No.14所示序列为上、下游引物P13、P14，整合成功的转化子PCR产物为2631bp，对照菌株PCR产物为2476bp。阳性转化子菌株命名为W303-1A(tpk1△::URA3)。
进一步将菌株W303-1A(tpk1△::URA3)平板生长菌体涂布于5’-FOA(5’-乳清酸)平板上，从而筛选出通过整合在染色体上的两个相同序列(TPK1t)之间同源重组而弹出URA3片段的菌落。用同上的引物对P13、P14验证，成功弹出URA3的目的菌株的PCR产物长1102bp，没有弹出URA3的对照PCR片段长2631bp。对1102bp PCR产物进行了测序，测序结果证明发生了预期的变化：TPK1基因的起始密码子前的70个碱基、整个ORF区和终止密码子之后的116个碱基被完全缺失。这样，最终得到TPK1基因缺失、URA3基因弹出的阳性转化子菌株W303-1A(tpk1△)。
二、W303-1A(tpk3△)构建
TPK3基因缺失用质粒的构建：以实施例1中(1)pUC18-URA3质粒为基础构建质粒pUC18–TPK3p-TPK3t-URA3-TPK3t的过程与实施例1中经过(2)、(3)和(4)步骤最终得到pUC18-TPK2p-TPK2t-URA3-TPK2t的过程一样，使用的引物对：扩增TPK3t为表2中的SEQ ID No.15、SEQ ID No.16所示序列为上、下游引物P15、P16，产物566bp；扩增TPK3p为表2中的SEQ ID No.17、SEQ ID No.18所示序列为上、下游引物P17、P18，产物545bp。
将质粒pUC18-TPK3p-TPK3t-URA3-TPK3t用Sac I和Pst I双酶酶切，得到线性化DNA片段TPK3p-TPK3t-URA3-TPK3t；然后转化酿酒酵母W303-1A菌株感受态细胞，同实施例1中“W303-1A(tpk2△)菌株构建”方法筛选、鉴定得到W303-1A(tpk3△::URA3)菌株和W303-1A(tpk3△)菌株。PCR方法验证用引物对为表2中的SEQ ID No.19、SEQ ID No.20所示序列为上、下游引物P19、P20，整合成功、含有URA3基因的转化子PCR产物为2695bp，整合成功、URA3基因弹出的转化子PCR产物为1116bp，对照菌株PCR产物为2400bp。对1116bp PCR产物进行了测序，测序结果证明发生了预期的变化：TPK3基因的整个ORF区和终止密码子之后的93个碱基被完全缺失。这样，最终得到TPK3基因缺失、URA3基因弹出的阳性转化子菌株W303-1A(tpk3△)。
实施例3：构建缺失YAK1基因的酵母菌株
YAK1基因缺失用质粒的构建：以实施例1中(1)pUC18-URA3质粒为基础构建质粒pUC18–YAK1p-YAK1t-URA3-YAK1t的过程与实施例1中经过(2)、(3)和(4)步骤最终得到pUC18-TPK1p-TPK1t-URA3-TPK1t的过程一样，使用的引物对：扩增YAK1t为表2中的SEQ ID No.21、SEQ ID No.22所示序列为上、下游引物P21、P22，产物506bp；扩增YAK1p为表2中的SEQ ID No.23、SEQ ID No.24所示序列为上、下游引物P23、P24，产物504bp。
YAK1基因缺失菌株的构建：将质粒pUC18–YAK1p-YAK1t-URA3-YAK1t用Sac I和Pst I双酶酶切，得到线性化DNA片段YAK1p-YAK1t-URA3-YAK1t；然后转化酿酒酵母W303-1A菌株感受态细胞，同实施例1中“W303-1A(tpk2△)菌株构建”方法筛选、鉴定得到W303-1A(yak1△::URA3)菌株和W303-1A(yak1△)菌株。PCR方法验证用引物对为表2中的SEQ ID No.25、SEQ ID No.26所示序列为上、下游引物P25、P26，整合成功、含有URA3基因的转化子PCR产物为2529bp，整合成功、URA3基因弹出的转化子PCR产物为1010bp，对照菌株PCR产物为3614bp。对1010bp PCR产物进行了测序，测序结果证明发生了预期的变化：YAK1基因的起始密码子之前40个碱基、整个ORF区和终止密码子之后的146个碱基被完全缺失。这样，最终得到YAK1基因缺失、URA3基因弹出的阳性转化子菌株W303-1A(yak1△)。
实施例4：构建同时缺失TPK1、TPK2、TPK3、YAK1基因的酵母菌株
下面分四个步骤构建得到同时缺失四个基因的菌株。事实上本领域的技术人员知道，除了在单倍体酵母细胞中同时失活TPK1、TPK2、TPK3的组合(此组合因生长缺陷而不能存活)以外，上述四个基因可以不分先后、任一组合的被修饰。
一、缺失YAK1基因
同实施例3方法构建得到W303-1A(yak1△)菌株。
二、缺失YAK1和TPK1基因
将实施例2中构建的缺失用质粒pUC18-TPK1p-TPK1t-URA3-TPK1t用Sac I和Pst I双酶酶切，得到线性化DNA片段TPK1p-TPK1t-URA3-TPK1t；然后转化步骤一中的W303-1A(yak1△)菌株感受态细胞，使用尿嘧啶(URA)营养缺陷型选择标记进行筛选，即使用CMG-URA平板进行筛选，对得到的转化子菌株用同实施例2中相同的方法鉴定得到TPK1缺失菌株W303-1A(yak1△)(tpk1△::URA3)，并进一步反选得到W303-1A(yak1△)(tpk1△)菌株。
三、缺失TPK2和TPK3基因
从W303-1A经过交配型转换获得与W303-1A菌株除交配型外基因型完全相同的W303-1B菌株(MATαleu2-3,112ura3-1trp1-92his3-11,15ade2-1can1-100)，以W303-1B为宿主，顺次敲除TPK2、TPK3两个基因。
将实施例1中构建的质粒pUC18-TPK2p-TPK2t-URA3-TPK2t用Sac I和Pst I双酶酶切，得到线性化DNA片段TPK2p-TPK2t-URA3-TPK2t；然后转化酿酒酵母W303-1B菌株感受态细胞，同实施例1中“W303-1A(tpk2△)菌株构建”方法筛选、鉴定得到W303-1B(tpk2△::URA3)菌株和W303-1B(tpk2△)菌株。
将实施例2中构建的质粒pUC18-TPK3p-TPK3t-URA3-TPK3t用Sac I和Pst I双酶酶切，得到线性化DNA片段TPK3p-TPK3t-URA3-TPK3t；然后转化酿酒酵母W303-1B(tpk2△)菌株感受态细胞，同实施例1中“W303-1A(tpk2△)菌株构建”方法筛选、鉴定得到W303-1B(tpk2△ tpk3△::URA3)菌株和W303-1B(tpk2△ tpk3△)菌株。
四、同时缺失TPK1、TPK2、TPK3、YAK1基因
将W303-1A(tpk1△ yak1△)菌株与W303-1B(tpk2△ tpk3△)菌株进行杂交，得到二倍体菌株，然后通过产孢、拆孢使等位基因分离，筛选、鉴定得到W303-1A(tpk1△ tpk2△ tpk3△ yak1△)和 W303-1B(tpk1△ tpk2△ tpk3△ yak1△)。鉴定四个基因敲除的验证引物同前。对鉴定所得PCR产物的测序结果证明两个菌株中染色体上的四个基因核酸序列均发生了预期的变化，全部被缺失了ORF区。
五、四个基因缺失菌株的生长和发酵生产胞外cAMP评价
对上述四个基因缺失菌株进行了生长和发酵生产胞外cAMP评价，操作如下：
1、种子液培养：挑取YPAD固体培养基平板上生长的菌落，接入装有5mL YPAD培养液的试管中，30℃、220rpm过夜培养，必要时进行二次扩大培养；
2、发酵：发酵用培养基：酵母提取物10g/L，蛋白胨20g/L，葡萄糖20g/L，自然pH值；将新鲜种子液接种到装有25ml发酵培养基的100ml容量瓶中，控制初始OD600值在0.1左右，30℃、220rpm下发酵；
3、发酵液的OD600测定：将发酵样品适当稀释后测定OD600，检测生长情况；
4、HPLC分析发酵上清液cAMP浓度即胞外cAMP浓度：HPLC分析cAMP的方法参照《中华人民共和国药典：2010年版》(国家药典委员会编)第419页的方法而有调整。具体操作如下：1)将发酵样品在13000rpm、1min条件下离心，取上清进行适当稀释，用孔径0.22μm的滤膜过滤，滤液用于色谱检测：检测波长258nm，Thermo Syncronis C18色谱柱，流动相为(5.78g/L KH2PO4，2.72g/L四丁基溴化铵):乙腈＝85:15
Figure PCTCN2016074067-appb-000005
，用磷酸调节pH至4.3，流速1mL/min，柱温35℃；2)cAMP标准样品的配置及标准曲线的测定：先将cAMP标准品用灭菌后的去离子水配置成浓度为50mmol/L的标准溶液，再用去离子水对其进行稀释，得到终浓度分别为1、3、5、7.5和10μmol/L的标准样品，用0.2μm滤膜过滤标准样品后进行HPLC分析，以峰面积对cAMP浓度做标准曲线；3)利用标准曲线由外标定量法计算出发酵液样品中cAMP的浓度。
96h内的生长和发酵生产胞外cAMP评价结果见表3。
表3、四个基因缺失菌株的生长和cAMP生产评价
Figure PCTCN2016074067-appb-000006
由表3可知：两个缺失菌株的最大胞外cAMP浓度分别是对照菌株W303-1A最大胞外cAMP浓度的144.0和155.1倍。需要说明的是：两个缺失菌株在0-24h内cAMP浓度迅速升高，24h达到最高值以后，cAMP浓度略有下降，但一直到96h时仍然保持在180μmol/L以上。
实施例5：构建同时缺失TPK1、TPK2、TPK3、YAK1、PDE1基因的酵母菌株
一、缺失PDE1基因
以W303-1A染色体为模板进行PCR，扩增PDE1基因的终止子区(简称为PDE1t)，使用表2中的SEQ ID No.27、SEQ ID No.28所示序列为上、下游引物P27、P28，全式金公司生产的Fast Pfu聚合酶，50℃退火1min，72℃延伸0.5min，共32个循环。得到500bp的PCR片段，使用限制性内切酶KpnI和BamHI双酶酶切此片段和载体质粒pUC18-URA3，T4DNA连接酶连接后转化大肠杆菌Top10感受态细胞，提取转化子质粒进行酶切鉴定，得到质粒pUC18-PDE1t-URA3。
以W303-1A染色体为模板进行PCR，扩增PDE1基因的启动子区(简称为PDE1p)，使用表2中的SEQ ID No.29、SEQ ID No.30所示序列为上、下游引物P29、P30，全式金公司生产的Fast Pfu聚合酶，50℃退火1min，72℃延伸0.5min，共32个循环。得到544bp的PCR片段，使用限制性内切酶Sac I和Kpn I双酶酶切此片段和载体质粒pUC18-PDE1t-URA3，T4DNA连接酶连接后转化大肠杆菌Top10感受态细胞，提取转化子质粒进行酶切鉴定，得到质粒pUC18-PDE1p-PDE1t-URA3。
将上述扩增的含PDE1t序列的PCR产物用Sal I和Pst I双酶酶切，与同样双酶酶切的pUC18-PDE1p-PDE1t-URA3大片段连接，得到质粒pUC18-PDE1p-PDE1t-URA3-PDE 1t。
将质粒pUC18-PDE1p-PDE1t-URA3-PDE 1t用Sac I和Pst I双酶酶切，用与实施例1中“W303-1A(tpk2△)菌株构建”中相同的方法，以实施例4中构建的W303-1A(tpk1△ tpk2△ tpk3△ yak1△)为宿主，构建得到W303-1A(tpk1△ tpk2△ tpk3△ yak1△ pde1△::URA3)和W303-1A(tpk1△ tpk2△ tpk3△ yak1△ pde1△)。PCR方法验证用引物对为表2中的SEQ ID No.31、SEQ ID No.32所示序列为上、下游引物P31、P32，整合成功、含有URA3基因的转化子PCR产物为2958bp，整合成功、URA3基因弹出的转化子PCR产物为1445bp，对照菌株PCR产物为2635bp。对1445bp PCR产物进行了测序，测序结果证明发生了预期的变化：PDE1基因的起始密码子之前40个碱基、整个ORF区和终止密码子之后的46个碱基被完全缺失。这样，最终得到PDE1基因缺失、URA3基因弹出的阳性转化子菌株。
二、W303-1A(tpk1△ tpk2△ tpk3△ yak1△ pde1△::URA3)和W303-1A(tpk1△ tpk2△ tpk3△ yak1△ pde1△)的cAMP生产对比
为对比考察一下选择标记基因URA3存在对cAMP生产可能的影响，进行了两个菌株的cAMP生产初步评价。1、种子液培养同实施例4；2、发酵：1)发酵用培养基：酵母提取物10g/L，蛋白胨20g/L，葡萄糖20g/L，自然pH值；2)发酵条件为：新鲜种子液接种到装有25ml发酵培养基的100ml容量瓶中，控制初始OD600值在0.1左右，30℃、220rpm下发酵。发酵液分析同实施例4中的HPLC分析方法。120h内的胞外cAMP生产结果见图2。图2结果显示：1、两个缺失菌株120h内的最大胞外cAMP浓度分别为252.0、953.8μmol/L，分别为对照菌株W303-1A最大胞外cAMP浓度(1.45μmol/L)的173.8、657.8倍，为实施例4里W303-1B(tpk1△ tpk2△ tpk3△ yak1△)菌株最大胞外cAMP浓度的1.17、4.42倍；2、W303-1A(tpk1△ tpk2△ tpk3△ yak1△ pde1△::URA3)的cAMP浓度48h起基本稳定，而W303-1A(tpk1△ tpk2△ tpk3△ yak1△ pde1△)的cAMP浓度则一直上升；3、不保留选择标记URA3可以明显提高cAMP产量。
三、W303-1A(tpk1△ tpk2△ tpk3△ yak1△ pde1△)的生长和cAMP生产评价
进行了不带URA3的五个基因缺失菌株的生长和发酵生产胞外cAMP评价：1、种子液培养同实施例4；2、发酵：1)发酵用培养基：酵母提取物10g/L，蛋白胨20g/L，葡萄糖20～150g/L，自然pH值；2)发酵条件为：新鲜种子液接种到装有25ml发酵培养基的100ml容量瓶中，控制初始OD600值在1左右，30℃、220rpm下发酵。发酵液分析同实施例4中的HPLC分析方法。120h内的生长和发酵生产胞外cAMP评价结果见表4。
表4、五个基因缺失菌株的生长和cAMP生产评价
Figure PCTCN2016074067-appb-000007
实施例6：BAS1-BAS2融合共表达
将BAS1和BAS2基因进行融合共表达，并整合到酵母细胞的染色体上。为此首先需要构建带有左、右同源臂的融合共表达整合载体。这里选择的整合位点是被文献报道(Bai Flagfeldt D,Siewers V,Huang L,et al.Characterization of chromosomal integration sites for heterologous gene expression in Saccharomyces cerevisiae.Yeast,2009,26(10):545-551)有较高基因表达效率的YNRCΔ9，位于染色体XIV上。
一、BAS1-BAS2融合共表达质粒构建
共4个连接得到BAS1-BAS2融合共表达质粒pUC18-H1YNRCΔ9-BAS1-BAS2-H2YNRCΔ9-URA3-H2YNRCΔ9(示意图见图3)。
1、pUC18-H1YNRCΔ9-SacI-SalI-H2YNRCΔ9质粒构建
以W303-1A的染色体DNA为模板进行PCR扩增YNRCΔ9的部分序列作为整合的左、右同源臂。使用表2中的SEQ ID No.33、SEQ ID No.34所示序列为上、下游引物P33、P34，全式金公司生产的Fast Pfu聚合酶，50℃退火1min，72℃延伸45sec，共32个循环，得到521bp的PCR1片段(对应序列用作整合的左同源臂H1YNRCΔ9)。使用表2中的SEQ ID No.35、SEQ ID No.36所示序列为上、下游引物P35、P36，同法扩增得到448bp的PCR2片段(对应序列用作整合的右同源臂H2YNRCΔ9)。再以PCR1、PCR2产物为模板，以P33、P36为引物对，交叉重叠延伸PCR反应扩增得到949bp的PCR12片段。此片段的5’端添加有酶切位点EcoRI，3’端添加有酶切位点BamHI，中间添加有酶切位点SacI、Sal I。将PCR产物用EcoRI和BamHI双酶酶切，与同样双酶酶切的pUC18大片段连接，转化大肠杆菌Top10感受态细胞，提取转化子质粒进行酶切鉴定，得到质粒pUC18-H1YNRCΔ9-SacI-SalI-H2YNRCΔ9。测序证明克隆片段没有突变。
2、pUC18-H1YNRCΔ9-SacI-SalI-H2YNRCΔ9-URA3-H2YNRCΔ9质粒构建
以YCplac33为模板进行PCR扩增URA3基因用作整合时的选择标记基因。使用表2中的SEQ ID No.37、SEQ ID No.38所示序列为上、下游引物P37、P38，全式金公司生产的Fast Pfu聚合酶，50℃退火1min，72℃延伸1min，共32个循环，得到1060bp的PCR1片段。仍以W303-1A的染色体DNA为模板进行PCR扩增H2YNRCΔ9片段，但上、下游引物对换成表2中的SEQ ID No.39、SEQ ID No.40所示序列为P39、P40，扩增得到438bp的PCR2片段。再以PCR1、PCR2产物为模板，以P37、P40为引物对，交叉重叠延伸PCR反应扩增得到1478bp的PCR12片段。此片段的5’端添加有酶切位点BamHI，3’端添加有酶切位点HindIII。将PCR产物用BamHI和HindIII双酶酶切，与同样双酶酶切的pUC18-H1YNRCΔ9-SacI-SalI-H2YNRCΔ9大片段连接，转化大肠杆菌Top10感受态细胞，提取转化子质粒进行酶切鉴定，得到质粒pUC18-H1YNRCΔ9-SacI-SalI-H2YNRCΔ9-URA3-H2YNRCΔ9。测序证明克隆片段没有突变。
3、pGEM-T easy-BAS1-BAS2质粒构建
以W303-1A的染色体DNA为模板进行PCR扩增基因BAS1，使用表2中的SEQ ID No.41、SEQ ID No.42所示序列为上、下游引物P41、P42，全式金公司生产的Fast Pfu聚合酶，50℃退火1min，72℃延伸2.5min，共32个循环。得到2907bp的PCR1片段，此片段5’端添加有酶切位点SacI，包含BAS1基因起始密码子ATG上游762bp序列和包括ATG在内的2112bp ORF序列，删除了ORF的3′端324bp(含终止密码子)序列。
以W303-1A的染色体DNA为模板进行PCR扩增基因BAS2，使用表2中的SEQ ID No.43、SEQ ID No.44所示序列为上、下游引物P43、P44，全式金公司生产的Fast Pfu聚合酶，50℃退火1min，72℃延伸1.5min，共32个循环。得到1692bp的PCR2片段，其3’端添加有酶切位点Sal I；包含BAS2基因ORF全部序列(含终止密码子)。
以PCR1、PCR2产物为模板，以P41、P44为引物对，交叉重叠延伸PCR反应扩增得到4578bp的PCR12片段。将此PCR片段进行加A尾反应后，琼脂糖凝胶电泳分离，切胶回收，按说明书操作要求与购自Promega公司的pGEM-T easy载体连接，转化大肠杆菌Top10感受态细胞，提取转化子质粒进行酶切鉴定，得到质粒pGEM-T easy-BAS1-BAS2。测序证明克隆片段没有突变。
4、pUC18-H1YNRCΔ9-BAS1-BAS2-H2YNRCΔ9-URA3-H2YNRCΔ9质粒构建
用SacI、Sal I双酶酶切质粒pGEM-T easy-BAS1-BAS2，回收大片段SacI-BAS1-BAS2-Sal I，与同样双酶酶切的pUC18-H1YNRCΔ9-SacI-SalI-H2YNRCΔ9-URA3-H2YNRCΔ9大片段连接，得到整合质粒pUC18-H1YNRCΔ9-BAS1-BAS2-H2YNRCΔ9-URA3-H2YNRCΔ9。
二、菌株W303-1A(tpk1△ tpk2△ tpk3△ yak1△ pde1△BAS1BAS2)构建
将质粒pUC18-H1YNRCΔ9-BAS1-BAS2-H2YNRCΔ9-URA3-H2YNRCΔ9用Pvu II酶切，得到线性化DNA片段H1YNRCΔ9-BAS1-BAS2-H2YNRCΔ9-URA3-H2YNRCΔ9；然后转化W303-1A(tpk1△ tpk2△ tpk3△ yak1△ pde1△)菌株感受态细胞，同实施例1中“W303-1A(tpk2△)菌株构建”方法筛选、鉴定得到W303-1A(tpk1△ tpk2△ tpk3△ yak1△ pde1△ BAS1BAS2-URA3)菌株和W303-1A(tpk1△ tpk2△ tpk3△ yak1△  pde1△BAS1BAS2)菌株。PCR方法验证用引物对为表2中的SEQ ID No.45、SEQ ID No.46所示序列为上、下游引物P45、P46，整合成功、含有URA3基因的转化子PCR产物为7389bp，整合成功、URA3基因弹出的转化子PCR产物为5929bp，对照菌株PCR产物为1687bp。对5929bp PCR产物的测序结果证明染色体上发生了预期的变化：YNRCΔ9位点有321bp序列被敲除，替代插入的是BAS1BAS2融合片段，此片段没有发生突变；不含URA3基因。
三、菌株W303-1A(tpk1△ tpk2△ tpk3△ yak1△ pde1△BAS1BAS2)的生长和cAMP生产评价
实施例5已经证明了葡萄糖浓度显著影响cAMP的生产。鉴于cAMP合成所需组分(分子式为C10Hl2N5O6P)，在葡萄糖浓度提高的同时，推测培养基中氮、磷的含量尤其是氮的含量可能成为cAMP合成的首要制约因素。另外，作为cAMP合成的前提物，胞外腺嘌呤的状况也极大地影响嘌呤合成途径各基因的表达水平，进而影响cAMP生产。因此，这里对比考察了上述菌株在两个酵母提取物/蛋白胨含量水平(1*YP、2*YP)、添加腺嘌呤下的cAMP生产情况。具体操作如下：1、种子液培养同实施例4；2、发酵：1)发酵用培养基：酵母提取物10g/L、蛋白胨20g/L，简记为1*YP，葡萄糖150g/L，自然pH值；酵母提取物20g/L，蛋白胨40g/L，简记为2*YP，葡萄糖150g/L，自然pH值；腺嘌呤的添加量为0.625、1.25g/L，简称为A0.625、A1.25；2)发酵条件为：新鲜种子液接种到装有25ml发酵培养基的100ml容量瓶中，控制初始OD600值在1左右，30℃、220rpm下发酵；3)发酵液分析：同实施例4中的HPLC分析方法，同时补充腺嘌呤的HPLC分析：a、样品处理和HPLC分析与cAMP分析的样品处理和HPLC分析相同；b、腺嘌呤标准曲线制作：将腺嘌呤标准品用灭菌后的去离子水配置成浓度为5mg/mL的标准溶液，再用去离子水对其进行稀释，得到终浓度分别为0.1、0.2、0.3、0.4、0.5mg/mL的标准样，过滤除菌后进行色谱分析，做标准曲线；c、利用标准曲线由外标定量法计算出发酵液样品中腺嘌呤的浓度。
发酵结果见表5。表5结果表明：1、同为1*YP、葡萄糖15％的发酵条件下，融合共表达BAS1-BAS2菌株最大胞外cAMP浓度较实施例5里的W303-1A(tpk1△ tpk2△ tpk3△ yak1△pde1△)菌株最大胞外cAMP浓度(3925.6μmol/L)有所提高，前者为后者的1.108倍；2、提高酵母粉和蛋白胨浓度效果极为显著；3、添加腺嘌呤能进一步提高胞外cAMP产量。
表5、W303-1A(tpk1△ tpk2△ tpk3△ yak1△ pde1△BAS1-BAS2)菌株的cAMP生产评价
Figure PCTCN2016074067-appb-000008

Claims (17)

  1. 一种酵母，其特征在于，所述酵母包括第一种和第二种基因修饰，其中，所述第一种基因是蛋白激酶A(protein kinase A，PKA)催化亚基编码基因TPK1，TPK2和TPK3，通过修饰第一种基因使PKA活性或表达被完全抑制，从而消除对环磷酸腺苷(cyclic adenosine monophosphate，cAMP)的反馈抑制，但同时使得酵母的生长被抑制，第二种基因修饰消除了第一种基因修饰导致的生长抑制，从而使得酵母能够正常生长，所述酵母的cAMP产量上升，其中，所述cAMP产量上升，是相对于未经基因修饰的酵母的cAMP产量而言。
  2. 根据权利要求1所述的酵母，其特征在于，所述第二种基因包括蛋白激酶Rim15编码基因RIM15、转录因子Msn1/Msn2编码基因MSN1/MSN2、蛋白激酶Yak1编码基因YAK1或蛋白激酶Sch9编码基因SCH9，优选YAK1。
  3. 根据权利要求1-2任一所述的酵母，其特征在于，所述第二种基因修饰使得所修饰基因编码酶的活性或表达被完全抑制或者编码酶的活性提高或者过表达。
  4. 根据权利要求1-3任一所述的酵母，其特征在于，所述酵母还包括第三种基因修饰，以减少cAMP的降解，从而提高cAMP产量。
  5. 根据权利要求4所述的酵母，其特征在于，所述第三种基因为cAMP磷酸二酯酶编码基因PDE1和/或PDE2，优选PDE1。
  6. 根据权利要求4-5任一所述的酵母，其特征在于，所述第三种基因修饰使得所修饰基因编码酶的活性或表达被完全抑制。
  7. 根据权利要求1-6任一所述的酵母，其特征在于，所述使得编码酶的活性或表达被完全抑制的基因修饰方式包括点突变、缺失、插入、反义polynucleotides、siRNA、microRNA、CRISPR；使得编码酶的活性提高或者过表达的基因修饰方式包括点突变、连接强启动子、连接增强子、提高拷贝数。
  8. 根据权利要求1-7任一所述的酵母，其特征在于，所述酵母还包括第四种基因修饰，以增加嘌呤合成途径中cAMP前体物的合成的正调控，使cAMP前体物的合成上升，从而提高cAMP产量。
  9. 根据权利要求8所述的酵母，其特征在于，所述第四种基因包括转录因子Bas1和Bas2编码基因。
  10. 根据权利要求9所述的酵母，其特征在于，所述基因修饰使Bas1/Bas2复合物表达上升。
  11. 根据权利要求8-10任一所述的酵母，其特征在于，所述第四种基因的基因修饰方式包括点突变、连接强启动子、连接增强子、提高拷贝数、融合共表达，优选融合共表达。
  12. 根据权利要求1-11任一中的酵母，其特征在于，所述酵母为酿酒酵母(Saccharomyces cerevisiae)、巴斯德酵母(Saccharomyces pastorianus),树干毕赤酵母(Pichia stipitis),Saccharomyces Bayanus和休哈塔假丝酵母(Candida shehatae)中的任意一种，优选酿酒酵母(Saccharomyces cerevisiae)。
  13. 一种构建权利要求1-12任一所述的酵母的方法。
  14. 一种利用权利要求1-12任一的酵母生产cAMP的发酵方法。
  15. 一种利用权利要求1-12任一的酵母发酵生产的发酵液。
  16. 权利要求1-12任一所述的酵母或者权利要求15所述的发酵液在生产cAMP中的用途。
  17. 权利要求1-12任一所述的酵母或者权利要求15所述的发酵液在医药、畜牧业、食品或化工领域的应用。
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