CN107312723B

CN107312723B - The yeast strain of high yield cAMP a kind of and its application

Info

Publication number: CN107312723B
Application number: CN201710726437.7A
Authority: CN
Inventors: 邹少兰; 高文萱; 胡芸; 杨顺华; 马平生
Original assignee: Tianjin University
Current assignee: Tianjin University
Priority date: 2017-08-17
Filing date: 2017-08-22
Publication date: 2019-02-15
Anticipated expiration: 2037-08-22
Also published as: CN107312723A

Abstract

The present invention provides a kind of yeast strain that can excessively synthesize extracellular cAMP and its construction method, zymotechnique and the application in medicine, animal husbandry, food, health care product or chemical field.The yeast strain includes the first and second of gene modification, wherein, the first described gene is protein kinase A (protein kinase A, PKA) catalytic subunit encoding gene TPK1, TPK2 and TPK3, PKA activity or expression are totally constrained by modifying the first gene, to eliminate to cyclic adenosine monophosphate (cyclic adenosine monophosphate, cAMP feedback inhibition), but it is suppressed the growth of yeast, second of gene modification eliminates growth inhibition caused by the first gene modification, so that yeast being capable of normal growth, the cAMP yield of the yeast rises.The yeast strain further includes the third, the 4th kind of gene modification and/or the 5th kind of gene modification.Restructuring yeast strains of the invention can be stablized, continue, efficiently producing extracellular cAMP.

Description

The yeast strain of high yield cAMP a kind of and its application

Technical field

The invention belongs to genetic engineerings and technical field of microbial fermentation, and in particular to a kind of excessively to synthesize extracellular cAMP Yeast strain and its construction method, zymotechnique and application in medicine, animal husbandry, food, health care product or chemical field.

Background technique

Cyclic adenosine monophosphate (cyclic adenosine monophosphate, abbreviation cAMP) is widely present in human body A kind of important substance with physiological activity, as intracellular second messenger, glycometabolism, fat metabolism, nucleic acid are synthesized, Protein synthesis etc. plays important adjustment effect.CAMP is widely used in terms of medicine at present: clinically cAMP is for treating Angina pectoris, myocardial infarction, myocarditis and cardiogenic shock etc.；To the palpitaition for improving rheumatic heart disease, the symptoms such as out of breath, uncomfortable in chest It has certain effect；The curative effect of acute leukemia combination chemotherapy can be improved, also can be used for the inducer remission of acute leukemia；This Outside, also there is certain curative effect to senile chronic bronchitis, various hepatitis and psoriasis.CAMP also can be used as pharmaceutical intermediate preparation Dibutyryl adenosine cyclophosphate and adenosine cyclophosphate meglumine, raising is fat-soluble, to play more effective physiology and pharmacological action.cAMP Animal foods additive is also acted as, in the effect of isolated condition Imitating growth hormone, promotes growth of animals or poultry, increases high-quality fowl Product yield.

The production method of cAMP has chemical synthesis, enzymatic synthesis and three kinds of fermentation method.Domestic and international industry metaplasia at present The chemical synthesis all used with adenylic acid (Adenosine-5 '-monophosphate, abbreviation AMP) for raw material is produced, It, which is used, efficiently separates column progress intermediate separation, complicated for operation, and related expensive reagents, using a large amount of pyridine conduct Solvent also results in serious environmental pollution.Enzymatic synthesis is i.e. with adenyl cyclase (E.C.4.6.1.1, Cyr1p) for enzyme source It is catalyzed one step of ATP raw material and produces cAMP, but that there are substrates is expensive, adenyl cyclase content is low, purification difficult, stability for this method The limitation such as difference, therefore is also limited to laboratory stage at present, and from industrialization, there are also with a distance from comparable.In contrast, microbial fermentation Method production cAMP has the advantages that following some: mild condition, simple process can utilize cheap carbon source, and by-product is few, environment Pollute small, it can be achieved that continuous production etc., is technique that is potential, being worth Devoting Major Efforts To Developing.

It is produced currently with bacterial fermentation process such as brevibacterium liquefaciens, microbacterium, corynebacteria, arthrobacterium, Escherichia coli CAMP has certain research, but face poor repeatability, yield it is unstable, it is at high cost, be difficult to the problems such as industrializing.And yeast is outstanding It is saccharomyces cerevisiae (Saccharomyces cerevisiae) as one of most important industrial microorganism, not only has industry Change that application technology is mature, resistance is strong, the advantages of being foodsafety GRAS microorganism, while realizing full-length genome as earliest The microorganism fungus kind of sequencing, and be one of the most important model organism of molecular biology and genetics research, cAMP signal path And its relevant basic research of regulation is goed deep into, Genetic Manipulative Technology means are perfect.Regrettably research both domestic and external all collects at present In in the signals-modulating function of cAMP intracellular, there are no as a product to pay close attention to specially grinding for extracellular production situation Study carefully.If studying the extracellular cAMP of yeast generates rule, and realization is transformed by genetic engineering means and produces extracellular cAMP ferment with excessive Mother is that the fermentation method of strain produces, and will greatly reduce cost, simplifies technique, opens up newly to produce cAMP for microbial fermentation Approach, push high level medicine and biogenetic products green clean manufacturing, have important economic value and social effect.

And up to the present cAMP rests on research rank because cAMP can only be injected use in the application in animal husbandry field Section.It such as can solve application method problem, will be conducive to give full play to its extensive Nutrition and Metabolism adjustment effect, and actually become The safe noresidue of non-hormone is pollution-free thus has the Animal feed-additive of applications well prospect, for being directly used as feed addition Agent, or it is prepared into feed addictive.

Summary of the invention

The purpose of the present invention is overcoming above-mentioned the shortcomings of the prior art, one kind is provided and is easy culture, is suitble on a large scale Fermenting and producing and yeast strain being easily achieved industrialization, can excessively synthesizing by genetic modification extracellular cAMP, construct this The recombination method of bacterial strain and application.Specifically,

The present invention provides a kind of yeast strain, this yeast strain have passed through the first and second of gene modification, wherein institute Stating the first gene is protein kinase A (protein kinase A, PKA) catalytic subunit encoding gene TPK1, TPK2 and TPK3, It is totally constrained PKA activity or expression by modifying the first gene, to eliminate the feedback inhibition to cAMP, but is made simultaneously The growth for obtaining yeast is suppressed, and second of gene modification eliminates growth inhibition caused by the first gene modification, so that Yeast can normal growth, the cAMP yield of the yeast rises, wherein the cAMP yield rises, and is relative to without base For the cAMP yield of the yeast of modification.

Preferably, second of gene include but are not limited to protein kinase Rim15 encoding gene RIM15, transcription because Sub- Msn1/Msn2 encoding gene MSN1/MSN2, protein kinase Yak1 encoding gene YAK1 and/or protein kinase Sch9 encode base Because of SCH9, preferably YAK1.It is furthermore preferred that second of gene modification makes the activity or expression quilt of institute's modifier codase The activity of complete inhibition or codase is improved or is overexpressed.

Preferably, second of gene modification makes protein kinase Rim15 encoding gene RIM15, transcription factor Msn1/Msn2 The activity or expression of encoding gene MSN1/MSN2, and/or protein kinase Yak1 encoding gene YAK1 codase are totally constrained； The activity of protein kinase Sch9 encoding gene SCH9 codase is either made to improve or be overexpressed.

Further, any of the above-described yeast further includes the third gene modification, to reduce the degradation of cAMP, to improve CAMP yield.

Preferably, the third described gene is cAMP di-phosphate ester enzyme coding gene PDE1 and/or PDE2, preferably PDE1. It is furthermore preferred that the third described gene modification is totally constrained the activity of institute's modifier codase or expression.

Preferably, the gene modification mode that the above-mentioned activity for making codase or expression are totally constrained include point mutation, Missing, insertion, antisense polynucleotides, siRNA, microRNA, CRISPR, more preferably missing and point mutation；So that compiling The gene modification mode that the activity of code enzyme is improved or is overexpressed includes point mutation, connection strong promoter, link enhancement, improves Copy number.

Further, any of the above-described yeast further includes the 4th kind of gene modification, to increase cAMP precursor in purine route of synthesis The positive regulation of the synthesis of object rises the synthesis of cAMP precursor, to improve cAMP yield.

Preferably, the 4th kind of gene includes but are not limited to transcription factor Bas1, Bas2 encoding gene.More preferably , the gene modification rises the expression of Bas1/Bas2 compound.

Preferably, the modification mode of above-mentioned 4th kind of gene includes but are not limited to point mutation, connection strong promoter, connection Enhancer improves copy number, fusion coexpression, more preferably fusion coexpression.

Further, any of the above-described yeast further includes the 5th kind of gene modification, to increase to the intracellular of cAMP and its precursor The regulation of outer transport and accumulation makes cAMP synthesize and secrete and rises, to improve cAMP yield.

Preferably, the 5th kind of gene includes but are not limited to plasma membrane carrier protein Fcy2 encoding gene and Snq2 coding Gene.It is furthermore preferred that the gene modification rises Fcy2 and/or Snq2 activity or expression.

Preferably, the modification mode of above-mentioned 5th kind of gene includes but are not limited to point mutation, connection strong promoter, connection Enhancer improves copy number.

Further, the modification mode of the gene includes to use selectable marker gene.Preferably, the selected marker base Because including but are not limited to URA3, LEU2, HIS3, TRP1, LYS2.More preferably use URA3 selectable marker gene.Further Preferably, the modification mode of the gene is that selectable marker gene is recycled without retaining selectable marker gene.

Yeast strain described in the invention has higher extracellular cAMP yield compared with the yeast without gene modification.For example, The extracellular cAMP yield of yeast strain described herein may be up to 11581.6 μm of ol/L.

Yeast strain described in the invention is saccharomyces cerevisiae (Saccharomyces cerevisiae), Pasteur ferment Female (Saccharomyces pastorianus), pichia stipitis (Pichia stipitis), Saccharomyces Any one in Bayanus and shehatae candida (Candida shehatae).Preferably saccharomyces cerevisiae (Saccharomyces cerevisiae)。

It is an object of the present invention to provide a kind of sides for constructing the above-mentioned yeast strain that extracellular cAMP yield greatly improves Method.

The first gene modification is introduced in yeast first, eliminates the feedback inhibition to cAMP, but make yeast simultaneously Growth is suppressed, secondly, introducing second of gene modification, second of gene modification eliminates raw caused by the first gene modification It is long to inhibit, so that yeast normal growth.

It further, further include that the third gene modification is carried out to yeast in the method to reduce the degradation of cAMP, from And further increase cAMP yield.

It further, further include carrying out the 4th kind of gene modification to yeast to increase in purine route of synthesis in the method The positive regulation of the synthesis of cAMP precursor rises the synthesis of cAMP precursor, improves cAMP yield.

It further, further include carrying out the 5th kind of gene modification to yeast to increase to cAMP and its precursor in the method The regulation of the outer transport and accumulation intracellular of object, makes cAMP synthesize and secrete and rises, to improve cAMP yield.

It is an object of the present invention to provide a kind of method using above-mentioned yeast production cAMP, the method includes Above-mentioned yeast is cultivated in fermentation medium.

Further, the method includes fermentation medium components, such as carbon source, nitrogen source, microelement.

Preferably, in the method, the concentration of glucose of fermentation culture medium is 20-150g/L, yeast extract 10- 20g/L, peptone 20-40g/L, adenine additive amount are 0-1.25g/L.

It is an object of the present invention to provide a kind of fermentation liquids using above-mentioned yeast fermenting and producing.

It is an object of the present invention to provide the purposes of any of the above-described yeast or fermentation liquid in production cAMP.

It is an object of the present invention to provide any of the above-described yeast or fermentation liquid in medicine, animal husbandry, food, health care The application of product or chemical field.

It is an object of the present invention to provide any of the above-described yeast or fermentation liquid preparation medicine, animal husbandry, food, Application in the product in the fields such as health care product or chemical industry.

Further, the application of any of the above-described yeast strain or fermentation liquid in animal husbandry field includes being used as animal Growth accelerator is directly used as feed or feed addictive or is prepared into feed or feed addictive.

The fields such as above-mentioned medicine, animal husbandry, food, health care product or chemical industry are prepared it is an object of the present invention to provide a kind of Product method, the method includes preparing the said goods using any of the above-described yeast or fermentation liquid.

Further, the method includes the yeast or fermentation liquid are further processed into any other dosage form, preferably For edible dosage form.The more preferably forms such as pulvis, granule, tablet, capsule or liquid.

Correspondingly, the present invention also provides the medicine comprising any of the above-described yeast or fermentation liquid, animal husbandry, food, guarantor The product of strong product or chemical field, such as drug, feed, feed addictive, food, health care product etc..

Preferably, the drug, feed, feed addictive, food, health care product can be direct bacterial strain or fermentation liquid, Or the yeast or fermentation liquid are further processed into any other dosage form, preferably edible dosage form.More preferably powder The forms such as agent, granule, tablet, capsule or liquid.

Detailed description of the invention

The present invention is to provide it is a kind of by genetic modification, can excessively synthesize the yeast strain of extracellular cAMP, construct this The recombination method of bacterial strain and application.More specifically,

As described above, yeast of the invention includes the first gene modification and second of gene modification.Further, described Yeast further includes the third, the 4th kind and/or the 5th kind of gene modification.Wherein, the first gene modification is eliminated to the anti-of cAMP Feedback inhibits, but is suppressed the growth of yeast；Second of gene modification eliminates raw caused by the first gene modification It is long to inhibit, so that yeast being capable of normal growth；The third gene modification reduces the degradation of cAMP, and the 4th kind of gene modification increases The positive regulation for adding the synthesis of cAMP precursor in purine route of synthesis rises the synthesis of cAMP precursor, improves cAMP yield. 5th kind of gene modification makes cAMP synthesize and secrete with the regulation for increasing the outer transport intracellular to cAMP and its precursor and accumulating Rise, to improve cAMP yield.

More specifically, the first gene modification can pass through modification protein kinase A (protein kinase A, PKA) Catalytic subunit encoding gene TPK1, TPK2 and TPK3 are totally constrained PKA activity or expression, feedback inhibition of the PKA to cAMP It is eliminated, but is suppressed the growth of yeast；Second of gene modification eliminates raw caused by the first gene modification It is long to inhibit, so that yeast being capable of normal growth；Second of gene modification can make the activity or table of protein kinase Yak1 etc. Up to being totally constrained, to eliminate because inhibiting cell growth, therefore bacterial strain caused by PKA is active or expression is totally constrained It being capable of normal growth；The third gene modification is totally constrained the phosphodiesterase Pde activity or expression for hydrolyzing cAMP；4th Kind gene modification keeps positive regulation such as transcription factor Bas1, Bas2 for being synthesized cAMP precursor AMP in purine route of synthesis living Property or expression rise, increase AMP synthesis, it is final to improve cAMP yield；5th kind of gene modification carries cAMP precursor plasma membrane Body protein such as Fcy2 and/or cAMP plasma membrane carrier protein such as Snq2 activity or expression rise, and make in cAMP synthesis and secretion It rises, to improve cAMP yield.

The present invention describes the feedback suppression for eliminating PKA to cAMP with second of gene modification by the first of yeast strain System synthesizes extracellular cAMP yield to improve.PKA is currently the only known cAMP target in yeast, it is considered that in cAMP concentration Fluctuation effect is applied by PKA approach；In other words, stringent negative-feedback tune of the cAMP signal path by PKA in yeast cells Control.The tetramer albumen that yeast PKA is made of the adjusting subunit of two catalytic subunits and a dimer, in shortage intracellular When cAMP or cAMP is horizontal lower, PKA exists in the form of being passivated complex.When cAMP level intracellular rises, cAMP and PKA's Subunit is adjusted to combine, the catalytic subunit of PKA is disintegrated down from adjusting subunit, a series of protein phosphorylation cascade reactions are activated, Cause the activity of these albumen to change, and then regulates and controls the level of cAMP intracellular on different level；Primary effect therein Enzyme is adenyl cyclase Cyr1, and Cyr1 is catalyzed ATP and synthesizes cAMP.The adjusting subunit of PKA is encoded by gene BCY1, catalytic subunit It is encoded by gene TPK1, TPK2 and TPK3.

When three catalytic subunit Tpk1, Tpk2 and Tpk3 activity or expression while when being totally constrained, PKA activity also just by Complete inhibition, PKA are eliminated the feedback inhibition of cAMP.On the other hand, when PKA activity is totally constrained, protein kinase Rim15 is rested in nucleus, and activating transcription factor Msn1/Msn2, Msn1/Msn2 are activated in turn has grown negative regulation to cell The protein kinase Yak1 of effect, make cellular retention in the G1 phase and can not continued growth, therefore the yeast that PKA activity is totally constrained Bacterial strain needs further gene modification to ensure bacterial strain normal growth.

The catalytic subunit of protein kinase Sch9 and PKA have homology, and under normal physiological conditions, Sch9 and PKA have There is different substrates；But growth caused by the activity for increasing any kinases can all be compensated due to lacking another kinases lacks It falls into.

The first gene modification in the present invention eliminates PKA to the feedback inhibition of cAMP, makes PKA activity or has expressed It is complete to inhibit, second of gene modification eliminate yeast strain because of growth inhibition caused by the first gene modification, so as to just It is frequently grown.Bacterial strain of the PKA to the feedback inhibition of cAMP and in the case of keeping PKA activity to be totally constrained is eliminated normally to give birth to There are many long strategies.The feedback inhibition for making PKA activity or expression be totally constrained, eliminate PKA to cAMP, can compile It is carried out in the nucleic acid sequence of code catalytic subunit Tpk1, Tpk2 and Tpk3, this nucleic acid sequence can be control region, code area.

The nucleic acid sequence of Tpk1 can be found in such as GenBank Accession No.NC_001142.9.

The nucleic acid sequence of Tpk2 can be found in such as GenBank Accession No.NC_001148.4.

The nucleic acid sequence of Tpk3 can be found in such as GenBank Accession No.NC_001143.9.

The strategy of bacterial strain normal growth in the case of keeping PKA activity to be totally constrained includes but is not limited to protein kinase Rim15 encoding gene RIM15, transcription factor Msn1/Msn2 encoding gene MSN1/MSN2 and/or protein kinase Yak1 encode base Because YAK1 carries out gene modification, the result of modification is totally constrained corresponding enzymatic activity or expression, and bacterial strain is to be able to normal life It is long.The strategy of bacterial strain normal growth in the case of keeping PKA activity to be totally constrained further includes but is not limited to protein kinase Sch9 encoding gene SCH9 carries out gene modification, and the result of modification is enhanced corresponding enzymatic activity or expression, and bacterial strain is to be able to Normal growth.

The nucleic acid sequence of Yak1 can be found in such as GenBank Accession No.NC_001142.9.

The nucleic acid sequence of Rim15 can be found in such as GenBank Accession No.NC_001138.5.

The nucleic acid sequence of Msn1/Msn2 can be in such as GenBank Accession No.NC_001147.6, NC_ It is found in 001145.3.

The nucleic acid sequence of Sch9 can be found in such as GenBank Accession No.NC_001140.6.

It is totally constrained enzymatic activity or expression no matter carrying out the first and second of gene modification in which way, or is made Enzymatic activity or expression are enhanced, the modification step of TPK1, TPK2, TPK3 and second of gene totally four genes in yeast strain, In addition to (this combination cannot be deposited because of growth defect for the combination that inactivates TPK1, TPK2, TPK3 simultaneously in haploid yeast cell It is living) other than, aforementioned four gene can in no particular order, any combination is modified.

The yeast strain by above-mentioned the first and second of gene modification that the present invention describes is relative to without the first More extracellular cAMP are generated with the yeast strain of second of gene modification.

On the other hand, for cAMP once generating, the mode for uniquely inactivating it is urging in cyclic nucleotide phosphodiesterase Pde 5 '-AMP are hydrolyzed under change.Therefore, Pde also affects the level of cAMP intracellular.Therefore, the present invention passes through the third gene Modification is totally constrained Pde activity or expression, to reduce the degradation of cAMP, improve cAMP contents level.Saccharomyces cerevisiae is thin Born of the same parents itself include two cAMP phosphodiesterases: Pde1 encodes a low-affinity (Km=20-250 μM), specific lower Enzyme, mainly mediate quick cAMP signal transduction；Pde2 encodes a high-affinity (Km=170nM), specific very strong Enzyme, the main foundation level for controlling cAMP.

The nucleic acid sequence of Pde1 can be found in such as GenBank Accession No.NC_001139.9.

The nucleic acid sequence of Pde2 can be found in such as GenBank Accession No.NC_001147.6.

The first described in the invention, second and the third gene modification can be by known in the art any A variety of methods are realized.So that the gene modification mode that the activity of codase or expression are totally constrained can be the core to gene Acid sequence carries out point mutation, missing or is inserted into one or more nucleotide, and (purine is to fast for the conversion of point mutation such as mononucleotide Purine or pyrimidine are to pyrimidine) or transversion (purine to pyrimidine or pyrimidine to purine).The mutation of nucleic acid can cause in its expression The displacement of the conservative or non-conservative amino acid of one or more in polypeptide, it is this to replace the conformation that may result in polypeptide It changes, it is also possible to lose some or all of function, it may occur however that frameshift mutation will lead to entire polypeptide chain from the point Start to encode an entirely different polypeptide, it is also possible to form a terminator codon in advance and make polypeptide chain incompleteness, or even make Gene silencing.

Missing or insertion can be obtained with nucleic acid sequence with PCR or chemically synthesized method, and it is multiple also to can use cell System amplification obtains.The gene modification for being totally constrained gene enzymatic activity or expression can also be by offer or antisence widow's core Thuja acid antisense polynucleotides, siRNA, microRNA or other can prevent the mRNA of gene to be finished from turning over The method of the nucleic acid of protein is translated into realize.The class activating transcription factor effector nuclease developed in recent years (transcription activator-like effector nuclease, TALEN) and CRISPR technology --- rule at The short palindrome in the interval of cluster repeats (Clustered Regularly Interspaced Short Palindromic Repeats) It can also be used to inactivated gene.

The gene modification mentioned in the present invention for being totally constrained gene enzymatic activity or expression refers to make this Enzyme/polypeptide activity reduction (compared with yeast or wild-type yeast without gene modification) at least 95% (for example, at least 96%, 97%, 98%, 99% or 100%).

So that the gene modification mode that the activity of codase is improved or is overexpressed includes but is not limited to point mutation, connects by force Promoter, improves copy number at link enhancement.For example, point mutation improves polypeptide active；Improve the copy number of nucleic acid sequence；It is logical Crossing gene modification connect nucleic acid sequence with an effable strong promoter or enhancer；Change nucleic acid sequence promoter or Other regulatory factors of person are to improve its expression (such as the bond strength for improving promoter sequence and transcription initiation factor)； Improve the half-life period of the mRNA of nucleic acid sequence transcription；Inhibit the degradation to mRNA or polypeptide chain.

The gene modification mentioned in the present invention for being enhanced gene enzymatic activity or expression refers to that one is repaired by gene Enzyme/polypeptide activity of the nucleic acid sequence encoding of decorations improves at least 20% (example to the functioning efficiency of substrate compared with wild type Such as at least 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or higher).

The present invention describe by it is above-mentioned the first, the yeast strain of second and the third gene modification, relative to not Through the first, the yeast strain of second and the third gene modification generate more extracellular cAMP.Even with respect to the first More extracellular cAMP are generated with the yeast strain of second of gene modification.

It is horizontal to improve the extracellular cAMP of yeast cells, the gene of cAMP-PKA signal path approach is modified outside, can also It is realized with synthesizing the synthetic quantity of precursor AMP by regulation purine route of synthesis, increase cAMP.The purine of saccharomyces cerevisiae synthesizes In approach, collaboration of AMP de novo synthesis gene, that is, ADE gene expression between by 2 transcription regulatory factors Bas1 and Bas2 The positive regulation of effect.Bas1 is Myb race transcription regulatory factor, Bas2 also known as Pho2, is homeodomain (Homeodomain) transcriptional control The factor, for the two with composite form in conjunction with specific promoter region, synergistic effect activates the expression of 10 ADE genes.Work as born of the same parents When interior ADP, ATP excessive concentration, first enzyme Ade4p of ADP, ATP feedback inhibition de novo synthesis (PRPP amide transferase, Abbreviation PRPPAT) activity so that the synthesis of intermediate product SAICAR declines, Bas2p can perceive the concentration variation of SAICAR, when When this concentration is down to a certain degree, Bas2p no longer forms compound with Bas1p.When there is considerable amount of adenine to deposit in culture medium When, adenine enters intracellular, forms AMP by remedial pathway, and AMP is further converted to ADP and ATP, also cause entirely from Head route of synthesis synthesis is suppressed.

4th kind of gene modification of the invention synthesizes AMP by increasing the positive regulation that AMP is synthesized in purine route of synthesis Rise, improves cAMP yield.The core just regulated and controled for increasing AMP synthesis is to promote the formation of Bas1/Bas2 compound, raising multiple The positive regulation activity of object is closed, the feedback inhibition of adenine, ADP and ATP are inhibited.And Bas1/Bas2 compound is promoted to be formed, improved There are many strategies of the positive regulation activity of compound, and the activity or table of single positive regulative transcription factor Bas1 or Bas2 can be improved It reaches, can also directly merge coexpression Bas1 and Bas2, or further increase on the basis of fusion co-expresses Bas1 and Bas2 Activity or expression quantity.The polypeptide active of nucleic acid encode can be made to improve there are many kinds of method.It lives for example, point mutation improves polypeptide Property；Improve the copy number of nucleic acid sequence；Make nucleic acid sequence and an effable strong promoter or enhancer by gene modification Connection；Change promoter or other regulatory factors of nucleic acid sequence to improve its expression and (such as improve promoter sequence The bond strength of column and transcription initiation factor)；Improve the half-life period of the mRNA of nucleic acid sequence transcription；Inhibit to mRNA or more The degradation of peptide chain.Assert that the raising of the polypeptide active of a nucleic acid sequence encoding Jing Guo gene modification needs its work to substrate Improve at least 20% compared with wild type with efficiency (for example, at least 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or higher).The overexpression of the nucleic acid sequence of single copy or multicopy can pass through connection It realizes, can also be realized by being integrated into the genome of yeast into carrier.Carrier suitable for nucleic acid sequence overexpression is It is commercialized or can be obtained by gene recombination technology commonly used in the art.Have in the art some in yeast The method of nucleic acid sequence is integrated in genome.For example, Methods in Enzymology:Guide to Yeast Genetics and Molecular and cell Biology,Vol.194,2004,Abelson et al.,eds.,Academic Press.Carrier containing nucleic acid sequence can some pairs expression it is necessary to element and purpose nucleic acid sequence carry out can operate Connection: can take expression selected marker nucleic acid sequence (such as auxotrophic selection marker gene, antibiotic resistance base Cause)；Or take the gene (such as 6xHis tag) that can be used to purified polypeptide；It can also be with one or more replication orgins. To expression it is necessary to element include the nucleic acid sequence that can instruct or regulate and control purpose nucleic acid sequence, such as promoter sequence. Representative promoter includes (not limited to this) PGK promoter, TPI1 promoter, ADH1 promoter.To expression it is necessary to Element further includes enhancer sequence, response element, or the inducible factor of regulation coded sequence expression.

To expression it is necessary to element can come from bacterium, yeast, insect, plant, mammal, fungi or virus, Expression vector can have the element of separate sources.To expression it is necessary to element may refer in Goeddel, 1990, Gene Expression Technology:Methods in Enzymology,185,Academic Press,San Diego,CA。 In some cases, can choose to the optimum nucleic acid sequence of specific codon or to expression it is necessary to element so as in ferment Best expression is obtained in mother.Referring to Bennetzen&Hall, 1982, J.Biol.Chem., 257:3026-31.

The nucleic acid sequence of Bas1 can be found in such as GenBank Accession No.NC_001143.9.

The nucleic acid sequence of Bas2 can be found in such as GenBank Accession No.NC_001136.10.

The present invention describe by it is above-mentioned the first, second, the yeast strain phase of the third and the 4th kind of gene modification For without the first, second, the yeast strain of the third and the 4th kind of gene modification generate more extracellular cAMP.Even Relative to the first and second gene modification yeast strain or relative to the first, second and the third gene modification Yeast strain generate more extracellular cAMP.

It is horizontal to improve the extracellular cAMP of yeast cells, the outer transport intracellular and product of regulation cAMP and its precursor can also be passed through Tire out to realize.Yeast cells outer matter transportation intracellular needs many plasma membrane carrier proteins to participate in.It is responsible for from extracellular to transmitter loss The carrier protein of base has Fcy2, Fur4 and Fui1, and wherein Fcy2 is purine-cytimidine permease, mediates adenine, guanine With the transport and accumulation of hypoxanthine and cytimidine from extracellular to intracellular.And plasma membrane ATP combines (ABC) channel protein Snq2 to participate in The transport of a variety of drugs includes mutagens, fungicide, steroids and anticancer by a variety of exogenous compounds of dependency ATP outlet Drug etc.；Primary Study shows that Snq2 directly may be adjusted by PKA and be participated in the feedback inhibition to cAMP, is responsible for simultaneously CAMP is to extracellular discharge.

5th kind of gene modification of the invention is by increasing the outer tune transported and accumulate intracellular to cAMP and its precursor Control makes cAMP synthesize and secrete and rises, to improve cAMP yield.Increase the outer transport intracellular and product to cAMP and its precursor The mode of tired regulation is modified relevant carriers protein coding gene, increase carrier protein activity or expression.It is repaired The gene of decorations includes but is not limited to Fcy2 encoding gene and Snq2 encoding gene, and the result of modification makes in corresponding enzymatic activity or expression It rises, bacterial strain increases outer transport and accumulation intracellular to cAMP and its precursor, so that cAMP is synthesized and is secreted and rises, to improve CAMP yield.

So that the gene modification mode that the activity of codase is improved or is overexpressed includes but is not limited to point mutation, connects by force Promoter, improves copy number at link enhancement.

The nucleic acid sequence of Fcy2 can be found in such as GenBank Accession No.BK006939.2.

The nucleic acid sequence of Snq2 can be found in such as GenBank Accession No.BK006938.2.

The present invention describe by it is above-mentioned the first, second, the yeast strain phase of the third and the 5th kind of gene modification For without the first, second, the yeast strain of the third and the 5th kind of gene modification generate more extracellular cAMP.Even Relative to the first and second gene modification yeast strain or relative to the first, second and the third gene modification Yeast strain generate more extracellular cAMP.

In genetical modification steps of the invention, nucleic acid sequence (such as expression vector) can be transferred to yeast by a variety of methods In cell or other host cells.These methods include (not limited to this) electroporation, calcium phosphate precipitation, heat shock method, Lipofection, microinjection, lithium chloride method, Li-acetate method, mercaptoethanol method and virus mediated gene transfer method.

In addition to yeast cells, " host cell " can also be that any standard molecular biology that can be used for operates and generates core The cell of acid and polypeptide.It includes but are not limited to bacterial cell (such as E.coli), insect cell, plant cell and mammal are thin Born of the same parents' (such as CHO or COS cell)." yeast cells " includes recombinant yeast cell described herein.It is as referred to herein that " host is thin Born of the same parents " not only include the mother cell that nucleic acid transduction is carried out to it, further include its daughter cell.

In genetical modification steps of the invention, the selectable marker gene for being transferred to yeast cells Select to use includes but not It is only limitted to the available any auxotroph gene of yeast, such as URA3, LEU2, HIS3, TRP1, LYS2.To the greatest extent may be used to can be carried out Gene modification more than energy, while auxotroph gene expression bring itself being avoided to may interfere with, it preferably can be easily anti- URA3 gene multiple, being recycled.Still more preferably, the selectable marker genes such as URA3 be recycled and be not kept in by In the bacterial strain of gene modification.

Although strategy applied by the present invention is directed to the gene and polypeptide of S.cerevisiae bacterial strain, identical strategy Other fermentable yeast are equally applicable to, are preferably able to carry out the yeast strain of industrial fermentation.In addition to S.cerevisiae bacterium Strain is outer, and suitable bacterial strain further includes saccharomyces pastorianus (Saccharomyces pastorianus), pichia stipitis (Pichia stipitis), Saccharomyces Bayanus and shehatae candida (Candida shehatae).? Access or Gene Name are perhaps slightly different in these bacterial strains, but can modify using same strategy and technology corresponding Access and homologous gene.Gene engineering microzyme strain described herein is known to researcher in this field.

The method for the yeast strain production cAMP that invention further describes above-mentioned by gene modification.

Preferably, in the fermentation process, the concentration of glucose of fermentation culture medium is 20-150g/L, yeast extract 10-20g/L, peptone 20-40g/L, adenine additive amount are 0-1.25g/L.

It is furthermore preferred that fermentation culture medium glucose, yeast extract and peptone can be same with having in the method Function, a greater amount of cheap raw materials are replaced or are substituted, including but not limited to molasses, corn pulp/Dried Corn Steep Liquor Powder, corn egg White powder, bean cake powder.

Invention further describes the purposes of the above-mentioned yeast strain by gene modification, can be used for fermentating metabolism carbon aquation It closes object and generates extracellular cAMP.Further, above-mentioned yeast strain cell by gene modification and/or it is fermented obtain include The fermentation liquid of extracellular cAMP, can be directly as the substitute of cAMP.And as cAMP applies generally to every field, for example, doctor Medicine, animal husbandry, food, health care product or chemical field etc..Further, the application includes in the product for preparing above-mentioned field In application, such as drug, food, health care product and feed etc..Correspondingly, the present invention also provides pass through gene modification comprising above-mentioned Yeast strain cell and/or the fermented obtained fermentation liquid comprising extracellular cAMP medicine, animal husbandry, food, health care product Or the product, such as drug, food, health care product, feed, feed addictive in the fields such as chemical industry etc..

It is described in medicine, animal husbandry, the application of field of food, health care product, including by above-mentioned yeast strain cell, and/or The fermented obtained fermentation liquid comprising extracellular cAMP, Nutrition and Metabolism adjusting, health care and the disease for being directly used in humans and animals are controlled It treats, forms the product in these fields, such as drug, food and feed/feed addictive etc. and the application of these products.Or The cell, fermentation liquid are further processed into any other dosage form, preferably edible dosage form.More preferably pulvis, The forms such as granule, tablet, capsule or liquid；And the processing method, it is processing method commonly used in the art, is not necessarily to this field Technical staff makes the creative labor again；In animal husbandry field, it is preferred that prepare powder for low temperature drying or spray drying process Agent.

The animal, animal such as homing pigeon including model animal, fowl poultry kind, aquatic livestock, pet and various specific uses, Pigeon for meat etc., preferably fowl poultry kind animal, it is furthermore preferred that including but not limited to chicken and duck goose, pig, cattle and sheep horse donkey, further preferred conduct Chicken, pig.The feed that is added with above-mentioned use as feed addictive, can be at present that any commonly employed animal is raised on the market Material is not limited by the conventional animal feed type cooperated.However conventional animal feed therein is preferably daily ration, into one Step is preferably chicken daily ration, pig daily ration, ruminant, aquatic livestock daily ration.The usage amount as feed addictive, For (0.05-50) mg cAMP/ (kg weight), preferably (0.5-20) mgcAMP/ (kg weight), more preferable 0.5- 10.0mgcAMP/ (kg weight).

If not specified otherwise, present document relates to all technical terms, technological concept be all well known to those skilled in the art 's.It can be used to construct and measure yeast strain involved in the present invention in spite of a variety of similar or equivalent method, hereinafter A kind of suitable method and material will be described.These materials, method and example are intended only as putting to the proof, it is not in any degree upper limit The system present invention.All documents, patent and other references referred to herein respect its integrality.Side of the present invention Method and material will be provided hereinafter together with detailed description and chart.

The advantages of invention and benefit

Gained restructuring yeast strains of the invention have low nutrition demand, are easy culture, are suitble to large scale fermentation production and easy In the performance for realizing industrialization；Related gene modification is carried out by genetic engineering means, cAMP access feedback inhibition is being eliminated, is subtracting While few cAMP degradation, increase the metabolic fluxes for leading to cAMP precursor AMP, improves the outer transport intracellular of cAMP and its precursor And accumulation, so that gained restructuring yeast strains is stablized, is continued, efficiently produce extracellular cAMP；By improving carbon source concentration, optimization The measures such as carbon/nitrogen ratio, addition precursor, make metabolic fluxes more effectively flow to purpose product, extracellular cAMP optimized, high Zymotechnique, cAMP concentration can reach 11581.6 μm of ol/L.The present invention overcomes conventional chemical synthesis complex process, original It is steady to also overcome enzymatic synthesis substrate valuableness, adenyl cyclase for the shortcomings that material is expensive, by-product is more, environmental pollution is serious The deficiencies of qualitative difference, have technological operation is simple, product can be easily separated extraction, cost of material is low, safety and environmental protection, can industrial metaplasia The advantages of production, produces cAMP for microbial fermentation and opens new approach, the green of high level medicine and biogenetic products will be pushed clear Clean production can be widely used in medicine, animal husbandry, food or chemical field, have important economic value and social effect.

As application in animal husbandry, by directly adding, diet oral way use the high yeast cells containing cAMP Or its fermentation liquid and its preparation, can overcome the shortcomings of to study at present and application in can only use the drawn game of cAMP pure preparation by injection Limit, while yeast cell culture and cAMP health care, growth promotion (collaboration) effect are played, it is a kind of novel yeast source biology feeding Feed additives and the Animal feed-additive that non-hormone is safe and non-toxic, pollution-free, no chemical residues have no drug resistance, can substitute feeding With the antibiotic usage for using and reducing in breeding process of antibiotic, meet current animal husbandry development trend, while also advantageous In reducing cost to the maximum extent, so that more simple yeast culture or simple cAMP preparation and its derivative have more extensively Wealthy application prospect.

Detailed description of the invention

Fig. 1 is 1 plasmid schematic diagram of embodiment, and wherein Figure 1A is plasmid pUC18-URA3, and Figure 1B is plasmid pUC18- TPK2p-TPK2t-URA3-TPK2t。

Fig. 2 is that 5 five gene deletion strains cAMP of embodiment produce preliminary assessment.

Fig. 3 is 6 plasmid pUC18-H1 of embodiment_YNRCΔ9-BAS1-BAS2-H2_YNRCΔ9-URA3-H2_YNRCΔSchematic diagram.

Specific embodiment

Technical solution of the present invention is further described combined with specific embodiments below.It is understood that The particular implementation of this description indicates by way of example, is not intended as limitation of the present invention.If not referring in particular to Conventional means bright, that technological means used in embodiment is well known to those skilled in the art.Model of the present invention is not being deviated from In the case where enclosing, main feature of the invention can be used for various embodiments.It will be appreciated by those skilled in the art that or It is able to confirm that, routine experiment is only used only, and many equivalents can be applied in particular step described herein.These are equivalent Object is considered place within the scope of the present invention, and is covered by claim.

Several culture mediums involved in embodiment are as follows:

1) yeast strain activation and seed liquor culture culture medium YPAD

Yeast extract (Yeast extracts) 10g/L, peptone (Peptone) 20g/L, glucose (Glucose) 20g/L, adenine (Adenine) 0.05g/L, natural ph；Separately add agar powder 15g/L when solid culture.

2) transformation and selection culture medium C MG when uracil (URA) auxotrophic selection marker is used^-URA

Use the default minimal medium CMG plate abbreviation CMG of uracil^-URAPlate is screened.Component is as follows: ammonia Base soda acid based mixtures, 0.83g/L；Yeast nitrogen Yeast Nitrogen Base, abbreviation YNB, 6.7g/ without amino acid L；Glucose, 20g/L；Agar powder, 15g/L.Amino acids basic mixture therein is shown in Table 1.

1. amino acids basic mix ingredients of table

Adenine 50mg/L	Leucine 100mg/L	Arginase 12 0mg/L	Lysine 30mg/L
				Aspartic acid 100mg/L	Methionine 20mg/L	Glutamic acid 100mg/L	Phenylalanine 50mg/L
Histidine 100mg/L	Serine 150mg/L	Isoleucine 30mg/L	Threonine 150mg/L
				Tryptophan l00mg/L	Tyrosine 30mg/L	Uracil 50mg/L	Valine 150mg/L

Note: saving specific aminoacid ingredient, can be made into Selective agar medium.Adjust pH value 5.6,110 DEG C of glucose sterilizings 15min, 121 DEG C of sterilizing 21min of other ingredients, uses preceding mixing.

3) 5 '-FOA plates (5 '-orotic acid plate)

The preparation method is as follows:

A, 5 '-FOA solution of 100ml is prepared, comprising:

5 '-FOA:0.2g of YNB w/o AA:1.4g dropout powder:0.17g

Uracil: 20mg adenine: 10mg leucine: 40mg

Histidine: 30mg tryptophan: 20mg glucose: 4g

It is kept stirring at 45 DEG C with magnetic stirring apparatus, dissolves its each component all, filtration sterilization later；

B, 100ml agar powder Agar solution is prepared, its ultimate density is made to reach 3.0%, after 121 DEG C of sterilizing 15min, by it It is cooled to 45 DEG C；

C, 5 '-FOA of 100ml and 100ml Agar solution are mixed, prevents bubble, pours into sterile petri dish, it is cold But it forms.

4) Escherichia coli culture LB culture medium

Yeast extract (Yeast extracts) 5g/L, peptone (Peptone) 10g/L, sodium chloride (NaCl) 10g/ L adjusts pH value to 7.0；Separately add agar powder 15g/L when solid culture.Antibiotic is added when needing, the addition of ampicillin is dense eventually Spend 100 μ g/mL.

Primer sequence involved in embodiment is shown in Table 2.

The primer sequence used in 2 embodiment of table

In embodiment, Wine brewing yeast strain W303-1A (=ATCC208352) can be bought from U.S. ATCC.YCplac33 It can be bought from Invitrogen company.PUC18, YIplac211 can from Invitrogen company, Promega company, Biovector Co., LTD Deng Duo company buys.

Embodiment 1: the yeast strain of building missing TPK2 gene

Utilize the TPK2 gene on homologous recombination bialternative series missing yeast chromosomal, first building missing plasmid PUC18-TPK2p-TPK2t-URA3-TPK2t, this plasmid transformed yeast competent cell after linearization for enzyme restriction are with URA3 Riddled basins, which screen to obtain, occurs the yeast that double crossing over obtains with the TPK2t in the downstream TPK2p and URA3 for left and right homology arm Bacterial strain tpk2 △:: URA3；Bacterial strain tpk2 △ is obtained again with 5 '-FOA plate screenings:: URA3 arranged on left and right sides on URA3 chromosome Generation recombinates and pops up the bacterial strain tpk2 △ of the Saccharomycetes of URA3 gene between TPK2t.

Building and identification with primer are shown in Table 2, and schematic diagram is shown in Fig. 1.

One, pUC18-TPK2p-TPK2t-URA3-TPK2t is constructed

The building of this plasmid is related to four connections:

(1) pUC18-URA3 is constructed

PCR amplification URA3 gene is carried out by template of plasmid YCplac33, uses SEQ ID No.1, the SEQ ID in table 2 Sequence shown in No.2 is upstream and downstream primer P1, P2, the Fast Pfu polymerase of Quan Shi King Company production, 50 DEG C of annealing 1min, 72 DEG C extend 1.5min, totally 32 circulation.The PCR fragment of 1055bp is obtained, the bis- enzyme enzymes of restriction enzyme BamHI and SalI are used It cuts this segment and vector plasmid pUC18, T4DNA ligase is ligated and transformed into Escherichia coli Top10 competent cell, extract conversion Sub- plasmid carries out digestion identification, obtains plasmid pUC18-BamHI-URA3-SalI (abbreviation pUC18-URA3, see Figure 1A).

(2) pUC18-TPK2t-URA3 is constructed

Wine brewing yeast strain W303-1A chromosome is extracted, PCR is carried out by template of W303-1A chromosome, expands TPK2 base The terminator district (referred to as TPK2t) of cause is upstream and downstream using sequence shown in SEQ ID No.3, the SEQ ID No.4 in table 2 Primer P3, P4, the Fast Pfu polymerase of Quan Shi King Company production, 50 DEG C of annealing 1min, 72 DEG C of extension 0.5min, totally 32 are followed Ring.The PCR fragment of 526bp is obtained, bis- this segment of enzyme digestion of restriction enzyme KpnI and BamHI and vector plasmid are used PUC18-URA3, T4DNA ligase are ligated and transformed into Escherichia coli Top10 competent cell, extract transformant plasmid and carry out enzyme Identification is cut, plasmid pUC18-TPK2t-URA3 is obtained.

(3) pUC18-TPK2p-TPK2t-URA3 is constructed

W303-1A chromosome is extracted, PCR is carried out by template of W303-1A chromosome, expands the promoter region of TPK2 gene (referred to as TPK2p), using sequence shown in SEQ ID No.5, the SEQ ID No.6 in table 2 be upstream and downstream primer P5, P6, entirely The Fast Pfu polymerase of formula King Company production, 50 DEG C of annealing 1min, 72 DEG C of extension 0.5min, totally 32 recycle.Obtain 532bp PCR fragment, use bis- this segment of enzyme digestion of restriction enzyme Sac I and Kpn I and vector plasmid pUC18-TPK2t- URA3, T4DNA ligase are ligated and transformed into Escherichia coli Top10 competent cell, extract transformant plasmid and carry out digestion identification, Obtain plasmid pUC18-TPK2p-TPK2t-URA3.

(4) pUC18-TPK2p-TPK2t-URA3-TPK2t is constructed

By the PCR product bis- enzyme digestions of Sal I and Pst I of the sequence containing TPK2t expanded in above-mentioned (2), with same pair The pUC18-TPK2p-TPK2t-URA3 large fragment of enzyme digestion connects, and obtains plasmid pUC18-TPK2p-TPK2t-URA3-TPK2t (see Figure 1B).

Two, W303-1A (tpk2 △) strain construction

By the plasmid pUC18-TPK2p-TPK2t-URA3-TPK2t bis- enzyme digestions of Sac I and Pst I, linearized DNA fragmentation TPK2p-TPK2t-URA3-TPK2t；Then thin using Li-acetate method transformed saccharomyces cerevisiae W303-1A bacterial strain competence Born of the same parents are screened using uracil auxotrophy selected marker (URA), that is, use CMG^-URAPlate is screened, to obtaining Transformant bacterial strain carry out YPAD Liquid Culture, extract chromosomal DNA, using its for template progress the verifying of PCR method, primer pair is table Sequence shown in SEQ ID No.7, SEQ ID No.8 in 2 is upstream and downstream primer P7, P8, and successful integration contains URA gene Transformant PCR product is 2592bp, and control strain PCR product is 2299bp.Positive transformant Strain Designation is W303-1A (tpk2△::URA3)。

Further by bacterial strain W303-1A (tpk2 △:: URA3) plated growth thallus is coated on 5 '-FOA plates, thus Filter out the bacterium that URA3 segment is popped up by integrating homologous recombination between two identical sequences (TPK2t) on chromosome It falls.With primer pair P7, P8 verifying ibid, the long 1053bp of PCR product of the purpose bacterial strain of URA3 is successfully popped up, is not popped up URA3 host strain W303-1A (tpk2 △:: the long 2592bp of PCR fragment URA3).1053bp PCR product is surveyed Expected variation has occurred in sequence, sequencing result proof: 2 bases, the entire area ORF and end before the initiation codon of TPK2 gene Only 107 bases after codon are lacked completely.In this way, finally obtaining the sun of TPK2 gene delection, the pop-up of URA3 gene Property transformant bacterial strain W303-1A (tpk2 △).

Embodiment 2: the yeast strain of building missing TPK1 or TPK3 gene

One, W303-1A (tpk1 △) is constructed

The building of TPK1 gene delection plasmid pUC18-TPK1p-TPK1t-URA3-TPK1t: with (1) in embodiment 1 Plasmid pUC18-TPK1p-TPK1t-URA3-TPK1t is constructed based on pUC18-URA3 plasmid, is passed through in process and embodiment 1 (2), (3) finally obtain as the process of pUC18-TPK2p-TPK2t-URA3-TPK2t with (4) step, the primer pair used: It is upstream and downstream primer P9, P10, product that amplification TPK1t, which is sequence shown in SEQ ID No.9, SEQ ID No.10 in table 2, 516bp；Amplification TPK1p be sequence shown in SEQ ID No.11, SEQ ID No.12 in table 2 be upstream and downstream primer P11, P12, product 572bp.

By the plasmid pUC18-TPK1p-TPK1t-URA3-TPK1t bis- enzyme digestions of Sac I and Pst I, linearized DNA fragmentation TPK1p-TPK1t-URA3-TPK1t；Then transformed saccharomyces cerevisiae W303-1A bacterial strain competent cell, it is phonetic using urinating Pyridine (URA) auxotrophic selection marker is screened, that is, uses CMG^-URAPlate is screened, to obtained transformant bacterial strain It is further verified with PCR method, primer pair is that sequence shown in SEQ ID No.13, SEQ ID No.14 in table 2 is that upstream and downstream are drawn Object P13, P14, the transformant PCR product of successful integration are 2631bp, and control strain PCR product is 2476bp.Positive transformant Strain Designation be W303-1A (tpk1 △:: URA3).

Further by bacterial strain W303-1A (tpk1 △:: URA3) plated growth thallus is coated on 5 '-FOA (5 '-orotic acid) On plate, popped up to filter out by integrating homologous recombination between two identical sequences (TPK1t) on chromosome The bacterium colony of URA3 segment.With primer pair P13, P14 verifying ibid, the PCR product for successfully popping up the purpose bacterial strain of URA3 is long 1102bp does not pop up the long 2631bp of control PCR fragment of URA3.1102bp PCR product is sequenced, sequencing result Expected variation has occurred in proof: 70 bases, the entire area ORF and terminator codon before the initiation codon of TPK1 gene it 116 bases afterwards are lacked completely.In this way, finally obtaining the positive transformants daughter bacteria of TPK1 gene delection, the pop-up of URA3 gene Strain W303-1A (tpk1 △).

Two, W303-1A (tpk3 △) is constructed

The building of TPK3 gene delection plasmid: to construct plasmid based on (1) pUC18-URA3 plasmid in embodiment 1 The process of pUC18-TPK3p-TPK3t-URA3-TPK3t is finally obtained with embodiment 1 by (2), (3) and (4) step The process of pUC18-TPK2p-TPK2t-URA3-TPK2t is the same, the primer pair used: amplification TPK3t is the SEQ ID in table 2 Sequence shown in No.15, SEQ ID No.16 is upstream and downstream primer P15, P16, product 566bp；Expanding TPK3p is in table 2 Sequence shown in SEQ ID No.17, SEQ ID No.18 is upstream and downstream primer P17, P18, product 545bp.

By the plasmid pUC18-TPK3p-TPK3t-URA3-TPK3t bis- enzyme digestions of Sac I and Pst I, linearized DNA fragmentation TPK3p-TPK3t-URA3-TPK3t；Then transformed saccharomyces cerevisiae W303-1A bacterial strain competent cell, with embodiment 1 In " W303-1A (tpk2 △) strain construction " method screening, identification obtain W303-1A (tpk3 △:: URA3) bacterial strain and W303- 1A (tpk3 △) bacterial strain.PCR method verifying sequence shown in the SEQ ID No.19 that primer pair is in table 2, SEQ ID No.20 For upstream and downstream primer P19, P20, successful integration, the transformant PCR product containing URA3 gene are 2695bp, successful integration, The transformant PCR product of URA3 gene pop-up is 1116bp, and control strain PCR product is 2400bp.To 1116bp PCR product It is sequenced, expected variation has occurred in sequencing result proof: after the entire area ORF of TPK3 gene and terminator codon 93 bases are lacked completely.In this way, finally obtaining the positive transformant bacterial strain of TPK3 gene delection, the pop-up of URA3 gene W303-1A(tpk3△)。

Embodiment 3: the yeast strain of building missing YAK1 gene

The building of YAK1 gene delection plasmid: to construct plasmid based on (1) pUC18-URA3 plasmid in embodiment 1 The process of pUC18-YAK1p-YAK1t-URA3-YAK1t is finally obtained with embodiment 1 by (2), (3) and (4) step The process of pUC18-TPK1p-TPK1t-URA3-TPK1t is the same, the primer pair used: amplification YAK1t is the SEQ ID in table 2 Sequence shown in No.21, SEQ ID No.22 is upstream and downstream primer P21, P22, product 506bp；Expanding YAK1p is in table 2 Sequence shown in SEQ ID No.23, SEQ ID No.24 is upstream and downstream primer P23, P24, product 504bp.

The building of YAK1 gene deletion strains: by plasmid pUC18-YAK1p-YAK1t-URA3-YAK1t Sac I and Pst The bis- enzyme digestions of I, obtain linear DNA fragments YAK1p-YAK1t-URA3-YAK1t；Then transformed saccharomyces cerevisiae W303-1A bacterial strain Competent cell obtains W303-1A (yak1 with the screening of " W303-1A (tpk2 △) strain construction " method, identification in embodiment 1 △:: URA3) bacterial strain and W303-1A (yak1 △) bacterial strain.PCR method verifying with SEQ ID No.25 that primer pair is in table 2, Sequence shown in SEQ ID No.26 is upstream and downstream primer P25, P26, successful integration, the transformant PCR product containing URA3 gene For 2529bp, successful integration, the transformant PCR product of URA3 gene pop-up are 1010bp, and control strain PCR product is 3614bp.1010bp PCR product is sequenced, expected variation: the starting of YAK1 gene has occurred in sequencing result proof 146 bases before codon after 40 bases, the entire area ORF and terminator codon are lacked completely.In this way, final The positive transformant bacterial strain W303-1A (yak1 △) popped up to YAK1 gene delection, URA3 gene.

Embodiment 4: building while the yeast strain for lacking TPK1, TPK2, TPK3, YAK1 gene

Construct the bacterial strain for being lacked four genes simultaneously in four steps below.In fact those skilled in the art knows Road, in addition in haploid yeast cell simultaneously inactivate TPK1, TPK2, TPK3 combination (this combination because growth defect cannot Survival) other than, aforementioned four gene can in no particular order, any combination is modified.

One, YAK1 gene is lacked

It constructs to obtain W303-1A (yak1 △) bacterial strain with 3 method of embodiment.

Two, YAK1 and TPK1 gene is lacked

By the missing constructed in embodiment 2 plasmid pUC18-TPK1p-TPK1t-URA3-TPK1t Sac I and Pst I Double enzyme digestions, obtain linear DNA fragments TPK1p-TPK1t-URA3-TPK1t；Then the W303-1A in step of converting one (yak1 △) bacterial strain competent cell is screened using uracil (URA) auxotrophic selection marker, that is, uses CMG^-URA Plate is screened, and identifies to obtain TPK1 deletion mycopremna with identical method in same embodiment 2 to obtained transformant bacterial strain W303-1A (yak1 △) (tpk1 △:: URA3), and further anti-choosing obtains W303-1A (yak1 △) (tpk1 △) bacterial strain.

Three, TPK2 and TPK3 gene is lacked

It obtains with W303-1A bacterial strain from W303-1A by mating type conversion except mating type alia gene type is identical W303-1B bacterial strain (MAT α leu2-3,112ura3-1trp1-92his3-11,15ade2-1can1-100) is with W303-1B Host sequentially knocks out two genes of TPK2, TPK3.

By the plasmid pUC18-TPK2p-TPK2t-URA3-TPK2t constructed in embodiment 1 the bis- enzyme enzymes of Sac I and Pst I It cuts, obtains linear DNA fragments TPK2p-TPK2t-URA3-TPK2t；Then transformed saccharomyces cerevisiae W303-1B bacterial strain competence Cell, in embodiment 1 " W303-1A (tpk2 △) strain construction " method screening, identification obtain W303-1B (tpk2 △:: URA3) bacterial strain and W303-1B (tpk2 △) bacterial strain.

By the plasmid pUC18-TPK3p-TPK3t-URA3-TPK3t constructed in embodiment 2 the bis- enzyme enzymes of Sac I and Pst I It cuts, obtains linear DNA fragments TPK3p-TPK3t-URA3-TPK3t；Then transformed saccharomyces cerevisiae W303-1B (tpk2 △) bacterium Strain competent cell obtains W303-1B with the screening of " W303-1A (tpk2 △) strain construction " method, identification in embodiment 1 (tpk2 △ tpk3 △:: URA3) bacterial strain and W303-1B (tpk2 △ tpk3 △) bacterial strain.

Four, TPK1, TPK2, TPK3, YAK1 gene are lacked simultaneously

W303-1A (tpk1 △ yak1 △) bacterial strain is hybridized with W303-1B (tpk2 △ tpk3 △) bacterial strain, obtains two Then times body bacterial strain separates allele by producing spore, tearing spore open, screening, identification obtain W303-1A (tpk1 △ tpk2 △ Tpk3 △ yak1 △) and W303-1B (tpk1 △ tpk2 △ tpk3 △ yak1 △).Identify that the verifying primer of four gene knockouts is same Before.Four gene nucleic acid sequences in two bacterial strains on chromosome, which occur, to be proved to the sequencing result of identification gained PCR product Expected variation, has all been lacked the area ORF.

Five, the growth and the extracellular cAMP evaluation of fermenting and producing of four gene deletion strains

Growth and the extracellular cAMP evaluation of fermenting and producing have been carried out to aforementioned four gene deletion strains, operated as follows:

1, seed liquor culture: the bacterium colony grown in picking YPAD solid medium tablets, access are cultivated equipped with 5mL YPAD In the test tube of liquid, 30 DEG C, 220rpm be incubated overnight, carry out secondary expansion culture when necessary；

2, it ferments: fermentation culture medium: yeast extract 10g/L, peptone 20g/L, glucose 20g/L, natural ph； Fresh seeds liquid is inoculated into the 100ml volumetric flask equipped with 25ml fermentation medium, initial OD is controlled₆₀₀Value 0.1 or so, 30 DEG C, ferment under 220rpm；

3, the OD of fermentation liquid₆₀₀Measurement: OD is measured after fermented sample is suitably diluted₆₀₀, detect growing state；

4, HPLC analyze fermented supernatant fluid cAMP concentration, that is, extracellular cAMP concentration: HPLC analyze cAMP method referring to " in Magnificent people's republic's pharmacopeia: version in 2010 " (Chinese Pharmacopoeia Commission's volume) page 419 method and have adjustment.Concrete operations are such as Under: 1) fermented sample 13000rpm, 1min under the conditions of is centrifuged, supernatant is taken suitably to be diluted, with 0.22 μm of the filter in aperture Film filtering, filtrate are detected for chromatography: Detection wavelength 258nm, Thermo Syncronis C18 chromatographic column, mobile phase are (5.78g/L KH₂PO₄, 2.72g/L tetrabutylammonium bromide): acetonitrile=85:15With phosphorus acid for adjusting pH to 4.3, flow velocity 1mL/ Min, 35 DEG C of column temperature；2) it the configuration of cAMP standard sample and the measurement of standard curve: will first be gone after the sterilizing of cAMP standard items Ionized water is configured to concentration and is the standard solution of 50mmol/L, then is diluted with deionized water to it, obtains final concentration difference For the standard sample of 1,3,5,7.5 and 10 μm of ol/L, with HPLC analysis is carried out after 0.2 μm of membrane filtration standard sample, with peak face Product does standard curve to cAMP concentration；3) the dense of cAMP in fermentation broth sample is calculated by outer marking quantitative method using standard curve Degree.

The extracellular cAMP evaluation result of growth and fermenting and producing in 96h is shown in Table 3.

The growth of 3, four gene deletion strains of table and cAMP production evaluation

Strain name	Maximum OD₆₀₀	The extracellular cAMP concentration of highest (μm ol/L)
			W303-1A	21.1(48h)	1.39(60h)
W303-1A(tpk1△tpk2△tpk3△yak1△)	35.0(48h)	200.2(24h)
			W303-1B(tpk1△tpk2△tpk3△yak1△)	33.5(48h)	215.6(24h)

As shown in Table 3: the extracellular cAMP concentration of maximum of two deletion mycopremnas is that control strain W303-1A maximum is extracellular respectively 144.0 and 155.1 times of cAMP concentration.It should be understood that two deletion mycopremnas cAMP concentration in 0-24h increases rapidly, After reaching peak for 24 hours, cAMP concentration is declined slightly, but stills remain in 180 μm of ol/L or more when 96h.

Embodiment 5: building while the yeast strain for lacking TPK1, TPK2, TPK3, YAK1, PDE1 gene

One, PDE1 gene is lacked

PCR is carried out by template of W303-1A chromosome, expands the terminator district (referred to as PDE1t) of PDE1 gene, is used Sequence shown in SEQ ID No.27, SEQ ID No.28 in table 2 be upstream and downstream primer P27, P28, Quan Shi King Company production Fast Pfu polymerase, 50 DEG C of annealing 1min, 72 DEG C of extension 0.5min, totally 32 recycle.The PCR fragment of 500bp is obtained, is used Turn after bis- this segment of enzyme digestion of restriction enzyme KpnI and BamHI and the connection of vector plasmid pUC18-URA3, T4DNA ligase Change Escherichia coli Top10 competent cell, extracts transformant plasmid and carry out digestion identification, obtain plasmid pUC18-PDE1t- URA3。

PCR is carried out by template of W303-1A chromosome, expands the promoter region (referred to as PDE1p) of PDE1 gene, is used Sequence shown in SEQ ID No.29, SEQ ID No.30 in table 2 be upstream and downstream primer P29, P30, Quan Shi King Company production Fast Pfu polymerase, 50 DEG C of annealing 1min, 72 DEG C of extension 0.5min, totally 32 recycle.The PCR fragment of 544bp is obtained, is used Bis- this segment of enzyme digestion of restriction enzyme Sac I and Kpn I and vector plasmid pUC18-PDE1t-URA3, T4DNA ligase It is ligated and transformed into Escherichia coli Top10 competent cell, transformant plasmid is extracted and carries out digestion identification, obtain plasmid pUC18- PDE1p-PDE1t-URA3。

By the bis- enzyme digestions of Sal I and Pst I of the PCR product of the sequence containing PDE1t of above-mentioned amplification, with same double enzyme digestions PUC18-PDE1p-PDE1t-URA3 large fragment connection, obtain plasmid pUC18-PDE1p-PDE1t-URA3-PDE 1t.

By the plasmid pUC18-PDE1p-PDE1t-URA3-PDE 1t bis- enzyme digestions of Sac I and Pst I, with embodiment 1 In identical method in " W303-1A (tpk2 △) strain construction ", with W303-1A (the tpk1 △ tpk2 △ constructed in embodiment 4 Tpk3 △ yak1 △) be host, building obtain W303-1A (tpk1 △ tpk2 △ tpk3 △ yak1 △ pde1 △:: URA3) and W303-1A(tpk1△tpk2△tpk3△yak1△pde1△).PCR method verifying is with the SEQ ID that primer pair is in table 2 Sequence shown in No.31, SEQ ID No.32 is upstream and downstream primer P31, P32, successful integration, the transformant containing URA3 gene PCR product is 2958bp, and successful integration, the transformant PCR product of URA3 gene pop-up are 1445bp, control strain PCR product For 2635bp.1445bp PCR product is sequenced, sequencing result proves that expected variation has occurred: PDE1 gene rises 46 bases before beginning codon after 40 bases, the entire area ORF and terminator codon are lacked completely.In this way, final Obtain the positive transformant bacterial strain of PDE1 gene delection, the pop-up of URA3 gene.

Two, W303-1A (tpk1 △ tpk2 △ tpk3 △ yak1 △ pde1 △:: URA3) and W303-1A (tpk1 △ tpk2 △ tpk3 △ yak1 △ pde1 △) cAMP produce comparison

Selectable marker gene URA3 is investigated for comparison to exist on the possible influence of cAMP production, has carried out two bacterial strains CAMP produce preliminary assessment.1, seed liquor culture is the same as embodiment 4；2, it ferments: 1) fermentation culture medium: yeast extract 10g/ L, peptone 20g/L, glucose 20g/L, natural ph；2) fermentation condition are as follows: fresh seeds liquid is inoculated into ferment equipped with 25ml In the 100ml volumetric flask of culture medium, initial OD is controlled₆₀₀Value is fermented at 0.1 or so, 30 DEG C, 220rpm.Fermentation liquid analysis is same HPLC analysis method in embodiment 4.Extracellular cAMP production result in 120h is shown in Fig. 2.Fig. 2 is as the result is shown: 1, two missings The extracellular cAMP concentration of maximum in bacterial strain 120h is respectively 252.0,953.8 μm of ol/L, and respectively control strain W303-1A is maximum 173.8,657.8 times of extracellular cAMP concentration (1.45 μm of ol/L) are the inner W303-1B of embodiment 4 (tpk1 △ tpk2 △ tpk3 △ Yak1 △) 1.17,4.42 times of the maximum extracellular cAMP concentration of bacterial strain；2,W303-1A(tpk1△tpk2△tpk3△yak1△ Pde1 △:: cAMP concentration 48h URA3) are basicly stable, and W303-1A (tpk1 △ tpk2 △ tpk3 △ yak1 △ pde1 CAMP concentration △) then rises always；3, cAMP yield can be significantly improved by not retaining selected marker URA3.

Three, the growth of W303-1A (tpk1 △ tpk2 △ tpk3 △ yak1 △ pde1 △) and cAMP production evaluation

It has carried out without the growth of five gene deletion strains of URA3 and the extracellular cAMP evaluation of fermenting and producing: 1, seed liquor Culture is the same as embodiment 4；2, it ferments: 1) fermentation culture medium: yeast extract 10g/L, peptone 20g/L, glucose 20~ 150g/L, natural ph；2) fermentation condition are as follows: fresh seeds liquid is inoculated into the 100ml volumetric flask equipped with 25ml fermentation medium In, control initial OD₆₀₀Value is fermented at 1 or so, 30 DEG C, 220rpm.Fermentation liquid is analyzed with the analysis side HPLC in embodiment 4 Method.The extracellular cAMP evaluation result of growth and fermenting and producing in 120h is shown in Table 4.

The growth of 4, five gene deletion strains of table and cAMP production evaluation

Concentration of glucose (g/L)	Maximum OD₆₀₀	The extracellular cAMP concentration of highest (μm ol/L)
			20	31.1(24h)	981.0(48h)
50	35.0(48h)	2223.7(72h)
			100	63.7(72h)	3596.6(96h)
150	69.5(72h)	3925.6(96h)

According to the above results: the carbon source in culture medium can significantly affect the yield of extracellular cAMP, as glucose is dense Degree rises to 150g/L by 20g/L, and the yield of extracellular cAMP also rises to 3925.6 μm of ol/L by 981.0 μm of ol/L.

Embodiment 6:BAS1-BAS2 fusion coexpression

BAS1 and BAS2 gene is subjected to fusion coexpression, and is integrated on the chromosome of yeast cells.It needs first thus Construct the fusion coexpression integration vector with left and right homology arm.Here the integration site selected is by document report (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) there is YNRC Δ 9 compared with high gene expression efficiency, it is located at chromosome On XIV.

One, BAS1-BAS2 merges co-expression plasmid building

Totally 4 connections obtain BAS1-BAS2 fusion co-expression plasmid pUC18-H1_YNRCΔ9-BAS1-BAS2-H2_YNRCΔ9- URA3-H2_YNRCΔ9(schematic diagram is shown in Fig. 3).

1、pUC18-H1_YNRCΔ9-SacI-SalI-H2_YNRCΔ9Plasmid construction

It is left and right as what is integrated as the partial sequence of template progress PCR amplification YNRC Δ 9 using the chromosomal DNA of W303-1A Homology arm.It the use of sequence shown in SEQ ID No.33, the SEQ ID No.34 in table 2 is upstream and downstream primer P33, P34, full formula The Fast Pfu polymerase of King Company's production, 50 DEG C of annealing 1min, 72 DEG C of extension 45sec, totally 32 circulations, obtain 521bp's (corresponding sequence is used as the left homology arm H1 of integration to PCR1 segment_YNRCΔ9).Use SEQ ID No.35, the SEQ ID in table 2 Sequence shown in No.36 is upstream and downstream primer P35, P36, and expanding to obtain the PCR2 segment of 448bp with method, (corresponding sequence is used as whole The right homology arm H2 closed_YNRCΔ9).Again using PCR1, PCR2 product as template, using P33, P36 as primer pair, juxtaposition extension PCR Reaction amplification obtains the PCR12 segment of 949bp.5 ' ends of this segment are added with restriction enzyme site EcoRI, and 3 ' ends are added with digestion position Point BamHI, centre are added with restriction enzyme site SacI, Sal I.By the PCR product bis- enzyme digestions of EcoRI and BamHI, with same pair The pUC18 large fragment of enzyme digestion connects, and converts Escherichia coli Top10 competent cell, extracts transformant plasmid and carries out digestion mirror It is fixed, obtain plasmid pUC18-H1_YNRCΔ9-SacI-SalI-H2_YNRCΔ9.Sequencing proves that cloned sequence is not mutated.

2、pUC18-H1_YNRCΔ9-SacI-SalI-H2_YNRCΔ9-URA3-H2_YNRCΔ9Plasmid construction

PCR amplification URA3 gene is carried out as selectable marker gene when integrating by template of YCplac33.Using in table 2 SEQ ID No.37, SEQ ID No.38 shown in sequence be upstream and downstream primer P37, P38, Quan Shi King Company production Fast Pfu polymerase, 50 DEG C of annealing 1min, 72 DEG C of extension 1min, totally 32 circulations, obtain the PCR1 segment of 1060bp.Still with W303- The chromosomal DNA of 1A is that template carries out PCR amplification H2_YNRCΔ9Segment, but upstream and downstream primer pair changes the SEQ ID in table 2 into Sequence shown in No.39, SEQ ID No.40 is P39, P40, and amplification obtains the PCR2 segment of 438bp.Again with PCR1, PCR2 product For template, using P37, P40 as primer pair, the reaction amplification of juxtaposition extension PCR obtains the PCR12 segment of 1478bp.This segment 5 ' end be added with restriction enzyme site BamHI, 3 ' end be added with restriction enzyme site HindIII.By PCR product BamHI and HindIII Double enzyme digestions, the pUC18-H1 with same double enzyme digestions_YNRCΔ9-SacI-SalI-H2_YNRCΔ9Large fragment connection, converts large intestine bar Bacterium Top10 competent cell extracts transformant plasmid and carries out digestion identification, obtains plasmid pUC18-H1_YNRCΔ9-SacI-SalI- H2_YNRCΔ9-URA3-H2_YNRCΔ9.Sequencing proves that cloned sequence is not mutated.

3, pGEM-T easy-BAS1-BAS2 plasmid construction

PCR amplification gene BAS1 is carried out by template of the chromosomal DNA of W303-1A, uses the SEQ ID in table 2 Sequence shown in No.41, SEQ ID No.42 is upstream and downstream primer P41, P42, the Fast Pfu polymerization of Quan Shi King Company production Enzyme, 50 DEG C of annealing 1min, 72 DEG C of extension 2.5min, totally 32 recycle.Obtain the PCR1 segment of 2907bp, the addition of this segment 5 ' end There is restriction enzyme site SacI, includes BAS1 gene start codon ATG upstream 762bp sequence and the 2112bp including ATG ORF sequence deletes 3 ' end 324bp (containing terminator codon) sequences of ORF.

PCR amplification gene BAS2 is carried out by template of the chromosomal DNA of W303-1A, uses the SEQ ID in table 2 Sequence shown in No.43, SEQ ID No.44 is upstream and downstream primer P43, P44, the Fast Pfu polymerization of Quan Shi King Company production Enzyme, 50 DEG C of annealing 1min, 72 DEG C of extension 1.5min, totally 32 recycle.The PCR2 segment of 1692bp is obtained, 3 ' ends are added with enzyme Enzyme site Sal I；Include BAS2 gene ORF full sequence (containing terminator codon).

Using PCR1, PCR2 product as template, using P41, P44 as primer pair, the reaction amplification of juxtaposition extension PCR is obtained The PCR12 segment of 4578bp.This PCR fragment is carried out after adding A end reaction, agarose gel electrophoresis separation, gel extraction, normally Bright book operation requires to connect with the pGEM-T easy carrier purchased from Promega company, and conversion Escherichia coli Top10 competence is thin Born of the same parents extract transformant plasmid and carry out digestion identification, obtain plasmid pGEM-T easy-BAS1-BAS2.Sequencing proves cloned sequence It is not mutated.

4、pUC18-H1_YNRCΔ9-BAS1-BAS2-H2_YNRCΔ9-URA3-H2_YNRCΔ9Plasmid construction

With the bis- enzyme digested plasmid pGEM-T easy-BAS1-BAS2 of SacI, Sal I, large fragment SacI-BAS1- is recycled BAS2-Sal I, the pUC18-H1 with same double enzyme digestions_YNRCΔ9-SacI-SalI-H2_YNRCΔ9-URA3-H2_YNRCΔ9Large fragment connects It connects, obtains integrated plasmid pUC18-H1_YNRCΔ9-BAS1-BAS2-H2_YNRCΔ9-URA3-H2_YNRCΔ9。

Two, bacterial strain W303-1A (tpk1 △ tpk2 △ tpk3 △ yak1 △ pde1 △ BAS1BAS2) is constructed

By plasmid pUC18-H1_YNRCΔ9-BAS1-BAS2-H2_YNRCΔ9-URA3-H2_YNRCΔ9With PvuII digestion, obtain linear Change DNA fragmentation H1_YNRCΔ9-BAS1-BAS2-H2_YNRCΔ9-URA3-H2_YNRCΔ9；Then W303-1A (tpk1 △ tpk2 △ is converted Tpk3 △ yak1 △ pde1 △) bacterial strain competent cell, is sieved with " W303-1A (tpk2 △) strain construction " method in embodiment 1 Choosing, identification obtain W303-1A (tpk1 △ tpk2 △ tpk3 △ yak1 △ pde1 △ BAS1BAS2-URA3) bacterial strain and W303-1A (tpk1 △ tpk2 △ tpk3 △ yak1 △ pde1 △ BAS1BAS2) bacterial strain.PCR method verifying is with the SEQ that primer pair is in table 2 Sequence shown in ID No.45, SEQ ID No.46 is upstream and downstream primer P45, P46, successful integration, the conversion containing URA3 gene Sub- PCR product is 7389bp, and successful integration, the transformant PCR product of URA3 gene pop-up are 5929bp, and control strain PCR is produced Object is 1687bp.The sequencing result of 5929bp PCR product is proved expected variation: 9 site of YNRC Δ has occurred on chromosome There is 321bp sequence to be knocked, substitute insertion is BAS1BAS2 fusion segment, and there is no mutation for this segment；Without URA3 base Cause.

Three, the growth of bacterial strain W303-1A (tpk1 △ tpk2 △ tpk3 △ yak1 △ pde1 △ BAS1BAS2) and cAMP are raw Produce evaluation

Embodiment 5 has been proven that concentration of glucose significantly affects the production of cAMP.Component needed for being synthesized in view of cAMP (point Minor is C₁₀H_l2N₅O₆P), while concentration of glucose improves, thus it is speculated that nitrogen in culture medium, phosphorus content especially nitrogen content It is likely to become the primary restraining factors of cAMP synthesis.In addition, the preceding extract as cAMP synthesis, the situation of extracellular adenine also pole The earth influences the expression of each gene of purine route of synthesis, and then influences cAMP production.Therefore, comparison here has been investigated above-mentioned The cAMP condition of production of the bacterial strain under two yeast extracts/peptone contents level (1*YP, 2*YP), addition adenine.Tool Gymnastics is made as follows: 1, seed liquor culture is the same as embodiment 4；2, it ferments: 1) fermentation culture medium: yeast extract 10g/L, peptone 20g/L is abbreviated as 1*YP, glucose 150g/L, natural ph；Yeast extract 20g/L, peptone 40g/L, is abbreviated as 2* YP, glucose 150g/L, natural ph；The additive amount of adenine is 0.625,1.25g/L, referred to as A0.625, A1.25；2) Fermentation condition are as follows: fresh seeds liquid is inoculated into the 100ml volumetric flask equipped with 25ml fermentation medium, controls initial OD₆₀₀Value exists 1 or so, 30 DEG C, ferment under 220rpm；3) fermentation liquid is analyzed: with the HPLC analysis method in embodiment 4, while supplementing adenine HPLC analysis: a, sample treatment and HPLC analysis it is identical as the cAMP sample treatment analyzed and HPLC analysis；B, adenine mark Directrix curve production: the deionized water after the sterilizing of adenine standard items is configured to the standard solution that concentration is 5mg/mL, then is used Deionized water is diluted it, obtains the standard sample that final concentration is respectively 0.1,0.2,0.3,0.4,0.5mg/mL, crosses and filters out Chromatography is carried out after bacterium, does standard curve；C, to calculate gland in fermentation broth sample by outer marking quantitative method using standard curve fast The concentration of purine.

Fermentation results are shown in Table 5.Table 5 the result shows that: 1, be all 1*YP, glucose 15% fermentation condition under, merge altogether table Up to the maximum extracellular cAMP concentration of BAS1-BAS2 bacterial strain compared with W303-1A (the tpk1 △ tpk2 △ tpk3 △ yak1 △ in embodiment 5 Pde1 △) the maximum extracellular cAMP concentration (3925.6 μm of ol/L) of bacterial strain increase, and the former is 1.108 times of the latter；2, it improves Yeast powder and peptone concentration effect are extremely significant；3, addition adenine can be further improved extracellular cAMP yield.

The cAMP production of table 5, W303-1A (tpk1 △ tpk2 △ tpk3 △ yak1 △ pde1 △ BAS1-BAS2) bacterial strain is commented Valence

Component	Maximum OD₆₀₀	Highest cAMP concentration (μm ol/L)	Ratio	Ratio
					1*YP, glucose 150g/L	70.1(72h)	4348.8(120h)	1	—
2*YP, glucose 150g/L	71.6(96h)	8291.5(168h)	1.907	1
					2*YP, glucose 150g/L, A0.625	68.4(120h)	9265.3(168h)	2.131	1.117
2*YP, glucose 150g/L, A1.25	64.4(120h)	9721.6(168h)	2.235	1.172

Embodiment 7:FCY2 is overexpressed

Table will be carried out on the chromosome of FCY2 gene integration to yeast cells by yeast integration plasmid YIplac211 It reaches.Using W303-1A chromosomal DNA as template, 558bp, gene promoter containing PGK1 are synthesized by primer pair of primer P47 and P48 Area P_PGK1Product PCR1, using primer P49 and P50 as primer pair synthesize 1630bp, the gene containing FCY2 product PCR2, with P51 It is that primer pair synthesizes 481bp, the sub-district of gene end containing PGK1 T with P52_PGK1Product PCR3.It is with PCR1 and PCR2 segment again Template, using P47 and P50 as primer pair, juxtaposition extend amplification obtains product PCR12.Finally it is with PCR12 and PCR3 segment Template, using P47 and P52 as primer pair, amplification obtain product PCR123.By the product PCR123 bis- enzyme digestions of SphI and EcoRI Afterwards, it is connect with the carrier YIplac211 large fragment of same double enzyme digestions, obtains the plasmid YIplac211-P of 6.3kb size_PGK1- FCY2-T_PGK1.The sequencing result of this plasmid proves cloned sequence P_PGK1-FCY2-T_PGK1It is not mutated, with StuI linearization for enzyme restriction The bacterial strain W303-1A in Li-acetate method conversion embodiment 5 (tpk1 △ tpk2 △ tpk3 △ yak1 △ pde1 △) competence is thin afterwards Born of the same parents use CMG^-URAPlate is screened, and is carried out YPAD Liquid Culture to obtained transformant bacterial strain, is extracted chromosome, with P47, P52 primer pair carries out PCR identification, and the expected size of product is 2.6kb.Fermentation evaluation is carried out to obtained positive transformant bacterial strain: 1, seed liquor culture is the same as embodiment 4；2, ferment: fermentation culture medium in embodiment 6 (2*YP, glucose 150g/L, A0.625), fermentation condition is the same as embodiment 6；CAMP maximum output reaches 10197.8 μm of ol/L.

Embodiment 8:SNQ2 is overexpressed

Table will be carried out on the chromosome of SNQ2 gene integration to yeast cells by yeast integration plasmid YIplac211 It reaches.Using W303-1A chromosomal DNA as template, 4527bp, the ORF sequence of gene containing SNQ2 are synthesized by primer pair of primer P53 and P54 The PCR product of column is cut with SalI and BamHI are bis-, then with the 7 plasmid YIplac211-P of embodiment of same double enzyme digestions_PGK1- FCY2-T_PGK1Large fragment connection.The sequencing result of this plasmid proves that cloned sequence is not mutated, with vinegar after StuI linearization for enzyme restriction Bacterial strain W303-1A (tpk1 △ tpk2 △ tpk3 △ yak1 △ pde1 △ BAS1BAS2) impression in sour lithium method conversion embodiment 6 State cell, uses CMG^-URAPlate is screened, and is carried out YPAD Liquid Culture to obtained transformant bacterial strain, is extracted chromosome, uses P47, P52 primer pair in embodiment 6 carries out PCR identification, and the expected size of product is 5.5kb.To obtained positive transformants daughter bacteria Strain carries out fermentation evaluation: 1, seed liquor culture is the same as embodiment 4；2, ferment: fermentation culture medium is the same as (2*YP, the Portugal in embodiment 6 Grape sugar 100g/L, A 0.625), fermentation condition is the same as embodiment 6；CAMP maximum output reaches 11581.6 μm of ol/L.

Embodiment 9:cAMP produces bacterial strain fermentation liquor pulvis and raises small white mouse

Bacterial strain in embodiment 5 is activated step by step by the method in embodiment 6, amplifies culture and shake flask fermentation, hair Ferment culture medium is (2*YP, glucose 100g/L).Take the drying of 168h fermentation liquid directly freezed that pulvis is made.It is raised using experimental animal Material is used as daily ration, and pulvis is dissolved in drinking-water feeding small white mouse.Small white mouse is randomly divided into three groups, and every group 8, free water and feeding Start to add two days later；Additive amount is successively are as follows: 0,0.5,10mg cAMP/ (kg weight), addition in every 2 days is once and progress is primary Weighing is added 10 times altogether；Then elder generation fasting >=10h before adding is weighed, is recorded weight and calculate addition pulvis amount, single group uses Pulvis is dissolved in 20mL drinking-water.Blood sampling sampling carries out serum detection after last time is weighed, and internal organ are dissected and observed.As the result is shown: 1, increase weight: the 20th day average weight is respectively 37.54 ± 1.89g, 41.41 ± 0.48g and 40.12 ± 1.28g, and addition group is average 10.20% and 6.87%, dosage 0.5mg cAMP/ (kg weight) significant difference (P is respectively increased compared with control group average weight in weight <0.05)；2, toxicity: the dissection and analysis of small white mouse as the result is shown: maximum dose 10mg cAMP/ (kg weight) group and control group phase Than the internal organs such as heart, liver, spleen, lung, kidney see pathological change without obvious eye, show that pulvis does not have small white mouse Significant acute toxicity；3, serum analysis is as the result is shown: total protein average content is respectively 49.90 ± 1.34,57.50 ± 1.85 With 56.02 ± 1.05g/L, albumin average content is respectively 29.52 ± 0.63,34.25 ± 0.86 and 35.90 ± 0.62g/L, Sample sets are extremely significant to be higher than control group (p < 0.01), illustrates that protein in body content can really be effectively improved by adding cAMP preparation.

Embodiment 10:cAMP production bacterial strain fermentation liquor pulvis raises table hens

Bacterial strain in embodiment 6 is activated step by step by the method in embodiment 6 and amplifies culture, then in fermentor In use fermentation medium (2*YP, glucose 100g/L), 30 DEG C, ferment under 200-700rpm 120h, the direct low temperature of fermentation liquid Dry powder processed, uses as cAMP pulvis.

Young 180 plumages of 1 age in days health white meat-type chickens are selected, first carry out adaptability culture in 6 days, then each weight half-press of male and female It is randomly divided into three processing, 3 repetitions of each processing, each repetition 20.The pulvis additive amount of three processing is set to 0.5,2.5,0mgcAMP/ (kg weight).Carried out primary weighing and a cAMP pulvis addition every 5 days, front and back add altogether 7 times, Whole incubation time 42 days.Elder generation fasting >=10h and taboo water about 2h, then weighs and calculates addition pulvis amount before adding.CAMP powder Agent is directly appended in drinking-water, restores feed while supplying this drinking-water.The 25th day and the 35th day after adding for the first time (whole incubation time is respectively 32 days and 42 days) each repetition takes two chickens to be butchered, and analyzes organ index and serum refers to Mark.The Tianjin farm Tuan Bowa chicken farm carries out.Using online raising mode, domestic briquet stove heating temperature control.It is freely eaten and drinks Water.Using chicken mixed feed in the meat chicken mixed feed (0-21 age in days use) and meat of the production of Cangzhou Hong Yi feed corporation,Ltd (22-42 age in days use).

It the results are shown in Table 6, as shown in Table 6: 1, on growth performance, addition pulvis improves daily gain extremely significantly (p < 0.01), And feedstuff-meat ratio decline is also extremely significant (p < 0.01)；2, on organ index, heart, liver, stomach, intestines are not bright with control group Significant difference is different, shows that additive is small to internal organs stimulation, fanout free region；At the same time, it is significant to improve bursal index for addition pulvis (p < 0.05) shows that the development to broiler chicken immune organ has facilitation；3, on Serum Indexes, cholesterol in serum and highly dense Degree lipoprotein be all remarkably higher than control group (p < 0.05), illustrate add pulvis can promote body fat be metabolized and protein metabolism and It regulates and controls.

The influence of table 6, addition cAMP pulvis to growth of meat chicken, internal organs and Biochemical Indices In Serum

Index	Age in days	0.5mg cAMP/ (kg weight)	2.5mg cAMP/ (kg weight)	0mg cAMP/ (kg weight)
					Average daily gain g	42d	60.63±2.12^A	73.25±1.86^B	61.43±1.78^A
Average daily gain g	42d	105.96±6.46	120.82±3.81	111.49±5.50
					Feedstuff-meat ratio	42d	1.748±0.011^A	1.649±0.006^B	1.815±0.021^A
Bursal index	42d	2.89±0.31^a	3.81±0.40^b	2.93±0.38^a
					Cholesterol mmol/L	42d	3.665±0.108^a	3.666±0.165^a	3.178±0.227^b
High-density lipoprotein mmol/L	42d	2.739±0.055^a	2.653±0.125^a	2.248±0.140^b

Note: same column shoulder mark lowercase difference person indicates significant difference (P < 0.05), and capitalization difference person indicates difference Extremely significant (P < 0.01)

The fermentation by saccharomyces cerevisiae liquid or fermentation liquid that the embodiment of the present invention 4, embodiment 5, embodiment 7 and embodiment 8 are obtained Pulvis is also tested respectively, and substantially similar rule is as a result presented.

Embodiment 11:cAMP produces bacterial strain fermentation liquor pulvis and raises pig

It ferments with embodiment 10, fermented liquid spray drying powder is used as cAMP pulvis.

Healthy weanling pig 12 of 30kg weight are selected, male and female is fifty-fifty, is randomly divided into 3 groups, every group 4；3 groups of processing A, Handling B, handling the cAMP pulvis additive amount of C is respectively 0.75,1.5 and 0mg cAMP/ (kg weight).It was carried out every 5 days primary Weighing and a cAMP pulvis addition, until weight terminates when reaching 90kg.CAMP pulvis is directly appended in drinking-water, control before adding Pig empty stomach >=10h processed simultaneously weighs.Basal diet is fed, be freely eaten and is drunk water.As the result is shown: 1, three groups of daily gains are respectively 0.715 ± 0.057,0.770 ± 0.063 and 0.699 ± 0.139kg/d, processing A, B respectively relatively processing C improve 2.23%, 10.16%, and handle B and be significantly higher than processing C (p < 0.05)；2, three groups of feedstuff-meat ratios be respectively 2.95 ± 0.11,2.690 ± Relatively processing C reduces by 1.99%, 10.63% respectively by 0.09 and 3.01 ± 0.23kg/d, processing A, B, and handles B and substantially less than handle C (p < 0.05).As a result prove that yeast cAMP fermentation liquid pulvis is remarkably improved the growth performance of pig.

Using fermentation by saccharomyces cerevisiae liquid or fermentation liquid pulvis of the invention as the feed of ruminant animals and aquatic product animal Additive also observed similar result.

Sequence table

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<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 11

cccggggagc tcctccgtta atcctagtct gt 32

<210> 12

<211> 32

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 12

cccgggggta ccttctgtgc tacctttgaa gc 32

<210> 13

<211> 20

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 13

atccctccca tcctccttaa 20

<210> 14

<211> 20

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 14

gaaggagccg cagcattatt 20

<210> 15

<211> 38

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 15

cccggggtcg acggtaccga ttcttggtga gtctaaca 38

<210> 16

<211> 38

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 16

cccgggctgc agggatcctg gaacgctttt ttgcttgt 38

<210> 17

<211> 32

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 17

cccggggagc tcgtcaacgt ttcagatact ct 32

<210> 18

<211> 32

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 18

cccgggggta cctttgtgca ggctcgctct tt 32

<210> 19

<211> 22

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 19

gcgactatgc atttttgcaa aa 22

<210> 20

<211> 20

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 20

cggagccttc atgagataaa 20

<210> 21

<211> 38

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 21

cccggggtcg acggtaccaa agtttctgca ctagcttt 38

<210> 22

<211> 38

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 22

cccgggctgc agggatccag agagaggacc catggaat 38

<210> 23

<211> 32

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 23

cccggggagc tcctttcgcc ctcaaactca ac 32

<210> 24

<211> 32

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 24

cccgggggta ccatgttccc ttgcacaatg gc 32

<210> 25

<211> 21

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 25

caatacggat gaatatttgt g 21

<210> 26

<211> 20

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 26

acttttgatt gcgctgtgaa 20

<210> 27

<211> 38

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 27

cccggggtcg acggtaccaa attcattacc cgggagca 38

<210> 28

<211> 38

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 28

cccgggctgc agggatccat gctggaaccc tcatatga 38

<210> 29

<211> 32

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 29

cccggggagc tccagacata tagtctcgaa ga 32

<210> 30

<211> 32

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 30

cccgggggta cccctcgtta aaagccactt tc 32

<210> 31

<211> 19

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 31

aatcttactt tggcgaatg 19

<210> 32

<211> 21

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 32

acactatttc cttgttcata c 21

<210> 33

<211> 30

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 33

gcgcgcgaat tccgtattga ccattcctaa 30

<210> 34

<211> 39

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 34

tcgactcccg ggtaccgagc tctatcgtat cgcagccta 39

<210> 35

<211> 38

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 35

gctcggtacc cgggagtcga ctgtagacct aagttcat 38

<210> 36

<211> 30

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 36

atattaggat ccgccttgct tacctagatg 30

<210> 37

<211> 28

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 37

gcgttaggat ccgattcggt aatctccg 28

<210> 38

<211> 26

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 38

aggtctacag gggtaataac tgatat 26

<210> 39

<211> 28

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 39

gttattaccc ctgtagacct aagttcat 28

<210> 40

<211> 30

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 40

cgtacgaagc ttgccttgct tacctagatg 30

<210> 41

<211> 32

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 41

gggcccgagc tctctttagc cgtaattgcg aa 32

<210> 42

<211> 42

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 42

gtacgagaat tcttccatca tggatgtagt ccttgatatc tc 42

<210> 43

<211> 21

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 43

atgatggaag aattctcgta c 21

<210> 44

<211> 33

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 44

gggcccgtcg actcatatcc atctatgctc gtc 33

<210> 45

<211> 19

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 45

tcaactttgg gattactgc 19

<210> 46

<211> 19

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 46

ctcatcattt gcgtcatct 19

<210> 47

<211> 34

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 47

gggcccgcat gcctgcagaa gaaattaccg tcgc 34

<210> 48

<211> 30

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 48

acatgtcgac agacattgtt ttatatttgt 30

<210> 49

<211> 34

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 49

aacaatgtct gtcgacatgt tggaagaggg aaat 34

<210> 50

<211> 30

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 50

tttaggatcc tcctaacgac cgaagtattt 30

<210> 51

<211> 36

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 51

gtcgttagga ggatcctaaa ttgaattgaa ttgaaa 36

<210> 52

<211> 31

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 52

gggcccgaat tcgagctcgg tacccgggga c 31

<210> 53

<211> 32

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 53

gggcccgtcg acatgagcaa tatcaaaagc ac 32

<210> 54

<211> 32

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 54

gggcccggat ccctgcttct ttttccttat gt 32

Claims

1. a primary yeast, which is characterized in that the yeast includes the first, second of gene and the third gene modification, wherein The first described gene be protein kinase A (protein kinase A, PKA) catalytic subunit encoding gene TPK1, TPK2 and TPK3 is totally constrained PKA activity or expression by modifying the first gene, to eliminate to cyclic adenosine monophosphate (cyclic Adenosine monophosphate, cAMP) feedback inhibition, but make simultaneously yeast growth be suppressed, second of gene Modification eliminate growth inhibition caused by the first gene modification so that yeast can normal growth, the yeast CAMP yield rises, and second of gene includes that protein kinase Rim15 encoding gene RIM15, transcription factor Msn1/Msn2 are compiled Code gene M SN1/MSN2, protein kinase Yak1 encoding gene YAK1 and/or protein kinase Sch9 encoding gene SCH9, described the Three kinds of genes are cAMP di-phosphate ester enzyme coding gene PDE1 and/or PDE2, the third described gene modification makes modified base Because the activity of codase or expression are totally constrained, the degradation of cAMP is reduced, to improve cAMP yield, wherein the cAMP Yield rises, and is for the cAMP yield of the yeast without gene modification.

2. yeast according to claim 1, which is characterized in that second of gene is YAK1.

3. yeast according to claim 1, which is characterized in that second of gene modification encodes institute's modifier The activity of enzyme or expression are totally constrained or the activity of codase is improved or is overexpressed.

4. yeast according to claim 1, which is characterized in that the third described gene is PDE1.

5. yeast according to claim 1, which is characterized in that the yeast further includes the 4th kind of gene modification, to increase The positive regulation of the synthesis of cAMP precursor in purine route of synthesis rises the synthesis of cAMP precursor, to improve cAMP production Amount.

6. yeast according to claim 5, which is characterized in that the 4th kind of gene includes transcription factor Bas1 and Bas2 Encoding gene.

7. yeast according to claim 5, which is characterized in that the 4th kind of gene modification makes modifier codase Activity is improved or is overexpressed.

8. -7 any yeast according to claim 1, which is characterized in that the yeast further includes the 5th kind of gene modification, To increase the regulation of outer transport and accumulation intracellular to cAMP and its precursor, so that cAMP is synthesized and is secreted and rise, to improve CAMP yield.

9. yeast according to claim 8, which is characterized in that the 5th kind of gene includes that plasma membrane carrier protein Fcy2 is compiled Code gene and Snq2 encoding gene.

10. yeast according to claim 8, which is characterized in that the 5th kind of gene modification makes modifier codase Activity improve or be overexpressed.

11. yeast according to claim 1, which is characterized in that the activity for making codase or expression are pressed down completely The gene modification mode of system include point mutation, missing, insertion, antisense polynucleotides, siRNA, microRNA, CRISPR；So that the gene modification mode that the activity of codase is improved or is overexpressed includes point mutation, connection strong promoter, connects It connects enhancer, improve copy number or fusion coexpression.

12. yeast according to claim 1, which is characterized in that the yeast is saccharomyces cerevisiae (Saccharomyces Cerevisiae), saccharomyces pastorianus (Saccharomyces pastorianus), pichia stipitis (Pichia ), stipitis any one in Saccharomyces Bayanus and shehatae candida (Candida shehatae) Kind.

13. yeast according to claim 12, which is characterized in that the yeast is saccharomyces cerevisiae (Saccharomyces cerevisiae)。

14. a kind of method of any yeast of building claim 1-13, which is characterized in that the described method includes: first The first gene modification is introduced in yeast, eliminates the feedback inhibition to cAMP, but is suppressed the growth of yeast, It is secondary, second of gene modification is introduced, second of gene modification eliminates growth inhibition caused by the first gene modification, to make Yeast can normal growth, third introduces the third gene modification, reduces the degradation of cAMP, to improve cAMP yield.

15. a kind of method of the yeast production cAMP any using claim 1-13, which is characterized in that the method includes Above-mentioned yeast is cultivated in fermentation medium.

16. a kind of fermentation liquid of the yeast fermenting and producing any using claim 1-13.

17. use of the fermentation liquid in production cAMP described in claim the 1-13 any yeast or claim 16 On the way.

18. fermentation liquid described in claim the 1-13 any yeast or claim 16 is in medicine, animal husbandry, food The application of product, health care product or chemical field.

19. application as claimed in claim 18, which is characterized in that the application is the feed or feed addition as animal husbandry Agent.

20. a kind of method for the product for preparing medicine, animal husbandry, food, health care product or chemical field, which is characterized in that described Method prepares the said goods including the use of fermentation liquid described in claim 1-13 any yeast or claim 16.

21. method as claimed in claim 20, which is characterized in that the method includes by the yeast or fermentation liquid into one Step is processed into any other dosage form.

22. method as claimed in claim 21, which is characterized in that the dosage form includes edible dosage form.

23. the method as described in claim 21 or 22, which is characterized in that the dosage form be pulvis, granule, tablet, capsule, Or liquid form.

24. a kind of feed or feed addictive, which is characterized in that the feed or feed addictive include claim 1- Fermentation liquid described in the 13 any yeast or claim 16.

25. feed as claimed in claim 24 or feed addictive, which is characterized in that the yeast or fermentation liquid are into one Step is processed into any other dosage form.

26. feed as claimed in claim 25 or feed addictive, which is characterized in that the dosage form is edible dose Type.

27. feed or feed addictive as described in claim 25 or 26, which is characterized in that the dosage form be pulvis, Granula, tablet, capsule or liquid form.