CN107312723A

CN107312723A - The yeast strain of high yield cAMP a kind of and its application

Info

Publication number: CN107312723A
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: 2017-11-03
Anticipated expiration: 2037-08-22
Also published as: CN107312723B

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 products or chemical field.The yeast strain includes the first and second of genetic 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, so as to eliminate to CAMP (cyclic adenosine monophosphate, cAMP feedback inhibition), but cause the growth of yeast to be suppressed simultaneously, second of genetic modification eliminates growth inhibition caused by the first genetic modification, so that yeast being capable of normal growth, the cAMP yield of the yeast rises.The yeast strain also include the third, the 4th kind of genetic modification and/or the 5th kind of genetic modification.The restructuring yeast strains of the present invention can be stablized, continue, efficiently produce extracellular cAMP.

Description

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

Technical field

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

Background technology

CAMP (cyclic adenosine monophosphate, abbreviation cAMP) is widely present in human body A kind of important substance with physiologically active, its as intracellular second messenger, to glycometabolism, fat metabolism, nucleic acid synthesis, Protein synthesis etc. plays important adjustment effect.Current cAMP is widely used in terms of medicine：Clinically cAMP is used to treat Angina pectoris, myocardial infarction, myocarditis and cardiogenic shock etc.；To the palpitaition for improving rheumatic heart disease, the symptom such as out of breath, uncomfortable in chest Have certain effect；The curative effect of acute leukemia combination chemotherapy can be improved, also the inducer remission available for acute leukemia；This Outside, also there is certain curative effect to senile chronic bronchitis, various hepatitis and psoriasis.CAMP can also be prepared as pharmaceutical intermediate Dibutyryl adenosine cyclophosphate and adenosine cyclophosphate meglumine, improve fat-soluble, so as 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.

CAMP production method has three kinds of chemical synthesis, enzymatic synthesis and fermentation method.Current industry metaplasia both at home and abroad Production is all used with the chemical synthesis that adenylic acid (Adenosine-5 '-monophosphate, abbreviation AMP) is raw material, It, which is used, efficiently separates post progress intermediate separation, complex operation, and involved expensive reagents, using substantial amounts 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 The step production cAMP of ATP raw materials one is catalyzed, but there is substrate costliness, low adenyl cyclase content, purification difficult, stability in this method Difference etc. is limited, therefore is also limited to laboratory stage at present, is also had from industrialization with a distance from suitable.By contrast, microbial fermentation Method production cAMP has some following advantages：Mild condition, technique are simple, can utilize cheap carbon source, and accessory substance is few, environment Pollution is small, and continuous production etc. can be achieved, and is technique that is potential, being worth Devoting Major Efforts To Developing.

Produced currently with bacterial fermentation process such as brevibacterium liquefaciens, microbacterium, corynebacteria, arthrobacterium, Escherichia coli Research certain cAMP, but the problems such as face that poor repeatability, yield are unstable, cost is high, be difficult to industrialization.And yeast is outstanding It is saccharomyces cerevisiae (Saccharomyces cerevisiae) as one of most important industrial microorganism, not only with industry Change application technology maturation, strong stress resistance, the advantage for foodsafety GRAS microorganisms, full-length genome is realized earliest while being used as The microorganism fungus kind of sequencing, is one of most important model organism of molecular biology and genetics research, cAMP signal paths again And its related basic research of regulation and control is goed deep into, Genetic Manipulative Technology means are perfect.Regrettably research both domestic and external all collects at present In in intracellular cAMP signals-modulating function, also ground not as a product so as to pay close attention to the special of extracellular production situation Study carefully.If studying the extracellular cAMP of yeast produces rule, and transforms realization excessively to produce extracellular cAMP ferment by genetic engineering means Mother produces for the fermentation method of strain, will greatly reduce cost, simplifies technique, so as to be opened up newly for microbial fermentation production cAMP Approach, promote high level medicine and biogenetic products green clean manufacturing, with important economic value and social effect.

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

The content of the invention

Summary of the invention

The purpose of the present invention is to overcome the shortcomings of that above-mentioned prior art presence is easily cultivated there is provided one kind, is adapted on a large scale Fermenting and producing and be easily achieved industrialization, extracellular cAMP yeast strain can be excessively synthesized by genetic modification, build this The recombination method of bacterial strain and application.Specifically,

The present invention provides a kind of yeast strain, and this yeast strain have passed through the first and second of genetic modification, wherein, institute It is protein kinase A (protein kinase A, PKA) catalytic subunit encoding gene TPK1, TPK2 and TPK3 to state the first gene, PKA activity or expression are totally constrained by modifying the first gene, so as to eliminate the feedback inhibition to cAMP, but is made simultaneously The growth for obtaining yeast is suppressed, and second of genetic modification eliminates growth inhibition caused by the first genetic 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.

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

It is preferred that, second of genetic modification causes protein kinase Rim15 encoding genes RIM15, transcription factor Msn1/Msn2 The activity or expression of encoding gene MSN1/MSN2, and/or protein kinase Yak1 encoding gene YAK1 codases are totally constrained； Or the activity of protein kinase Sch9 encoding gene SCH9 codases is improved or is overexpressed.

Further, any of the above-described yeast also includes the third genetic modification, to reduce cAMP degraded, so as to improve CAMP yield.

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

It is preferred that, the above-mentioned activity for causing codase or the genetic modification mode that is totally constrained of expression include point mutation, Missing, insertion, antisense polynucleotides, siRNA, microRNA, CRISPR, more preferably missing and point mutation；So that compiling The activity raising of code enzyme or the genetic modification mode being overexpressed include point mutation, connection strong promoter, link enhancement, raising Copy number.

Further, any of the above-described yeast also includes the 4th kind of genetic modification, to increase cAMP precursors in purine route of synthesis The positive regulation and control of the synthesis of thing, rise the synthesis of cAMP precursors, so as to improve cAMP yield.

It is preferred that, the 4th kind of gene includes but are not limited to transcription factor Bas1, Bas2 encoding gene.More preferably , the genetic modification makes the expression rising of Bas1/Bas2 compounds.

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

Further, any of the above-described yeast also includes the 5th kind of genetic modification, to increase the intracellular to cAMP and its precursor The regulation and control of outer transport and accumulation, make cAMP synthesize and secretion rising, so as to improve cAMP yield.

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

It is preferred that, the modification mode of above-mentioned 5th kind of gene includes but are not limited to point mutation, connection strong promoter, connection Enhancer, raising copy number.

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

Yeast strain described in the invention, there is higher extracellular cAMP yield compared with the yeast without genetic 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 side for building the above-mentioned yeast strain that extracellular cAMP yield is greatly improved Method.

The first genetic modification is introduced in yeast first, the feedback inhibition to cAMP is eliminated, but causes yeast simultaneously Growth is suppressed, secondly, introduces second of genetic modification, and second of genetic modification eliminates life caused by the first genetic modification It is long to suppress, so that yeast normal growth.

Further, also include carrying out yeast the third genetic modification to reduce cAMP degraded in methods described, from And further improve cAMP yield.

Further, also include carrying out the 4th kind of genetic modification to yeast to increase in purine route of synthesis in methods described The positive regulation and control of the synthesis of cAMP precursors, rise the synthesis of cAMP precursors, improve cAMP yield.

Further, also include carrying out the 5th kind of genetic modification to yeast to increase to cAMP and its precursor in methods described The regulation and control transported and accumulated outside the intracellular of thing, make cAMP synthesize and secretion rising, so as to improve cAMP yield.

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

Further, methods described includes fermentation medium component, such as carbon source, nitrogen source, trace element.

It is preferred that, in methods described, fermentation is 20-150g/L, yeast extract 10- with the concentration of glucose of culture medium 20g/L, peptone 20-40g/L, adenine addition are 0-1.25g/L.

It is an object of the present invention to provide a kind of zymotic fluid 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 zymotic fluid in production cAMP.

It is an object of the present invention to provide any of the above-described yeast or zymotic fluid 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 zymotic fluid prepare medicine, animal husbandry, food, Application in the product in the field such as health products or chemical industry.

Further, the application of any of the above-described yeast strain or zymotic fluid 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 products or chemical industry are prepared it is an object of the present invention to provide one kind Product method, methods described prepares the said goods using any of the above-described yeast or zymotic fluid.

Further, methods described includes the yeast or zymotic fluid being further processed into any other formulation, preferably For edible formulation.The more preferably form such as pulvis, granule, tablet, capsule or liquid.

Accordingly, the present invention also provides the medicine comprising any of the above-described yeast or zymotic fluid, animal husbandry, food, guarantor The product of strong product or chemical field, such as medicine, feed, feed addictive, food, health products etc..

It is preferred that, the medicine, feed, feed addictive, food, health products can be direct bacterial strain or zymotic fluid, Or the yeast or zymotic fluid are further processed into any other formulation, preferably edible formulation.More preferably powder The forms such as agent, granule, tablet, capsule or liquid.

Detailed description of the invention

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

As described above, the yeast of the present invention includes the first genetic modification and second of genetic modification.Further, it is described Yeast also include the third, the 4th kind and/or the 5th kind of genetic modification.Wherein, the first genetic modification is eliminated to the anti-of cAMP Feedback suppresses, but causes the growth of yeast to be suppressed simultaneously；Second of genetic modification eliminates life caused by the first genetic modification It is long to suppress, so that yeast being capable of normal growth；The third genetic modification reduces cAMP degraded, and the 4th kind of genetic modification increases Plus in purine route of synthesis the synthesis of cAMP precursors positive regulation and control, the synthesis of cAMP precursors is increased, improve cAMP yield. 5th kind of genetic modification makes cAMP synthesize and secrete to increase the regulation and control transported and accumulated outside the intracellular to cAMP and its precursor Rise, so as to improve cAMP yield.

More specifically, the first genetic modification can pass through modified protein kinases A's (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 causes the growth of yeast to be suppressed simultaneously；Second of genetic modification eliminates life caused by the first genetic modification It is long to suppress, so that yeast being capable of normal growth；Second of genetic modification can make protein kinase Yak1 etc. activity or table Up to being totally constrained, thus eliminate be totally constrained because of PKA activity or expression caused by suppress cell growth, therefore bacterial strain Being capable of normal growth；The third genetic modification is totally constrained hydrolysis cAMP phosphodiesterase Pde activity or expression；4th Planting genetic modification makes such as transcription factor Bas1, Bas2 of the positive regulation and control through synthesizing cAMP precursors AMP in purine route of synthesis live Property or expression rise, make AMP synthesis risings, it is final to improve cAMP yield；5th kind of genetic modification carries cAMP precursors plasma membrane Body protein such as Fcy2 and/or cAMP plasma membranes carrier protein such as Snq2 activity or expression rise, and make in cAMP synthesis and secretion Rise, so as to improve cAMP yield.

The present invention is described to be pressed down by the first of yeast strain with second of genetic modification elimination PKA to cAMP feedback System synthesizes extracellular cAMP yield to improve.PKA is currently the only known cAMP targets in yeast, it is considered that in cAMP concentration Fluctuation effect is applied by PKA approach；In other words, cAMP signal paths are adjusted by PKA strict negative-feedback in yeast cells Control.The tetramer albumen that yeast PKA is made up of the regulation subunit of two catalytic subunits and a dimer, lacks in intracellular When cAMP or cAMP levels are relatively low, PKA exists in the form of being passivated complex.When intracellular cAMP levels rise, cAMP and PKA's Adjust subunit to combine, PKA catalytic subunit is disintegrated down from regulation subunit, activates a series of protein phosphorylation cascade reactions, Cause the activity of these albumen to change, and then regulate and control on varying level intracellular cAMP level；Primary effect therein Enzyme is adenyl cyclase Cyr1, Cyr1 catalysis ATP synthesis cAMP.PKA regulation subunit is encoded by gene BCY1, catalytic subunit Encoded by gene TPK1, TPK2 and TPK3.

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

Protein kinase Sch9 and PKA catalytic subunit have homology, under normal physiological conditions, and Sch9 and PKA has There are different substrates；But the activity of any kinases of increase, which can all compensate to grow caused by lacking another kinases, to be lacked Fall into.

The first genetic modification in the present invention eliminates feedback inhibition of the PKA to cAMP, makes PKA activity or has expressed It is complete to suppress, second of genetic modification eliminate yeast strain caused by the first genetic modification growth inhibition, so as to just It is frequently grown.The bacterial strain in the case of PKA is totally constrained cAMP feedback inhibition and holding PKA activity is eliminated normally to give birth to Long strategy has a variety of.PKA activity or expression are totally constrained, are eliminated feedback inhibitions of the PKA to cAMP, can compile Carried out in code catalytic subunit Tpk1, Tpk2 and Tpk3 nucleotide sequence, this nucleotide sequence can be control region, code area.

Tpk1 nucleotide sequence can be found in such as GenBank Accession No.NC_001142.9.

Tpk2 nucleotide sequence can be found in such as GenBank Accession No.NC_001148.4.

Tpk3 nucleotide sequence can be found in such as GenBank Accession No.NC_001143.9.

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

Yak1 nucleotide sequence can be found in such as GenBank Accession No.NC_001142.9.

Rim15 nucleotide sequence can be found in such as GenBank Accession No.NC_001138.5.

Msn1/Msn2 nucleotide sequence can be in such as GenBank Accession No.NC_001147.6, NC_ Found in 001145.3.

Sch9 nucleotide sequence can be found in such as GenBank Accession No.NC_001140.6.

Enzymatic activity or expression are totally constrained no matter carrying out the first and second of genetic 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, Except in haploid yeast cell simultaneously inactivate TPK1, TPK2, TPK3 combination (this combination can not be deposited because of growth defect It is living) beyond, aforementioned four gene can in no particular order, any combination is modified.

The yeast strain of process that the present invention is described above-mentioned the first and second of genetic modification is relative to without the first More extracellular cAMP are produced with the yeast strain of second of genetic modification.

On the other hand, cAMP is once produce, and it is urging in cyclic nucleotide phosphodiesterase Pde uniquely to make the mode that it is inactivated 5 '-AMP are hydrolyzed under change.Therefore, Pde also contributes to intracellular cAMP level.Therefore, the present invention passes through the third gene Modification is totally constrained Pde activity or expression, so as to reduce cAMP degraded, improve cAMP contents levels.Saccharomyces cerevisiae is thin Born of the same parents are in itself comprising two cAMP phosphodiesterases：It is Pde1 one low-affinity (Km=20-250 μM) of coding, specific relatively low Enzyme, mainly mediate quick cAMP signal transductions；It is Pde2 one high-affinity (Km=170nM) of coding, specific very strong Enzyme, major control cAMP foundation level.

Pde1 nucleotide sequence can be found in such as GenBank Accession No.NC_001139.9.

Pde2 nucleotide sequence can be found in such as GenBank Accession No.NC_001147.6.

The first described in the invention, second and the third genetic modification can be by known in the art any A variety of methods are realized.The genetic modification mode that the activity of codase or expression are totally constrained is the core to gene Acid sequence carries out point mutation, lacks or insert one or more nucleotides, 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, this displacement may result in the conformation of polypeptide Change, it is also possible to lose some or all of function, it may occur however that frameshift mutation will cause whole polypeptide chain at the point Start to encode an entirely different polypeptide, it is also possible to form a terminator codon in advance and cause polypeptide chain incomplete, or even make Gene silencing.

Missing or insertion can be obtained with nucleotide sequence with PCR or the method for chemical synthesis, can also be multiple using cell Amplification processed is obtained.The genetic modification for being totally constrained gene enzymatic activity or expression can also be by offer or the few core of antisence Thuja acid antisense polynucleotides, siRNA, microRNA or other can prevent the mRNA of gene to be finished from turning over The method for being translated into the nucleic acid of protein is realized.The class activating transcription factor effector nuclease developed in recent years (transcription activator-like effector nuclease, TALEN) and CRISPR technologies --- rule into The short palindrome in interval of cluster repeats (Clustered Regularly Interspaced Short Palindromic Repeats) It may also be used for inactivated gene.

Mentioned in the present invention make gene enzymatic activity or the genetic modification that is totally constrained of expression refer to making it is this Enzyme/polypeptide activity reduction (being compared with yeast or wild-type yeast without genetic modification) at least 95% (for example, at least 96%, 97%, 98%, 99%, or 100%).

So that the genetic modification mode that the activity of codase is improved or is overexpressed includes but is not limited to point mutation, connection by force Promoter, link enhancement, raising copy number.For example, point mutation improves polypeptide active；Improve the copy number of nucleotide sequence；It is logical Crossing genetic modification makes nucleotide sequence be connected with effable a strong promoter or enhancer；Change nucleotide sequence promoter or Other regulatory factors of person are so as to improve its expression (such as the bond strength for improving promoter sequence and transcription initiation factor)； Improve the mRNA of nucleotide sequence transcription half-life period；Suppress the degraded to mRNA or polypeptide chain.

The genetic modification for being enhanced gene enzymatic activity or expression mentioned in the present invention refers to that one is repaiied by gene The activity of enzyme/polypeptide 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).

Process that the present invention is described it is above-mentioned the first, the yeast strain of second and the third genetic modification, relative to not Through the first, the yeast strain of second and the third genetic modification produce more extracellular cAMP.Even with respect to the first More extracellular cAMP are produced with the yeast strain of second of genetic modification.

Improve the extracellular cAMP levels of yeast cells, the gene of cAMP-PKA signal path approach is modified outside, also may be used Realized with synthesizing precursor AMP synthetic quantity by regulating and controlling purine route of synthesis, increase cAMP.The purine synthesis of saccharomyces cerevisiae In approach, AMP de novo synthesis genes are the expression of ADE genes by the collaboration between 2 transcription regulatory factors Bas1 and Bas2 The positive regulation and control of effect.Bas1 is Myb races transcription regulatory factor, Bas2 also known as Pho2, is homeodomain (Homeodomain) transcriptional control The factor, the two is combined with composite form with specific promoter region, the expression of synergy 10 ADE genes of activation.Work as born of the same parents During interior ADP, ATP excessive concentration, first enzyme Ade4p of ADP, ATP feedback inhibition de novo synthesis (PRPP amide transferases, Abbreviation PRPPAT) activity so that intermediate product SAICAR synthesis declines, and Bas2p can perceive SAICAR change in concentration, when When this concentration is down to a certain degree, Bas2p no longer forms compound with Bas1p.Deposited when there is considerable amount of adenine in culture medium When, adenine enters intracellular, and by remedial pathway formation AMP, AMP is further converted to ADP and ATP, also cause it is whole from Head route of synthesis synthesis is suppressed.

The 4th kind of genetic modification of the present invention synthesizes AMP by increasing the positive regulation and control that AMP is synthesized in purine route of synthesis Rise, improve cAMP yield.The core of the positive regulation and control of increase AMP synthesis is to promote the formation of Bas1/Bas2 compounds, improves multiple The positive regulation activity of compound, suppresses adenine, ADP and ATP feedback inhibition.And promote the formation of Bas1/Bas2 compounds, improve The strategy of the positive regulation activity of compound has a variety of, can improve single positive regulative transcription factor Bas1 or Bas2 activity or table Reach, can also directly merge coexpression Bas1 and Bas2, or further improved on the basis of fusion coexpression Bas1 and Bas2 Activity or expression quantity.The polypeptide active that having a variety of methods can encode nucleic acid is improved.Lived for example, point mutation improves polypeptide Property；Improve the copy number of nucleotide sequence；Nucleotide sequence and effable a strong promoter or enhancer are made by genetic modification Connection；Change the promoter or other regulatory factors of nucleotide sequence and (for example improve promoter sequence so as to improve its expression The bond strength of row and transcription initiation factor)；Improve the mRNA of nucleotide sequence transcription half-life period；Suppress to mRNA or many The degraded of peptide chain.Assert the work that the raising of a polypeptide active by the nucleic acid sequence encoding of genetic modification needs it to substrate Improve compared with wild type at least 20% with efficiency (for example, at least 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or higher).The overexpression of the nucleotide sequence of single copy or multicopy can pass through connection Realize, can also be realized by being incorporated into the genome of yeast into carrier.Suitable for nucleotide sequence be overexpressed carrier Through being commercialized or can be obtained by gene recombination technology commonly used in the art.There are some in the art in yeast The method that nucleotide 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.The element that carrier containing nucleotide sequence can be necessary some to expression carries out operable with purpose nucleic acid sequence Connection：Nucleotide sequence (such as the auxotrophic selection marker gene, antibiotic resistance base of expression selected marker can be taken Cause)；Or take the gene (such as 6xHis tag) that can be used to purified polypeptide；One or more replication orgins can also be carried. The element that expression is necessary is included to instruct or regulating and controlling the nucleotide sequence of purpose nucleic acid sequence, such as promoter sequence. Representational promoter includes (being not limited to this) PGK promoters, TPI1 promoters, ADH1 promoters.Expression is necessary Element also includes the inducible factor of enhancer sequence, response element, or regulation and control coded sequence expression.

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

Bas1 nucleotide sequence can be found in such as GenBank Accession No.NC_001143.9.

Bas2 nucleotide sequence can be found in such as GenBank Accession No.NC_001136.10.

Process that the present invention is described it is above-mentioned the first, second, the yeast strain phase of the third and the 4th kind of genetic modification For without the first, second, the yeast strain of the third and the 4th kind of genetic modification produce more extracellular cAMP.Even Relative to the first and second genetic modification yeast strain or relative to the first, second and the third genetic modification Yeast strain produce more extracellular cAMP.

Improve transport and product outside the extracellular cAMP levels of yeast cells, intracellular that can also be by regulating and controlling cAMP and its precursor Tire out to realize.The outer matter transportation of yeast cells 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, mediation adenine, guanine With hypoxanthine and cytimidine from extracellular to the transport of intracellular and accumulation.And plasma membrane ATP combines (ABC) channel protein Snq2 and participated in The transport of multi-medicament, includes mutagens, bactericide, steroids and anticancer by arranging a variety of exogenous compounds outside dependency ATP Medicine etc.；Primary Study show Snq2 may by PKA direct regulation and participate in, to cAMP feedback inhibition, being simultaneously responsible for CAMP is to extracellular discharge.

The 5th kind of genetic modification of the present invention is by increasing the tune for transporting and accumulating outside the intracellular to cAMP and its precursor Control, makes cAMP synthesize and secretion rising, so as to improve cAMP yield.Increase is to transport outside the intracellular of cAMP and its precursor and product The mode of tired regulation and control is that relevant carriers protein coding gene is modified, and carrier protein activity or expression is increased.Repaiied The gene of decorations includes but is not limited to Fcy2 encoding genes and Snq2 encoding genes, and the result of modification makes in corresponding enzymatic activity or expression Rise, bacterial strain, which is added, to be transported and accumulate outside the intracellular to cAMP and its precursor, cAMP is synthesized and is secreted and is risen, so as to improve CAMP yield.

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

Fcy2 nucleotide sequence can be found in such as GenBank Accession No.BK006939.2.

Snq2 nucleotide sequence can be found in such as GenBank Accession No.BK006938.2.

Process that the present invention is described it is above-mentioned the first, second, the yeast strain phase of the third and the 5th kind of genetic modification For without the first, second, the yeast strain of the third and the 5th kind of genetic modification produce more extracellular cAMP.Even Relative to the first and second genetic modification yeast strain or relative to the first, second and the third genetic modification Yeast strain produce more extracellular cAMP.

In the genetical modification steps of the present invention, nucleotide sequence (such as expression vector) can be transferred to yeast by a variety of methods In cell or other host cells.These methods include (being 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.

Except yeast cells, " host cell " can also be that any standard molecular biology that can be used for operates and produces core The cell of acid and polypeptide.Bacterial cell (such as E.coli) is included but are not limited to, insect cell, plant cell and mammal are thin Born of the same parents' (such as CHO or COS cells)." 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, in addition to its daughter cell.

In the genetical modification steps of the present invention, include but not for being transferred to the selectable marker gene of yeast cells Select to use It is only limitted to the available any auxotroph gene of yeast, such as URA3, LEU2, HIS3, TRP1, LYS2.In order to be able to carry out to the greatest extent may be used Genetic modification more than energy, while avoid what auxotroph gene expression brought in itself from may interfere with, preferably can be easily anti- URA3 genes that are multiple, recycling.Still more preferably, the selectable marker gene such as URA3 be recycled and be not kept in by In the bacterial strain of genetic modification.

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

Invention further describes the method that the yeast strain of above-mentioned process genetic modification produces cAMP.

It is preferred that, in the fermentation process, fermentation is 20-150g/L, yeast extract with the concentration of glucose of culture medium 10-20g/L, peptone 20-40g/L, adenine addition are 0-1.25g/L.

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

Invention further describes the purposes of the yeast strain of above-mentioned process genetic modification, it can be used for fermentating metabolism carbon aquation Compound produces extracellular cAMP.Further, the yeast strain cell of above-mentioned process genetic modification, and/or it is fermented obtain include Extracellular cAMP zymotic fluid, can directly as cAMP substitute.And as cAMP applies generally to every field, for example, doctor Medicine, animal husbandry, food, health products or chemical field etc..Further, the application is included in the product for preparing above-mentioned field In application, such as medicine, food, health products and feed.Accordingly, the present invention also provides and includes above-mentioned process genetic modification Yeast strain cell, and/or the fermented obtained zymotic fluid comprising extracellular cAMP medicine, animal husbandry, food, health products Or the product in the field such as chemical industry, such as medicine, food, health products, feed, feed addictive.

It is described medicine, animal husbandry, field of food, health products application, including by above-mentioned yeast strain cell, and/or The fermented obtained zymotic fluid for including extracellular cAMP, Nutrition and Metabolism regulation, health care and the disease for being directly used in humans and animals is controlled Treat, form the product in these fields, such as medicine, food and feed/feed addictive, and these products application.Or Described cell, zymotic fluid are further processed into any other formulation, preferably edible formulation.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, without this area Technical staff pays creative work again；In animal husbandry field, it is preferred that be that low temperature drying or spray drying process prepare powder Agent.

The animal, including model animal, fowl poultry kind, aquatic livestock, the animal such as homing pigeon of 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.Can be that any commonly employed animal is raised on the market at present with above-mentioned used as feed addictive and the feed that is added Material, i.e., do not limited by the conventional animal feed species coordinated.But conventional animal feed therein is preferably daily ration, enters one Step is preferably chicken daily ration, pig daily ration, ruminant, aquatic livestock daily ration.The usage amount as feed addictive, It is preferably (0.5-20) mgcAMP/ (kg body weight), more preferably 0.5- for (0.05-50) mg cAMP/ (kg body weight) 10.0mgcAMP/ (kg body 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.Can be for building and determining the yeast strain being related in the present invention, hereinafter in spite of a variety of similar or equivalent method A kind of suitable method and material will be described.These materials, method and example are intended only as putting to the proof, and 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 hereinafter will be provided together with detailed description and chart.

The advantage and benefit of invention

Gained restructuring yeast strains of the invention have low nutrition demand, easily cultivate, are adapted to large scale fermentation production and easy In the performance for realizing industrialization；Related gene modification is carried out by genetic engineering means, cAMP paths feedback inhibition is being eliminated, is subtracting While few cAMP degradeds, cAMP precursors AMP metabolic fluxes are led in increase, improve and are transported outside the intracellular of cAMP and its precursor And accumulation, gained restructuring yeast strains is stablized, is continued, efficiently produces 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.Instant invention overcomes conventional chemical synthesis complex process, original Material is expensive, the shortcoming that accessory substance is more, environmental pollution is serious, also overcomes enzymatic synthesis substrate costliness, adenyl cyclase steady The deficiencies such as qualitative difference, with technological operation is simple, product can be easily separated extractions, low cost of material, safety and environmental protection, can industry metaplasia The advantage of production, is that microbial fermentation production cAMP opens new approach, and the green for promoting high level medicine and biogenetic products is clear Clean production, can be widely used in medicine, animal husbandry, food or chemical field, with important economic value and social effect.

As the application in animal husbandry, by directly adding, diet oral way use the high yeast cells containing cAMP Or its zymotic fluid and its preparation, it can overcome the shortcomings of that cAMP pure preparation drawn games can only be used by injection in research at present and application Limit, is that a kind of new yeast source biology is raised while playing yeast cell culture and cAMP health cares, growth promotion (collaboration) effect The Animal feed-additive that feed additives and non-hormone safety 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 favourable In reducing cost to greatest extent, so that more simple yeast culture or simple cAMP preparations and its derivative are with wider Wealthy application prospect.

Brief description of the drawings

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

Fig. 2 is 5 five gene deletion strains cAMP production preliminary assessments of embodiment.

Fig. 3 is the plasmid pUC18-H1 of embodiment 6_YNRCΔ9-BAS1-BAS2-H2_YNRCΔ9-URA3-H2_YNRCΔSchematic diagram.

Embodiment

Technical scheme of the present invention is further described with reference to specific embodiment.It is understood that The particular implementation of this description represents that it is not intended as limitation of the present invention by way of example.If not refer in particular to It is bright, the conventional meanses that technological means used is well known to those skilled in the art in embodiment.Model of the present invention is not being deviated from In the case of enclosing, principal character 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, only using only normal experiment, many equivalents can be applied in particular step described herein.These are equal Thing is considered as place within the scope of the present invention, and is covered by claim.

Involved several culture mediums are as follows in embodiment：

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 during solid culture.

2) using transformation and selection culture medium C MG during uracil (URA) auxotrophic selection marker^-URA

Use the default minimal medium CMG flat board abbreviations CMG of uracil^-URAFlat board 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.

The amino acids basic mix ingredients of table 1.

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：Save specific aminoacid ingredient, you can make Selective agar medium.Adjust pH value 5.6,110 DEG C of sterilizings of glucose 15min, other 121 DEG C of sterilizing 21min of composition, uses preceding mixing.

3) 5 '-FOA flat boards (5 '-orotic acid flat board)

Preparation method is as follows：

A, the preparation-FOA solution of 100ml 5 ', including：

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

Uracil：20mg adenines：10mg leucines：40mg

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

With magnetic stirring apparatus in not stopping stirring at 45 DEG C, make its each component all dissolving, afterwards filtration sterilization；

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

C ,-the FOA of 100ml 5 ' and 100ml Agar solution mixed, prevent bubble, pour into sterile petri dish, it is cold But it is molded.

4) LB culture mediums are used in Escherichia coli culture

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 during solid culture.Antibiotic is added when needing, the addition of ampicillin is dense eventually Spend 100 μ g/mL.

Involved primer sequence is shown in Table 2 in embodiment.

The primer sequence used in the embodiment of table 2

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

Embodiment 1：Build the yeast strain of missing TPK2 genes

The TPK2 genes on yeast chromosomal are lacked using homologous recombination bialternative series, missing plasmid is built first PUC18-TPK2p-TPK2t-URA3-TPK2t, this plasmid transformed yeast competent cell after linearization for enzyme restriction, using URA3 as Riddled basins screening obtains that the yeast that double crossing over is obtained occurs by left and right homology arm of the TPK2t in TPK2p and URA3 downstreams Bacterial strain tpk2 △::URA3；Again bacterial strain tpk2 △ are obtained with 5 '-FOA plate screenings::URA3 arranged on left and right sides on URA3 chromosomes The bacterial strain tpk2 △ for recombinating and ejecting the Saccharomycetes of URA3 genes occur between TPK2t.

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

First, pUC18-TPK2p-TPK2t-URA3-TPK2t is built

The structure of this plasmid is related to four connections：

(1) pUC18-URA3 is built

Enter performing PCR amplification URA3 genes by template of plasmid YCplac33, use 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 polymerases of Quan Shi King Companies production, 50 DEG C of anneal 1min, 72 DEG C extension 1.5min, totally 32 circulation.1055bp PCR fragment is obtained, the double enzyme enzymes of restriction enzyme BamHI and SalI are used Escherichia coli Top10 competent cells are converted after cutting this fragment and the connection of vector plasmid pUC18, T4DNA ligase, conversion is extracted Sub- plasmid carries out digestion identification, obtains plasmid pUC18-BamHI-URA3-SalI (abbreviation pUC18-URA3, see Figure 1A).

(2) pUC18-TPK2t-URA3 is built

Wine brewing yeast strain W303-1A chromosomes are extracted, performing PCR is entered by template of W303-1A chromosomes, TPK2 bases are expanded The terminator district (referred to as TPK2t) of cause, is upstream and downstream using sequence shown in the SEQ ID No.3 in table 2, SEQ ID No.4 Primer P3, P4, the Fast Pfu polymerases of Quan Shi King Companies production, 50 DEG C of annealing 1min, 72 DEG C of extension 0.5min, totally 32 are followed Ring.526bp PCR fragment is obtained, double this fragment of enzyme digestion of restriction enzyme KpnI and BamHI and vector plasmid is used Escherichia coli Top10 competent cells are converted after the connection of pUC18-URA3, T4DNA ligase, transformant plasmid is extracted and carries out enzyme Identification is cut, plasmid pUC18-TPK2t-URA3 is obtained.

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

W303-1A chromosomes are extracted, performing PCR is entered by template of W303-1A chromosomes, the promoter region of TPK2 genes is expanded (referred to as TPK2p), is upstream and downstream primer P5, P6 using sequence shown in the SEQ ID No.5 in table 2, SEQ ID No.6, entirely The Fast Pfu polymerases of formula King Company production, 50 DEG C of annealing 1min, 72 DEG C of extension 0.5min, totally 32 circulations.Obtain 532bp PCR fragment, use double this fragment of enzyme digestion of restriction enzyme Sac I and Kpn I and vector plasmid pUC18-TPK2t- Escherichia coli Top10 competent cells are converted after the connection of URA3, T4DNA ligase, transformant plasmid is extracted and carries out digestion identification, Obtain plasmid pUC18-TPK2p-TPK2t-URA3.

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

By the double enzyme digestions of Sal I and Pst I of the PCR primer of the sequence containing TPK2t of amplification in above-mentioned (2), with same pair The pUC18-TPK2p-TPK2t-URA3 large fragments connection of enzyme digestion, obtains plasmid pUC18-TPK2p-TPK2t-URA3-TPK2t (see Figure 1B).

2nd, W303-1A (tpk2 △) strain construction

By the plasmid pUC18-TPK2p-TPK2t-URA3-TPK2t double enzyme digestions of Sac I and Pst I, linearized DNA fragmentation TPK2p-TPK2t-URA3-TPK2t；Then it is 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), i.e., using CMG^-URAFlat board is screened, to obtaining Transformant bacterial strain carry out YPAD Liquid Cultures, extract chromosomal DNA, entered using it as template performing PCR method verify, primer pair is table Sequence shown in SEQ ID No.7, SEQ ID No.8 in 2 is upstream and downstream primer P7, P8, successful integration, contains URA genes Transformant PCR primer is 2592bp, and control strain PCR primer is 2299bp.Positive transformant Strain Designation is W303-1A (tpk2△::URA3)。

Further by bacterial strain W303-1A (tpk2 △::URA3) plated growth thalline is coated on 5 '-FOA flat boards, so that Filter out and eject the bacterium of URA3 fragments by integrating homologous recombination between two identical sequences (TPK2t) on chromosome Fall.Verified with primer pair P7, P8 ibid, successfully eject the long 1053bp of PCR primer of URA3 purpose bacterial strain, do not eject URA3 host strain W303-1A (tpk2 △::URA3 the long 2592bp of PCR fragment).1053bp PCR primers are surveyed Sequence, sequencing result proves to there occurs expected change：2 bases, whole ORF areas and end before the initiation codon of TPK2 genes Only 107 bases after codon are lacked completely.So, TPK2 gene delections, the sun of URA3 genes ejection are finally given Property transformant bacterial strain W303-1A (tpk2 △).

Embodiment 2：Build the yeast strain of missing TPK1 or TPK3 genes

First, W303-1A (tpk1 △) is built

TPK1 gene delections plasmid pUC18-TPK1p-TPK1t-URA3-TPK1t structure：With (1) in embodiment 1 Plasmid pUC18-TPK1p-TPK1t-URA3-TPK1t is built based on pUC18-URA3 plasmids, process in embodiment 1 with passing through (2), as (3) finally give pUC18-TPK2p-TPK2t-URA3-TPK2t process with (4) step, the primer pair used： Amplification TPK1t is that sequence shown in SEQ ID No.9, SEQ ID No.10 in table 2 is upstream and downstream primer P9, P10, product 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 double enzyme digestions of Sac I and Pst I, linearized DNA fragmentation TPK1p-TPK1t-URA3-TPK1t；Then transformed saccharomyces cerevisiae W303-1A bacterial strains competent cell, phonetic using urinating Pyridine (URA) auxotrophic selection marker is screened, i.e., using CMG^-URAFlat board is screened, to obtained transformant bacterial strain Further verified with PCR methods, 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 Thing P13, P14, the transformant PCR primer of successful integration is 2631bp, and control strain PCR primer is 2476bp.Positive transformant Strain Designation is W303-1A (tpk1 △::URA3).

Further by bacterial strain W303-1A (tpk1 △::URA3) plated growth thalline is coated on 5 '-FOA (5 '-orotic acid) On flat board, ejected so as to filter out by integrating homologous recombination between two identical sequences (TPK1t) on chromosome The bacterium colony of URA3 fragments.Verified with primer pair P13, P14 ibid, the PCR primer for successfully ejecting URA3 purpose bacterial strain is long 1102bp, does not eject the URA3 long 2631bp of control PCR fragment.1102bp PCR primers are sequenced, sequencing result Proof there occurs expected change：70 bases, whole ORF areas and terminator codon before the initiation codon of TPK1 genes it 116 bases afterwards are lacked completely.So, TPK1 gene delections, the positive transformants daughter bacteria of URA3 genes ejection are finally given Strain W303-1A (tpk1 △).

2nd, W303-1A (tpk3 △) is built

The structure of TPK3 gene delection plasmids：To build plasmid based on (1) pUC18-URA3 plasmids in embodiment 1 PUC18-TPK3p-TPK3t-URA3-TPK3t process in embodiment 1 by (2), (3) and (4) step with finally giving PUC18-TPK2p-TPK2t-URA3-TPK2t process is the same, the primer pair used：It is the SEQ ID in table 2 to expand TPK3t Sequence shown in No.15, SEQ ID No.16 is upstream and downstream primer P15, P16, product 566bp；TPK3p is expanded 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 double enzyme digestions of Sac I and Pst I, linearized DNA fragmentation TPK3p-TPK3t-URA3-TPK3t；Then transformed saccharomyces cerevisiae W303-1A bacterial strains competent cell, be the same as Example 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 checking is with sequence shown in the SEQ ID No.19, SEQ ID No.20 that primer pair is in table 2 For upstream and downstream primer P19, P20, successful integration, the transformant PCR primer containing URA3 genes are 2695bp, successful integration, The transformant PCR primer of URA3 genes ejection is 1116bp, and control strain PCR primer is 2400bp.To 1116bp PCR primers It is sequenced, sequencing result proves to there occurs expected change：After the whole ORF areas of TPK3 genes and terminator codon 93 bases are lacked completely.So, TPK3 gene delections, the positive transformant bacterial strain of URA3 genes ejection are finally given W303-1A(tpk3△)。

Embodiment 3：Build the yeast strain of missing YAK1 genes

The structure of YAK1 gene delection plasmids：To build plasmid based on (1) pUC18-URA3 plasmids in embodiment 1 PUC18-YAK1p-YAK1t-URA3-YAK1t process in embodiment 1 by (2), (3) and (4) step with finally giving PUC18-TPK1p-TPK1t-URA3-TPK1t process is the same, the primer pair used：It is the SEQ ID in table 2 to expand YAK1t Sequence shown in No.21, SEQ ID No.22 is upstream and downstream primer P21, P22, product 506bp；YAK1p is expanded in table 2 Sequence shown in SEQ ID No.23, SEQ ID No.24 is upstream and downstream primer P23, P24, product 504bp.

The structure of YAK1 gene deletion strains：By plasmid pUC18-YAK1p-YAK1t-URA3-YAK1t Sac I and Pst The double enzyme digestions of I, obtain linear DNA fragments YAK1p-YAK1t-URA3-YAK1t；Then transformed saccharomyces cerevisiae W303-1A bacterial strains The screening of " W303-1A (tpk2 △) strain construction " method, identification obtain W303-1A (yak1 in competent cell, be the same as Example 1 △::URA3) bacterial strain and W303-1A (yak1 △) bacterial strain.PCR method checking with the 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 primer containing URA3 genes For 2529bp, successful integration, the transformant PCR primer of URA3 genes ejection are 1010bp, and control strain PCR primer is 3614bp.1010bp PCR primers are sequenced, sequencing result proves to there occurs expected change：The starting of YAK1 genes 146 bases before codon after 40 bases, whole ORF areas and terminator codons are lacked completely.So, it is final to obtain The positive transformant bacterial strain W303-1A (yak1 △) ejected to YAK1 gene delections, URA3 genes.

Embodiment 4：Build the yeast strain for lacking TPK1, TPK2, TPK3, YAK1 gene simultaneously

Build and obtain while lacking the bacterial strain of four genes in four steps below.In fact those skilled in the art knows Road, except in haploid yeast cell simultaneously inactivate TPK1, TPK2, TPK3 combination (this combination because growth defect can not Survival) beyond, aforementioned four gene can in no particular order, any combination is modified.

First, YAK1 genes are lacked

The method of be the same as Example 3 builds and obtains W303-1A (yak1 △) bacterial strain.

2nd, YAK1 and TPK1 genes are lacked

By the missing built 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, i.e., using CMG^-URA Flat board is screened, and TPK1 deletion mycopremnas are obtained to identical method identification in obtained transformant bacterial strain be the same as Example 2 W303-1A(yak1△)(tpk1△::URA3), and further anti-choosing obtains W303-1A (yak1 △) (tpk1 △) bacterial strain.

3rd, TPK2 and TPK3 genes are lacked

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

By the plasmid pUC18-TPK2p-TPK2t-URA3-TPK2t built in embodiment 1 the double enzyme enzymes of Sac I and Pst I Cut, obtain linear DNA fragments TPK2p-TPK2t-URA3-TPK2t；Then transformed saccharomyces cerevisiae W303-1B bacterial strains competence The screening of " W303-1A (tpk2 △) strain construction " method, identification obtain W303-1B (tpk2 △ in cell, be the same as Example 1:: URA3) bacterial strain and W303-1B (tpk2 △) bacterial strain.

By the plasmid pUC18-TPK3p-TPK3t-URA3-TPK3t built in embodiment 2 the double enzyme enzymes of Sac I and Pst I Cut, obtain linear DNA fragments TPK3p-TPK3t-URA3-TPK3t；Then transformed saccharomyces cerevisiae W303-1B (tpk2 △) bacterium The screening of " W303-1A (tpk2 △) strain construction " method, identification obtain W303-1B in strain competent cell, be the same as Example 1 (tpk2△tpk3△::URA3) bacterial strain and W303-1B (tpk2 △ tpk3 △) bacterial strain.

4th, while lacking TPK1, TPK2, TPK3, YAK1 gene

W303-1A (tpk1 △ yak1 △) bacterial strains are hybridized with W303-1B (tpk2 △ tpk3 △) bacterial strain, two are obtained Times body bacterial strain, then separates allele by producing spore, tearing spore open, and screening, identification obtain W303-1A (tpk1 △ tpk2 △ Tpk3 △ yak1 △) and W303-1B (tpk1 △ tpk2 △ tpk3 △ yak1 △).Identify that the checking 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 primer Expected change, has all been lacked ORF areas.

5th, the growth and the extracellular cAMP evaluations of fermenting and producing of four gene deletion strains

Aforementioned four gene deletion strains have been carried out with growth and the extracellular cAMP of fermenting and producing is evaluated, has been operated as follows：

1st, seed liquor culture：The bacterium colony grown on picking YPAD solid medium flat boards, access is equipped with 5mL YPAD cultures In the test tube of liquid, 30 DEG C, 220rpm incubated overnights carry out secondary expand and cultivated if necessary；

2nd, ferment：Fermentation culture medium：Yeast extract 10g/L, peptone 20g/L, glucose 20g/L, natural ph； Fresh seeds liquid is inoculated into the 100ml volumetric flasks equipped with 25ml fermentation mediums, initial OD is controlled₆₀₀It is worth 0.1 or so, 30 DEG C, ferment under 220rpm；

3rd, the OD of zymotic fluid₆₀₀Determine：OD is determined after fermented sample is suitably diluted₆₀₀, detect growing state；

4th, it is extracellular cAMP concentration that HPLC, which analyzes fermented supernatant fluid cAMP concentration,：HPLC analyzes cAMP method reference《In Magnificent people's republic's pharmacopeia：Version in 2010》Method that (Chinese Pharmacopoeia Commission's volume) page 419 and have adjustment.Concrete operations are such as Under：1) fermented sample is centrifuged under the conditions of 13000rpm, 1min, takes supernatant suitably to be diluted, with 0.22 μm of the filter in aperture Membrane filtration, filtrate is detected for chromatogram：Detection wavelength 258nm, Thermo Syncronis C18 chromatographic columns, mobile phase is (5.78g/L KH₂PO₄, 2.72g/L TBABs):Acetonitrile=85:15With phosphorus acid for adjusting pH to 4.3, flow velocity 1mL/ Min, 35 DEG C of column temperature；2) measure of the configuration of cAMP standard samples and standard curve：Going after first cAMP standard items are sterilized Ionized water is configured to concentration and is 50mmol/L standard liquid, then it is diluted with deionized water, obtains final concentration difference For 1,3,5,7.5 and 10 μm of ol/L standard sample, with HPLC analyses are 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 results of growth and fermenting and producing in 96h are shown in Table 3.

Table 3, the growth and cAMP productions of four gene deletion strains are evaluated

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 maximum extracellular cAMP concentration of two deletion mycopremnas is that control strain W303-1A maximums are extracellular respectively 144.0 and 155.1 times of cAMP concentration.It should be noted that：Two deletion mycopremnas cAMP concentration in 0-24h is raised rapidly, 24h reaches after peak that cAMP concentration is declined slightly, but stills remain in 180 μm of more than ol/L when 96h.

Embodiment 5：Build the yeast strain for lacking TPK1, TPK2, TPK3, YAK1, PDE1 gene simultaneously

First, PDE1 genes are lacked

Enter performing PCR by template of W303-1A chromosomes, expand the terminator district (referred to as PDE1t) of PDE1 genes, use Sequence shown in SEQ ID No.27, SEQ ID No.28 in table 2 be upstream and downstream primer P27, P28, Quan Shi King Companies production Fast Pfu polymerases, 50 DEG C of annealing 1min, 72 DEG C of extension 0.5min, totally 32 circulations.500bp PCR fragment is obtained, is used Turn after double this fragment of enzyme digestion of restriction enzyme KpnI and BamHI and the connection of vector plasmid pUC18-URA3, T4DNA ligase Change Escherichia coli Top10 competent cells, extract transformant plasmid and carry out digestion identification, obtain plasmid pUC18-PDE1t- URA3。

Enter performing PCR by template of W303-1A chromosomes, expand the promoter region (referred to as PDE1p) of PDE1 genes, use Sequence shown in SEQ ID No.29, SEQ ID No.30 in table 2 be upstream and downstream primer P29, P30, Quan Shi King Companies production Fast Pfu polymerases, 50 DEG C of annealing 1min, 72 DEG C of extension 0.5min, totally 32 circulations.544bp PCR fragment is obtained, is used Double this fragment of enzyme digestion of restriction enzyme Sac I and Kpn I and vector plasmid pUC18-PDE1t-URA3, T4DNA ligase Escherichia coli Top10 competent cells are converted after connection, transformant plasmid is extracted and carries out digestion identification, obtain plasmid pUC18- PDE1p-PDE1t-URA3。

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

By the plasmid pUC18-PDE1p-PDE1t-URA3-PDE 1t double 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 △ built in embodiment 4 Tpk3 △ yak1 △) it is host, structure obtains W303-1A (tpk1 △ tpk2 △ tpk3 △ yak1 △ pde1 △::URA3) and W303-1A(tpk1△tpk2△tpk3△yak1△pde1△).PCR method checking 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 genes PCR primer is 2958bp, and successful integration, the transformant PCR primer of URA3 genes ejection are 1445bp, control strain PCR primer For 2635bp.1445bp PCR primers are sequenced, sequencing result proves to there occurs expected change：Of PDE1 genes 46 bases before beginning codon after 40 bases, whole ORF areas and terminator codons are lacked completely.So, finally Obtain PDE1 gene delections, the positive transformant bacterial strain of URA3 genes ejection.

2nd, W303-1A (tpk1 △ tpk2 △ tpk3 △ yak1 △ pde1 △::) and W303-1A (tpk1 △ tpk2 URA3 △ tpk3 △ yak1 △ pde1 △) cAMP production contrast

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

3rd, W303-1A (tpk1 △ tpk2 △ tpk3 △ yak1 △ pde1 △) growth and cAMP productions are evaluated

The growth without URA3 five gene deletion strains is carried out and the extracellular cAMP of fermenting and producing is evaluated：1st, seed liquor Cultivate be the same as Example 4；2nd, ferment：1) fermentation culture medium：Yeast extract 10g/L, peptone 20g/L, glucose 20~ 150g/L, natural ph；2) fermentation condition is：Fresh seeds liquid is inoculated into the 100ml volumetric flasks equipped with 25ml fermentation mediums In, control initial OD₆₀₀Value is fermented under 1 or so, 30 DEG C, 220rpm.HPLC analysis sides in zymotic fluid analysis be the same as Example 4 Method.The extracellular cAMP evaluation results of growth and fermenting and producing in 120h are shown in Table 4.

Table 4, the growth and cAMP productions of five gene deletion strains are evaluated

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)

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

Embodiment 6：BAS1-BAS2 fusion coexpressions

BAS1 and BAS2 genes are subjected to fusion coexpression, and are incorporated on the chromosome of yeast cells.Needed first for this Build 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 are the YNRC Δs 9 compared with high gene expression efficiency, positioned at chromosome On XIV.

First, BAS1-BAS2 merges co-expression plasmid and built

Totally 4 connections obtain BAS1-BAS2 fusion co-expression plasmids 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

The partial sequence for entering performing PCR amplification YNRC Δs 9 by template of W303-1A chromosomal DNA is used as the left and right of integration Homology arm.The use of sequence shown in the SEQ ID No.33 in table 2, SEQ ID No.34 is upstream and downstream primer P33, P34, full formula The Fast Pfu polymerases of King Company's production, 50 DEG C of annealing 1min, 72 DEG C of extension 45sec, totally 32 circulations, obtain 521bp's (correspondence sequence is used as the left homology arm H1 integrated to PCR1 fragments_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 the PCR2 fragments for obtaining 448bp with method amplification (correspond to sequence 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 949bp PCR12 fragments.5 ' ends of this fragment are added with restriction enzyme site EcoRI, and 3 ' ends are added with digestion position Point BamHI, centre is added with restriction enzyme site SacI, Sal I.By the PCR primer double enzyme digestions of EcoRI and BamHI, with same pair The pUC18 large fragments connection of enzyme digestion, converts Escherichia coli Top10 competent cells, 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

Enter selectable marker gene when performing PCR amplification URA3 genes are used as 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 Companies production Fast Pfu polymerase, 50 DEG C of annealing 1min, 72 DEG C of extension 1min, totally 32 circulations, obtain 1060bp PCR1 fragments.Still with W303- 1A chromosomal DNA is that template enters performing PCR amplification H2_YNRCΔ9Fragment, 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 438bp PCR2 fragments.Again with PCR1, PCR2 product For template, using P37, P40 as primer pair, the reaction amplification of juxtaposition extension PCR obtains 1478bp PCR12 fragments.This fragment 5 ' ends added with restriction enzyme site BamHI, 3 ' ends are added with restriction enzyme site HindIII.By PCR primer BamHI and HindIII Double enzyme digestions, the pUC18-H1 with same double enzyme digestions_YNRCΔ9-SacI-SalI-H2_YNRCΔ9Large fragment is connected, and converts large intestine bar Bacterium Top10 competent cells, extract transformant plasmid and carry out digestion identification, obtain plasmid pUC18-H1_YNRCΔ9-SacI-SalI- H2_YNRCΔ9-URA3-H2_YNRCΔ9.Sequencing proves that cloned sequence is not mutated.

3rd, pGEM-T easy-BAS1-BAS2 plasmid constructions

Enter performing PCR amplification gene BAS1 by template of W303-1A chromosomal DNA, use 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 polymerizations of Quan Shi King Companies production Enzyme, 50 DEG C of annealing 1min, 72 DEG C of extension 2.5min, totally 32 circulations.Obtain 2907bp PCR1 fragments, the addition of this fragment 5 ' end There is restriction enzyme site SacI, include BAS1 gene start codon ATG upstream 762bp sequences and the 2112bp including ATG ORF sequences, delete ORF 3 ' end 324bp (containing terminator codon) sequences.

Enter performing PCR amplification gene BAS2 by template of W303-1A chromosomal DNA, use 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 polymerizations of Quan Shi King Companies production Enzyme, 50 DEG C of annealing 1min, 72 DEG C of extension 1.5min, totally 32 circulations.1692bp PCR2 fragments are obtained, its 3 ' end is added with enzyme Enzyme site Sal I；Include BAS2 gene ORF full sequences (containing terminator codon).

Using PCR1, PCR2 product as template, using P41, P44 as primer pair, the reaction amplification of juxtaposition extension PCR is obtained 4578bp PCR12 fragments.This PCR fragment is carried out to add after A end reactions, agarose gel electrophoresis separation, gel extraction, normally Bright book operation requires to be connected with purchased from the pGEM-T easy carriers of Promega companies, 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 double enzyme digested plasmid pGEM-T easy-BAS1-BAS2 of SacI, Sal I, large fragment SacI-BAS1- is reclaimed BAS2-Sal I, the pUC18-H1 with same double enzyme digestions_YNRCΔ9-SacI-SalI-H2_YNRCΔ9-URA3-H2_YNRCΔ9Large fragment connects Connect, obtain integrated plasmid pUC18-H1_YNRCΔ9-BAS1-BAS2-H2_YNRCΔ9-URA3-H2_YNRCΔ9。

2nd, bacterial strain W303-1A (tpk1 △ tpk2 △ tpk3 △ yak1 △ pde1 △ BAS1BAS2) is built

By plasmid pUC18-H1_YNRCΔ9-BAS1-BAS2-H2_YNRCΔ9-URA3-H2_YNRCΔ9PvuII digestions are used, obtain linear Change DNA fragmentation H1_YNRCΔ9-BAS1-BAS2-H2_YNRCΔ9-URA3-H2_YNRCΔ9；Then W303-1A (tpk1 △ tpk2 △ are converted Tpk3 △ yak1 △ pde1 △) bacterial strain competent cell, " W303-1A (tpk2 △) strain construction " method is sieved in be the same as Example 1 Choosing, identification obtain W303-1A (tpk1 △ tpk2 △ tpk3 △ yak1 △ pde1 △ BAS1BAS2-URA3) bacterial strains and W303-1A (tpk1 △ tpk2 △ tpk3 △ yak1 △ pde1 △ BAS1BAS2) bacterial strain.PCR method checking 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 genes Sub- PCR primer is 7389bp, and successful integration, the transformant PCR primer of URA3 genes ejection are 5929bp, control strain PCR productions Thing is 1687bp.The sequencing result of 5929bp PCR primers is proved to there occurs expected change on chromosome：The site of YNRC Δs 9 There are 321bp sequences to be knocked, substitute insertion is BAS1BAS2 fusion fragments, and this fragment is not undergone mutation；Without URA3 bases Cause.

3rd, bacterial strain W303-1A (tpk1 △ tpk2 △ tpk3 △ yak1 △ pde1 △ BAS1BAS2) growth and cAMP lifes Production is evaluated

Embodiment 5 has been proven that concentration of glucose significantly affects cAMP production.In view of component needed for cAMP synthesis (point Minor is C₁₀H_l2N₅O₆P), while concentration of glucose is improved, thus it is speculated that nitrogen, the content of the content especially nitrogen of phosphorus in culture medium It is likely to become the primary restraining factors of cAMP synthesis.In addition, the preceding extract synthesized as cAMP, the situation of extracellular adenine also pole The expression of each gene of earth effect purine route of synthesis, and then influence cAMP productions.Therefore, contrast has investigated above-mentioned here 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：1st, seed liquor culture be the same as Example 4；2nd, ferment：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 addition of adenine is 0.625,1.25g/L, referred to as A0.625, A1.25；2) Fermentation condition is：Fresh seeds liquid is inoculated into the 100ml volumetric flasks equipped with 25ml fermentation mediums, controls initial OD₆₀₀Value exists 1 or so, 30 DEG C, ferment under 220rpm；3) zymotic fluid is analyzed：HPLC analysis methods in be the same as Example 4, while supplementing adenine HPLC analysis：A, sample treatment and HPLC analyses are identical with the cAMP sample treatments analyzed and HPLC analyses；B, adenine mark Directrix curve makes：Adenine standard items are configured to the standard liquid that concentration is 5mg/mL with the deionized water after sterilizing, then used Deionized water is diluted to it, obtain final concentration be respectively 0.1,0.2,0.3,0.4,0.5mg/mL standard sample, cross and filter out Chromatography is carried out after bacterium, standard curve is done；C, using standard curve by outer marking quantitative method to calculate gland in fermentation broth sample fast The concentration of purine.

Fermentation results are shown in Table 5.The result of table 5 shows：1st, it is all under 1*YP, the fermentation condition of glucose 15%, fusion table altogether Up to the maximum extracellular cAMP concentration of BAS1-BAS2 bacterial strains 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；2nd, improve Dusty yeast and peptone concentration effect are extremely notable；3rd, addition adenine can further improve extracellular cAMP yield.

Table 5, the cAMP productions of W303-1A (tpk1 △ tpk2 △ tpk3 △ yak1 △ pde1 △ BAS1-BAS2) bacterial strain are commented Valency

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 by yeast integration plasmid YIplac211 on the chromosome of FCY2 gene integrations to yeast cells Reach.Using W303-1A chromosomal DNAs template, 558bp, gene promoter containing PGK1 are synthesized by primer pair of primer P47 and P48 Area P_PGK1Product PCR1, by primer pair of primer P49 and P50 synthesize 1630bp, the product PCR2 of the gene containing FCY2, with P51 It is primer pair synthesis 481bp, the sub-district of gene end containing PGK1 T with P52_PGK1Product PCR3.Again using PCR1 and PCR2 fragments as Template, using P47 and P50 as primer pair, juxtaposition extension amplification obtain product PCR12.It is last using PCR12 and PCR3 fragments as Template, using P47 and P52 as primer pair, amplification obtain product PCR123.By the product PCR123 double enzyme digestions of SphI and EcoRI Afterwards, the carrier YIplac211 large fragments with same double enzyme digestions are connected, and obtain the plasmid YIplac211-P of 6.3kb sizes_PGK1- FCY2-T_PGK1.The sequencing result of this plasmid proves cloned sequence P_PGK1-FCY2-T_PGK1It is not mutated, uses StuI linearization for enzyme restriction Bacterial strain W303-1A (tpk1 △ tpk2 △ tpk3 △ yak1 △ pde1 △) competence afterwards in Li-acetate method conversion embodiment 5 is thin Born of the same parents, use CMG^-URAFlat board is screened, and YPAD Liquid Cultures are carried out to obtained transformant bacterial strain, extracts chromosome, with P47, P52 primer pairs enter performing PCR identification, and it is 2.6kb that product, which is expected size,.Fermentation evaluation is carried out to obtained positive transformant bacterial strain： 1st, seed liquor culture be the same as Example 4；2nd, ferment：In fermentation use culture medium be the same as Example 6 (2*YP, glucose 150g/L, A0.625), fermentation condition be the same as Example 6；CAMP maximum outputs reach 10197.8 μm of ol/L.

Embodiment 8：SNQ2 is overexpressed

Table will be carried out by yeast integration plasmid YIplac211 on the chromosome of SNQ2 gene integrations to yeast cells Reach.Using W303-1A chromosomal DNAs template, 4527bp, the ORF sequences of gene containing SNQ2 are synthesized by primer pair of primer P53 and P54 The PCR primer of row, is cut with SalI and BamHI is double, then with the plasmid YIplac211-P of embodiment 7 of same double enzyme digestions_PGK1- FCY2-T_PGK1Large fragment is connected.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) impressions in sour lithium method conversion embodiment 6 State cell, uses CMG^-URAFlat board is screened, and is carried out YPAD Liquid Cultures to obtained transformant bacterial strain, is extracted chromosome, uses P47, P52 primer pair in embodiment 6 enters performing PCR identification, and it is 5.5kb that product, which is expected size,.To obtained positive transformants daughter bacteria Strain carries out fermentation evaluation：1st, seed liquor culture be the same as Example 4；2nd, ferment：(2*YP, Portugal of the fermentation in culture medium be the same as Example 6 Grape sugar 100g/L, A 0.625), fermentation condition be the same as Example 6；CAMP maximum outputs reach 11581.6 μm of ol/L.

Embodiment 9：CAMP production bacterial strain fermentation liquor pulvis raises small white mouse

Bacterial strain in embodiment 5 is pressed to the method in embodiment 6 activate step by step, amplify culture and shake flask fermentation, hair Ferment culture medium is (2*YP, glucose 100g/L).Take 168h zymotic fluids directly freezed to dry and pulvis is made.Raised using experimental animal Pulvis is dissolved in drinking-water feeding small white mouse by material as daily ration.Small white mouse is randomly divided into three groups, every group 8, free water and feeding Start addition two days later；Addition is followed successively by：0th, 0.5,10mg cAMP/ (kg body weight), addition in every 2 days once with carry out once Weigh, add 10 times altogether；Elder generation fasting >=10h before addition, then weighs, records body weight and calculate addition pulvis amount, single group is used Pulvis is dissolved in 20mL drinking-water.Blood sampling sampling carries out Virus monitory, and anatomic observation internal organ after last time is weighed.As a result show： 1st, increase weight：20th day average weight is respectively 37.54 ± 1.89g, 41.41 ± 0.48g and 40.12 ± 1.28g, and addition group is averaged 10.20% and 6.87%, dosage 0.5mg cAMP/ (kg body weight) significant difference (P is respectively increased compared with control group average weight in body weight <0.05)；2nd, toxicity：The dissection and analysis result of small white mouse is shown：Maximum dose 10mg cAMP/ (kg body weight) are organized and control group phase Than the internal organs such as heart, liver, spleen, lung, kidney without substantially pathological change is observed, show that pulvis does not have to small white mouse Significant acute toxicity；3rd, serum analysis 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 pole is significantly higher than control group (p ＜ 0.01), illustrates that addition cAMP preparations can really effectively improve protein in body content.

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

Bacterial strain in embodiment 6 is pressed to the method in embodiment 6 activate step by step and amplify culture, then in fermentation tank In use fermentation medium (2*YP, glucose 100g/L), 30 DEG C, ferment under 200-700rpm 120h, the direct low temperature of zymotic fluid Powder processed is dried, is used as cAMP pulvis.

From young 180 plumages of the healthy white meat-type chickens of 1 age in days, the adaptability culture of 6 days is first carried out, then each half-press body weight of male and female Three processing are randomly divided into, each 3 repetitions of processing, it is each to repeat 20.The pulvis addition of three processing is set to 0.5th, 2.5,0mgcAMP/ (kg body weight).Once weighed every 5 days and a cAMP pulvis addition, front and rear addition 7 times altogether, Whole incubation time 42 days.Elder generation fasting >=10h and taboo water about 2h, then weighs and calculates addition pulvis amount before addition.CAMP powder Agent is directly appended in drinking-water, and feed is recovered while supplying this drinking-water.The 25th day and the 35th day after adding first (whole incubation time is respectively 32 days and 42 days) each repeats to take two chickens to be butchered, and analysis organ index and serum refer to Mark.Tianjin Tuan Bowa farms chicken farm is carried out.Using net-rearing mode, domestic briquet stove heating temperature control.Free choice feeding and drink Water.Chicken mixed feed in the meat chicken mixed feed (0-21 ages in days use) and meat that are produced using Cangzhou Hong Yi feed corporation,Ltds (22-42 ages in days use).

6 are the results are shown in Table, as shown in Table 6：1st, on growth performance, addition pulvis improves daily gain extremely significantly (p ＜ 0.01), And feedstuff-meat ratio declines also extremely significantly (p ＜ 0.01)；2nd, 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, addition pulvis raising bursal index is notable (p ＜ 0.05), shows that the development to broiler chicken immune organ has facilitation；3rd, on Serum Indexes, cholesterol in serum and highly dense Degree lipoprotein be all remarkably higher than control group (p ＜ 0.05), illustrate addition pulvis can promote body fat metabolism with 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 body weight)	2.5mg cAMP/ (kg body weight)	0mg cAMP/ (kg body 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
HDL 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 represents significant difference (P<0.05), capitalization difference person represents difference Extremely significantly (P<0.01)

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

Embodiment 11：CAMP production bacterial strain fermentation liquor pulvis raises pig

Be the same as Example 10 is fermented, fermented liquid spray drying powder, is used as cAMP pulvis.

The healthy weanling pig 12 of 30kg body weight is selected, male and female half and half is randomly divided into 3 groups, every group 4；3 groups of processing A, It is respectively 0.75,1.5 and 0mg cAMP/ (kg body weight) to handle B, processing C cAMP pulvis additions.Carried out once every 5 days Weigh and a cAMP pulvis addition, terminate when reaching 90kg to body weight.CAMP pulvis is directly appended in drinking-water, is controlled before addition Pig empty stomach >=10h processed simultaneously weighs.Feed basal diet, free choice feeding and drinking-water.As a result show：1st, 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)；2nd, three groups of feedstuff-meat ratios be respectively 2.95 ± 0.11,2.690 ± 0.09 and 3.01 ± 0.23kg/d, processing A, B relatively processing C reductions by 1.99%, 10.63% respectively, and handle B and be substantially less than processing C (p ＜ 0.05).As a result prove that yeast cAMP zymotic fluid pulvis is remarkably improved the growth performance of pig.

It regard the fermentation by saccharomyces cerevisiae liquid or zymotic fluid pulvis of the present invention as ruminant animals and the feed of aquatic product animal Additive, also observed similar result.

Sequence table

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<213>Artificial sequence (Artificial Sequence)

<400> 10

cccgggctgc agggatccat ccctgaaggc ttaaatag 38

<210> 11

<211> 32

<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, it is characterised in that the yeast includes the first and second of genetic modification, wherein, it is described the first Gene is protein kinase A (protein kinase A, PKA) catalytic subunit encoding gene TPK1, TPK2 and TPK3, passes through modification The first gene is totally constrained PKA activity or expression, so as to eliminate to CAMP (cyclic adenosine Monophosphate, cAMP) feedback inhibition, but simultaneously cause yeast growth be suppressed, second of genetic modification is eliminated Growth inhibition caused by the first genetic modification so that yeast can normal growth, the cAMP yield of the yeast rises, Wherein, the cAMP yield rises, and is for the cAMP yield of the yeast without genetic modification.

2. yeast according to claim 1, it is characterised in that second of gene is encoded including protein kinase Rim15 Gene RIM15, transcription factor Msn1/Msn2 encoding genes MSN1/MSN2, protein kinase Yak1 encoding genes YAK1 and/or egg White kinases Sch9 encoding gene SCH9, preferably YAK1.

3. according to any described yeast of claim 1-2, it is characterised in that second of genetic modification causes institute's modification base Because the activity of codase or expression are totally constrained or the activity raising of codase or overexpression.

4. according to any described yeast of claim 1-3, it is characterised in that the yeast also includes the third genetic modification, To reduce cAMP degraded, so as to improve cAMP yield.

5. yeast according to claim 4, it is characterised in that the third described gene is that cAMP phosphodiesterases encode base Because of PDE1 and/or PDE2, preferably PDE1.

6. according to any described yeast of claim 4-5, it is characterised in that the third described genetic modification causes institute's modification base Because the activity of codase or expression are totally constrained.

7. according to any described yeast of claim 1-6, it is characterised in that the yeast also includes the 4th kind of genetic modification, To increase the positive regulation and control of the synthesis of cAMP precursors in purine route of synthesis, the synthesis of cAMP precursors is set to increase, so as to improve CAMP yield.

8. yeast according to claim 7, it is characterised in that the 4th kind of gene includes transcription factor Bas1 and Bas2 Encoding gene.

9. according to any described yeast of claim 7-8, it is characterised in that the 4th kind of genetic modification compiles modifier The activity of code enzyme is improved or is overexpressed.

10. according to any described yeast of claim 1-9, it is characterised in that the yeast also includes the 5th kind of genetic modification, To increase the regulation and control to transporting and accumulating outside cAMP and its precursor intracellular, cAMP is set to synthesize and secretion rising, so as to improve CAMP yield.

11. yeast according to claim 10, it is characterised in that the 5th kind of gene includes plasma membrane carrier protein Fcy2 Encoding gene and Snq2 encoding genes.

12. according to any described yeast of claim 10-11, it is characterised in that the 5th kind of genetic modification makes modification base Because the activity of codase is improved or is overexpressed.

13. according to any described yeast of claim 1-12, it is characterised in that the activity for causing codase or expression quilt The genetic modification mode of complete inhibition include point mutation, missing, insertion, antisense polynucleotides, siRNA, microRNA、CRISPR；So that the genetic modification mode that the activity of codase is improved or is overexpressed includes point mutation, connects strong Promoter, link enhancement, raising copy number or fusion coexpression.

14. according to yeast of the claim 1-13 in any, it is characterised in that the yeast is saccharomyces cerevisiae (Saccharomyces cerevisiae), saccharomyces pastorianus (Saccharomyces pastorianus), pichia stipitis In (Pichia stipitis), Saccharomyces Bayanus and shehatae candida (Candida shehatae) Any one, preferably saccharomyces cerevisiae (Saccharomyces cerevisiae).

15. a kind of method for building any described yeast of claim 1-14, it is characterised in that methods described includes：First The first genetic modification is introduced in yeast, the feedback inhibition to cAMP is eliminated, but causes the growth of yeast to be suppressed simultaneously, its It is secondary, introduce second of genetic modification, second of genetic modification eliminate growth inhibition caused by the first genetic modification, so that Obtaining yeast being capable of normal growth.

16. yeast production cAMP any a kind of utilization claim 1-14 method, it is characterised in that methods described is included in Above-mentioned yeast is cultivated in fermentation medium.

17. a kind of zymotic fluid of yeast fermenting and producing any utilization claim 1-14.

18. use of the zymotic fluid described in any described yeast of claim 1-14 or claim 17 in production cAMP On the way.

19. the zymotic fluid described in any described yeast of claim 1-14 or claim 17 is in medicine, animal husbandry, food The application of product, health products or chemical field.

20. application as claimed in claim 19, it is characterised in that the application is to be added as the feed or feed of animal husbandry Agent.

21. a kind of method for the product for preparing medicine, animal husbandry, food, health products or chemical field, it is characterised in that described Method prepares the said goods using the zymotic fluid described in any claim 1-14 yeast or claim 17.

22. method as claimed in claim 21, it is characterised in that methods described includes the yeast or zymotic fluid entering one Step is processed into any other formulation, preferably edible formulation, more preferably pulvis, granule, tablet, capsule or liquid Etc. form.

23. a kind of feed or feed addictive, it is characterised in that the feed or feed addictive are included will using right The product for asking any described yeast of 1-14 or the zymotic fluid described in claim 17 to prepare, it is preferred that the yeast or Zymotic fluid is further processed into any other formulation, preferably edible formulation, more preferably pulvis, granule, tablet, glue The form such as capsule or liquid.