CN1599796A - Genetic strain optimization for improving the production of riboflavin - Google Patents

Abstract

The invention relates to the microbial production of riboflavin by culturing a microorganism of the genus Ashbya that is capable of producing riboflavin and that has at least in two of the genetic products from the group rib1, rib2, rib4 and rib7 higher activities than the wild type ATCC 10895, and subsequently isolating the riboflavin produced from the culture medium.

Description

Be used to improve the optimization of the hereditary bacterial strain of riboflavin production

The present invention relates to recombinate and produce the method for riboflavin.Because the riboflavin synthetic gene of AshbyaCif (Ashbya) biology or the combination and their expression of gene have been carried out special selection, and the output of riboflavin in these biologies is improved.

Wei ShengsuB2 is also referred to as riboflavin, and all plants and many microorganisms can both produce this material.Because the human and animal can not riboflavin biosynthesis, so riboflavin is essential.Riboflavin plays an important role in metabolism.For example it participates in the utilization of carbohydrate.The shortage of Wei ShengsuB2 can cause mouthful and inflammation, the skin folds in a garment of pharynx mucous membrane are scratched where it itches, inflammation and similar skin injury, conjunctivitis, visual sensitivity descend and corneal clouding.Lack B2 in the infants and can cause delayed growth and weight loss.Therefore Wei ShengsuB2 has important economic implications, for example it can be avitaminous the time as the vitamin supplement thing and can be used as fodder additives.It also can add in the numerous food product and goes.In addition, Wei ShengsuB2 also can be used as the tinting material as food such as mayonnaise, ice-creams and iced milks.

But Wei ShengsuB2 chemical preparation or produce (referring to as B.Kurth etc., 1996, see: Ullmann ' s Encyclopedia of industrial chemistry, the riboflavin in VCH Weinheim one book) with microorganism.In the method for chemosynthesis riboflavin, usually will be rapid and must use higher initial substance of price such as D-ribose could obtain highly purified product through multistep.

Using microbial fermentation to produce Wei ShengsuB2 is a kind of alternative approach of chemosynthesis riboflavin.The initial substance of this method is reproducible starting material, as sugar or vegetables oil.The fermentation that is used for producing riboflavin is known just like the false capsule yeast (Eremothecium ashbyii) of A Shu or ashbya gossypii (Ashbya gossypii) (The Merck Index, editors such as Windholz, Merck﹠amp with fungi; Co., 1183 pages, 1983), but yeast such as mycocandida (Candida), Pichia (Pichia) and yeast belong (Saccharomyces), or bacterium such as bacillus (Bacillus), fusobacterium (Clostridium), bar shaped bacteria belong to the producer that (Coryne bacteria) etc. also is described as riboflavin.EP-A-0405370 and EP-A-0821063 have described and have used the recombinant bacteria bacterial strain to carry out the production of riboflavin, and this recombinant bacterial strain transforms subtilis (Bacillus subtilis) with riboflavin biosynthesis gene and obtains.

WO 95/26406 and WO 94/11515 described the process how gene that the riboflavin biosynthesizing is special is cloned from eukaryote ashbya gossypii and yeast saccharomyces cerevisiae (Saccharomyces cerevisiae), also described the purposes that microorganism and this quasi-microorganism with these gene transformation are used for riboflavin biosynthesis.

The selection that WO 99/61623 has described riboflavin biosynthesis gene (rib3, rib4, rib5) is how to be used for improving riboflavin production.

In above-mentioned biology, begin to have the generation of 6 kinds of enzyme catalysis riboflavin from guanosine triphosphate (GTP) and ribulose-5-phosphate.Wherein, GTP cyclization hydrolase-II (rib1 gene product) is converted into 2 with GTP, 5-diamino-6-ribosyl amino-4-(3H)-pyrimidone-5-phosphoric acid.2,5-diamino-6-ribosyl amino-4-(3H)-pyrimidone-5-phosphoric acid reduction enzyme (rib7 gene product) is next with 2,5-diamino-6-ribosyl amino-4-(3H)-pyrimidone-5-phosphoric acid reduction generates 2,5-diamino-6-ribosyl amino-2,4-(1H, 3H)-pyrimidone-5-phosphoric acid, subsequently 2,5-diamino-6-ribosyl amino-2,4-(1H, 3H)-pyrimidone-5-phosphoric acid generated 5-amino-6-ribosyl amino-2 by special desaminase (rib2 gene product) deaminize, 4-(1H, 3H)-pyrimidone-5-phosphoric acid.This phosphoric acid ester is again by non-specific Phosphoric acid esterase dephosphorylation.

Ribulose-5-phosphate is another initial substance except that GTP in the last enzymatic step of riboflavin biosynthesizing, ribulose-5-phosphate is by 3,4-dihydroxyl-2-butanone-4-phosphate synthase (rib3 gene product) is converted into 3,4-dihydroxyl-2-butanone-4-phosphoric acid (DBP).

DBP and 5-amino-6-ribosyl amino-2, and 4-(1H, 3H)-pyrimidone all is that enzymatic synthesizes 6,7-dimethyl-8-ribosyl-2, the parent material of 4-lumazine.This reaction is by rib4 gene product (DMRL synthase) catalysis.DMRL then is converted into riboflavin by riboflavin synthase (rib5 gene product), and (Bacher etc. (1993), Bioorg.Chem.Front.3 rolls up, SpringerVerlag).

Although on riboflavin produces, obtained these progress, still be necessary to improve and the productivity that improves riboflavin with the increasing of adaption demand, and promote the efficient that riboflavin produces.

An object of the present invention is further to improve the productivity of Wei ShengsuB2.Method by microorganisms riboflavin, the inventor has realized this purpose, this method is by cultivating a kind of microorganism that can produce the AshbyaCif of riboflavin, separate the riboflavin that is produced from substratum subsequently, the microorganism of described AshbyaCif is compared with wild-type ATCC 10895 at the product of two kinds of genes that are selected from rib1, rib2, rib4 and rib7 demonstrate higher activity at least.

The method of this raising riboflavin production preferably demonstrates the active biology that improves (numeral all represent discussed rib gene product) with the combination of can riboflavin biosynthesis and wherein having a for example following rib gene product: 1+2,1+4 under each situation, 1+7,2+4,2+7,4+7.

Particularly preferably be such biology, the combination of wherein following rib gene product demonstrates active raising (numeral under each situation all the rib gene product discussed of expression): 1+2+4,1+2+7,1+4+7,2+4+7.

The activity that these rib gene products improve is assessed, and this is to implement by comparing with ashbya gossypii strains A TCC 10895 with biology for referencial use.The methods involving of measuring rib gene product activity that is enzymic activity is conventionally known to one of skill in the art, and describes to some extent in the literature.

For the productivity that improves Wei ShengsuB2, more beneficial method is that importing with the raising of natural enzymic activity and said gene combination combines and improves genetic expression.

Suitable biology of the inventive method or host living beings can be the biology that all can riboflavin biosynthesis in the AshbyaCif in principle.

Term rib gene product not only refers to SEQ ID NO:2 in the sequence table, 4,6,8 described peptide sequences, also refer to the peptide sequence that those can obtain by replacement, insertion or the deletion of these sequences being carried out amino acid code, wherein replace, insert or delete in amino acid code to being no more than 5%, preferably be no more than 3%, particularly preferably be no more than 2% peptide sequence.For example allelic naturally variation can produce above-mentioned sequence, and they also can obtain by original strain being carried out mutagenic treatment (for example mutagenic compound or electromagnetic radiation) and carry out the screening that riboflavin productivity improves subsequently.

The activity that the combination of rib1, rib2, rib4 and rib7 gene and/or these genes and their gene product improve according to the present invention significantly improves riboflavin productivity.Above-mentioned gene can well known to a person skilled in the art that at present method imports in the biology by all in principle; Advantageously, with prior aries such as particle gun or microinjections these gene transformation, transfection, electroporation are imported in biology or their cell.With regard to microorganism, those skilled in the art can find suitable method from following textbook: Sambrook J. etc., (1989) molecular cloning: laboratory manual, Cold SpringHarbor Laboratory Press; F.M.Ausubel etc., (1994) modern molecular biology technique (Current protocols in molecular biology), John Wiley and Sons; D.M.Glover etc., dna clone (DNA Cloning) volume 1, (1995), IRL Press (ISBN019-963476-9); Kaiser etc. (1994) yeast genetics method (Methods in YeastGenetics), Cold Spring Harbor Laboratory Press or Guthrie etc., yeast genetics and molecular biology guide (Guide to Yeast Genetics and Molecular Biology), Enzymology method, 1994, Academic Press.The example of the useful method that can mention is for example at the ura-3 gene particularly under the assistance of AshbyaCif ura-3 gene, import DNA by homology or allos reorganization, German application DE 19801120.2 and/or following REMI method (=" integration of restriction enzyme-mediated ") are described this.

The REMI technology is to turn to the basis with two ends with the linear DNA construct of same restrictions endonuclease digestion and the corotation that is used for the restriction enzyme of restricted this DNA construct of cutting.Therefore, this restriction enzyme is sheared the genomic dna of following biology, and wherein said biology is for importing the biology of above-mentioned DNA construct and Restriction Enzyme.This causes the homology repair mechanism to be activated.These repair mechanisms are repaired the splitting of chain that is caused by restriction endonuclease on the genomic dna, meanwhile, also can the DNA construct of cotransformation be mixed genome with certain frequency.Usually, the restriction enzyme site at DNA two ends obtains keeping.

B lker etc. in the insertion mutagenesis of fungi, described this technology (Mol Gen Genet, 248,1995:547-552).In order to illustrate the existence of the reorganization of allos in the yeast, and Schiestl and Petes (Proc.Natl.Acad.Sci.USA, 88,1991:7585-7589) used this method.(Mol.Gen.Genet.251 1996:75-80) has described stable conversion and the adjustable expression that this method is used for the induction type reporter gene to Brown etc.Up to now, this system did not also use as the genetic engineering instrument of optimizing pathways metabolism or proteic commercial overexpression.

The biosynthesizing of relevant riboflavin, shown that biosynthesis gene can be integrated in the genome of above-mentioned biology by the REMI method, and can optimize the production process of the meta-bolites that the primary and secondary metabolism produced, biosynthetic pathway particularly, wherein said meta-bolites for example amino acid (as Methionin, methionine(Met), Threonine or tryptophane), VITAMIN is (as vitamin A, B2, B6, B12, C, D, E, F, S-adenosylmethionine, vitamin H, pantothenic acid or folic acid), carotenoid is (as β-Hu Luobusu, lycopin (lycopin), canthaxanthin, astaxanthin or zeaxanthin), or protein such as lytic enzyme are (such as lipase, esterase, Ntn hydrolase, nitrilase, proteolytic enzyme), medium such as cytokine, for example lymphokine is (as MIF, MAF, TNF), interleukin-such as interleukin 1, Interferon, rabbit (as gamma-interferon), tPA, hormone is (as proteohormone, C-21 cortico-steroid), oligopeptides hormone or polypeptide hormone are (as antidiuretic hormone, endorphin, Endostatin (endostatin), angiostatin (angiostatin), the somatomedin erythropoietin, transcription factor, integral protein is (as GPIIb/IIIa or α _vβ and III), acceptor (as multiple glutamate receptor) or angiogenesis factor such as Angiotensin.

Utilize the REMI method nucleic acid fragment of nucleic acid fragment of the present invention or said gene can be placed the transcriptional activity site of genome.

It is useful that nucleic acid is cloned on the DNA construct with at least one reporter gene, and this DNA construct is with in the quiding gene group.This reporter gene should be easy to detect by growth analysis, fluorometric analysis, chemiluminescence analysis, bioluminescent assay or by photometry.The example of the reporter gene that can mention has antibiotics resistance gene, hydrolase gene, fluorescence protein gene, bioluminescent gene, alpha-glucosidase gene, peroxidase gene or biosynthesis gene (as riboflavin gene, luciferase genes, beta-galactosidase gene, gfp gene, lipase gene, esterase gene, peroxidase gene, β-Nei Xiananmei gene, acetyl transferase gene, phosphoric acid transferase gene or adenylyl transferase gene).These genes make the detection of transcriptional activity and quantitatively be easy to carry out, and therefore expression of gene are detected and quantitatively also are easy to carry out.Therefore, the positioning instant on genome can be determined, and its difference that shows productivity is no more than 2 times of (see figure 1)s.Fig. 1 shows clone LU21#1 and the LU21#2 that obtains by integration, and their different Wei ShengsuB2 (=riboflavin) productivity.

And if biosynthesis gene itself is easy to detect, such as the such situation of riboflavin among the application, also reporter gene that can be extra.

If import more than one of biological intravital gene, these all genes can be cloned into common importing organism in the carrier with a reporter gene, each gene also can be cloned on other carrier of branch with a reporter gene respectively, and these are divided other carrier while or import organism successively.The coding active gene fragment of discussing also can use the REMI technology to operate.

In principle, all known Restriction Enzymes all are applicable to the inventive method biosynthesis gene is integrated in the biological gene group.But only discern the Restriction Enzyme of 4 base pair restriction enzyme sites because they shear overfrequency in genome that desire is integrated or carrier not by preferably; Preferably discern 6,7,8 or more base pairs as the Restriction Enzyme of restriction enzyme site, such as BamHI, EcoRI, BglII, SphI, SpeI, XbaI, XhoI, NcoI, SalI, ClaI, KpnI, HindIII, SacI, PstI, BpnI, NotI, SrfI or SfiI, and other possible Restriction Enzyme of not mentioning.When used Restriction Enzyme no longer includes restriction enzyme site on the DNA that desire imports is favourable, can improve integration efficiency like this.Usually in the REMI mixture, the Restriction Enzyme consumption is 5-500U, preferred 10-250U, preferred especially 10-100U.Restriction Enzyme is advantageously used in the aqueous solution that comprises the seepage stability material, wherein said seepage stability material such as carbohydrate (such as sucrose, trehalose or glucose), polyol (as glycerine or polyoxyethylene glycol), buffer reagent with favourable buffering range pH5-9, preferred buffering range pH6-8, preferred especially pH7-8, wherein said buffer reagent such as Tris, MOPS, HEPES, the material of MES or PIPES and/or stable nucleic acid is as inorganic salt or the organic salt of Mg, Cu, Co, Fe, Mn or Mo.As required, also can there be other material such as EDTA, EDDA, DTT, beta-mercaptoethanol or nucleic acid inhibitor.But the REMI technology can not carried out when having these additives yet.

Method of the present invention is carried out in 5-80 ℃ temperature range, and preferred 10-60 ℃, preferred 20-40 ℃ especially.All known unsettled methods of cytolemma that make all are applicable to method of the present invention, for example electroporation, merge with the delivery vesica or go to stablize, preferred lithium salts wherein with multiple an alkali metal salt or alkaline earth salt such as lithium salts, rubidium salt or calcium salt.

Nucleic acid can be directly used in after the separation or purifying after be used for reaction of the present invention.

In principle, available all methods that well known to a person skilled in the art import plant with the combination that the present invention of rib gene mentions.

Foreign gene is transferred to Plant Genome is called conversion.In this application, described conversion and can be used for carrying out instantaneous or stable conversion from the method for plant tissue or vegetable cell aftergrowth.Suitable method has the protoplast transformation taken in by the polyoxyethylene glycol inducing DNA, uses particle gun, electroporation, cultivation, microinjection and the agriculture bacillus mediated transgenosis of dry embryo in containing the solution of DNA.Aforesaid method is at for example B.Jenes etc., gene transfer technique (Techniques forGene Transfer), see: transgenic plant, volume 1, Engineering and Utilization, S.D.Kung and R.Wu edit, Academic Press (1993) 128-143 and in Potrykus Annu.Rev.Plant Physiol.Plant Molec.Biol.42 (1991) 205-225 description is arranged.Preferably, construct to be expressed is cloned into the carrier that is suitable for transforming agrobacterium tumefaciens (Agrobacterium tumefaciens), as pBin19 (Bevan etc., Nucl.AcidsRes.12 (1984) 8711).Transform plant at for example H fgen and Willmitzer with agrobacterium tumefaciens, Nucl.Acid Res. (1988) 16, in 9,877 one literary compositions description arranged.

According to the present invention, the Agrobacterium of carrying expression vector also can be by known method, for example immerse in the Agrobacterium solution by leaf and blade with cutting, in suitable medium, cultivate subsequently and transform plant, crop plants particularly is as cereal, corn, soybean, paddy rice, cotton, beet, rape, Sunflower Receptacle, flax, hemp, potato, tobacco, tomato, rape, alfalfa, lettuce, multiple trees, nut, grapevine and beans.

Genetically modified vegetable cell can well known to a person skilled in the art that method regenerates by all.Suitable method finds in the paper that Potrykus or H fgen and Willmitzer deliver at above-mentioned S.D.Kung and R.Wu.

There are a lot of possibilities can improve the enzymic activity of rib gene product in the cell.

To such an extent as to being 1,2,4 or 7 activity by the enzyme of modifying their codings of endogenous rib1,2,4,7 genes someway, a kind of possibility shows compared with the higher activity of beginning enzyme.By the conversion that the change of for example catalytic center improves substrate, in perhaps passing through and the effect of enzyme inhibitors, the raising that can produce different aspect to enzymic activity, that is their specific activity is enhanced or their activity is not suppressed.At one more in the advantageous embodiments, the raising of enzymic activity also may realize by the raising of enzymic synthesis in the cell, for example by removing the inhibitory enzyme synthetic factor, perhaps improves the factor of promotion enzymic synthesis or the activity of regulatory element, perhaps preferably, by importing more gene copy.These measures improve the overall activity of gene product in the cell and do not change its specific activity.These methods also can be used in combination, that is specific activity and overall activity all are improved.Basically, can modify the nucleotide sequence of these genes, controlling element or its promotor by well known to a person skilled in the art method.For this purpose, sequence can be carried out for example mutagenesis, such as carrying out site-directed mutagenesis, and D.M.Glover etc., DNA Cloning, volume 1, (1995), and IRL Press (ISBN 019-963476-9), the 6th chapter, 193 pages have description to this.

Spee etc. (3 phases, 1993:777-778) described and utilize dITP to carry out the PCR method of random mutagenesis by Nucleic Acids Research, 21 volumes.

(Proc.Natl.Acad.Sci.USA, 91 roll up Stemmer, 1994:10747-10751) have described and have utilized extracorporeal recombination to carry out the research of molecular evolution.

(Nature Biotechnology, 14 roll up Moore etc., 1996:458-467) have described the combination of PCR method and recombination method.

Nucleotide sequence after the modification returns in the organism by carrier subsequently.

In order to improve enzymic activity, the upstream that also promoter region after modifying can be placed natural gene to be improving the expression of gene level, and therefore improves enzymic activity.Thereby also can import and for example improve the sequence that mRNA stability improves translation skill at 3 ' end.This also can bring the raising of enzymic activity.

Preferably, with in extra rib1,2, the 7 and 4 gene copies associating transfered cell.Can carry out natural adjusting to these gene copies, can improve the modified adjusting of genetic expression, maybe can adopt heterologous gene or reality to be the adjusting sequence of different plant species gene to such an extent as to can carry out being modified them at natural regulatory region.

Aforesaid method is combined especially favourable.

In the methods of the invention, sequence SEQ ID No.1, SEQ ID No.3, SEQ ID No.5 and SEQ ID No.7 or their function equivalent are carried out in conjunction with being favourable.

In order to realize the optimum expression of heterologous gene in biology, it is favourable according to the specific codon use of biology nucleotide sequence being modified.The codon use is easy to by computer other biological knowns of discuss to be calculated assessment and determines.

The positive regulating factor that increases rib1,2,7 and 4 gene copy number and/or strengthen rib1,2,7,4 genetic expressions all will advantageously improve rib1,2,7,4 expression of gene.Therefore, transcribe signal such as promotor and enhanser by force to improve regulatory element on transcriptional level be preferred by using.For example the stability by improving rib1,2,7,4 mRNA or by improving the code efficiency of reading of this mRNA on the rrna also may realize the raising of translation skill but in addition.

In order to improve gene copy number, for example rib gene 1,2,7,4 or homologous gene can be mixed nucleic acid fragment or carrier, wherein said nucleic acid fragment or carrier preferably comprise the regulatory gene sequence of rib gene or the promoter activity with similar effect.

Employed adjusting sequence especially improves the sequence of genetic expression for those.But, alternatively, each said gene can be imported separate carrier and be transformed into the production of being discussed and use in the biology.

Nucleic acid fragment of the present invention is interpreted as rib gene order SEQ ID No.1, SEQ ID No.3, SEQ ID No.5 and SEQ ID No.7 or their function equivalent that operationally is connected with one or more conditioning signals, and wherein said conditioning signal preferably can improve the conditioning signal of genetic expression.These regulate sequence, for example with elicitor or repressor bonded sequence, therefore can regulate expression of nucleic acids.Except these new adjusting sequences, be not these sequences in other words, the natural adjusting of these sequences still can be present in the upstream of practical structure gene in addition, and as required, can carry out genetic modification to them, thereby remove natural adjusting, improve genetic expression.But, gene construct also can be structurally simpler, that is to say does not have extra conditioning signal to be inserted into the sequence upstream of SEQ IDNo.1, SEQ ID No.3, SEQ ID No.5 or SEQ ID No.7 or their function equivalent, and natural promotor and its adjusting are not removed.The substitute is, natural adjusting sequence is suddenlyd change, thereby makes adjusting no longer take place and improve genetic expression.The promotor of these modifieds also can be inserted into their natural gene upstream to improve activity.In addition, gene construct can advantageously comprise one or more known enhancer sequence that are operably connected with promotor, the expression that might improve nucleotide sequence like this.Extra favourable sequence such as other regulatory element or terminator can insert 3 ' end of dna sequence dna.One or more rib gene copies can appear in the gene construct.

The example that is used for the favourable adjusting sequence of the inventive method is for example promotor cos, tac, trp, tet, trp-tet, lpp, lac, lpp-lac, lacI ^q, T7, T5, T3, gal, trc, ara, SP6, λ-P _ROr λ-P _LPromotor, these sequences are advantageously used in gram negative bacterium.And more favourable adjusting sequence is for example Gram-positive promotor amy and SPO2, yeast or fungal promoters ADC1, MF α, AC, P-60, CYC1, GAPDH, TEF, rp28, ADH, or plant promoter CaMV 35S[Franck etc., Cell 21 (1980) 285-294], PRP1[Ward etc., Plant.Mol.Biol.22 (1993)], SSU, OCS, lib4, usp, STLS1, B33, LEB4, no, perhaps on ubiquitin or phaseollin promotor.Among the application, also be favourable for example from pyruvic carboxylase in the Hansenula and methanol oxidase promotor.And more favourable plant promoter has for example benzene fulfonic amide inductive promotor (EP 388186), tsiklomitsin inductive promotor (Gatz etc., (1992) Plant J.2,397-404)), dormin inductive promotor (EP 335528) or ethanol or pimelinketone inductive promotor (WO 9321334).Those guarantee that the plant promoter of expressing is especially favourable in the tissue of purine or its precursor biosynthesizing happening part or plant part.Guarantee that the specific expressed promotor of leaf must mention especially.Mention potato kytoplasm FBPase promotor or potato ST-LSI promotor (Stockhaus etc., EMBO J.8 (1989) 2,445 245) especially.Glycine max phosphoribosyl pyrophosphate (PRPP) amide transferase promotor (also seeing Genbank accession number U87999) or another tubercle specific promoter of mentioning in EP 249676 also can advantageously obtain utilizing.

Basically, all natural promoters and its are regulated sequence, for example above-mentioned those, may be used to method of the present invention.In addition, the synthetic promotor also can advantageously obtain utilizing.

Import biological other interior gene and can as foregoing description, place (=gene construct) on the nucleic acid fragment extraly.These genes can be subjected to independently regulating or placing under the regulatory region identical with the rib gene.These genes can be for example to improve other biosynthesis gene of synthetic.

For the expression in above-mentioned host living beings, nucleic acid fragment advantageously inserts on the carrier of the genetic expression optimization that can make in the host bacterium, for example plasmid, phage or other DNA.The example of suitable plasmid has, for example the pLG338 in the intestinal bacteria, pACYC184, pBR322, pUC18, pUC19, pKC30, pRep4, pHS1, pHS2, pPLc236, pMBL24, pLG200, pUR290, pIN-III ¹¹³-B1, λ gt11 or pBdCI, pIJ101 in the streptomycete, pIJ364, pIJ702 or pIJ361, pUB110 in the bacillus, pC194 or pBD214, pSA77 in the coryneform bacteria or pAJ667, pALS1 in the fungi, pIL2 or pBB116,2 μ M, pAG-1, YEp6, YEp13 or pEMBLYe23 in the yeast, the pLGV23 in the plant, pGHlac+, pBIN19, pAK2004 or pDH51, or the derivative of above-mentioned plasmid.The above-mentioned plasmid of mentioning only constitutes alternative less plasmid.Other plasmid is conventionally known to one of skill in the art, and can find in for example cloning vector (Cloning Vectors) (Amsterdam-New York-Oxford, 1985, ISBN 0 444 904018 for editor such as Pouwels P.H, an Elsevier) book.Suitable plant vector especially at " molecular biology of plants and biotechnological means (Methods in PlantMolecular Biology and Biotechnology) " (CRC Press) 6/7 chapter, is described in the 71-119 page or leaf.

In order to express the gene of other existence, this nucleic acid fragment preferably also comprises in addition and is used to improve 3 ' of expression-and/or 5 '-terminal sequence of regulating, and this sequence is selected to optimize the relation of selected host living beings and gene and expression.

These are regulated sequence and are used for making have the gene of control and protein expression to become possibility.This means, depend on host living beings, for example, gene is only expressed after inducing and/or is crossed and express, or expresses and/or cross expression immediately.

Among the application, regulate the sequence or the factor and can preferably have the forward regulating effect, and the genetic expression that therefore improves quiding gene.Thereby, preferably, transcribe signal such as promotor and/or enhanser are realized regulatory element at transcriptional level enhancing by force by using.But in addition, the enhancing of translation also may realize by the stability that for example improves mRNA.

In another embodiment of carrier, gene construct of the present invention advantageously imports in the microbe with the form of linear DNA and is integrated into the genome of host living beings by allos or homologous recombination.This linear DNA can be by linearizing plasmid or only is made up of the nucleic acid fragment as carrier.

Any can be in cell the plasmid (but particularly carrying the plasmid of yeast saccharomyces cerevisiae 2 μ m plasmid replication starting points) of self-replicating, all can be used as carrier equally, but equally as previously mentioned, the linear DNA fragment that can be incorporated in the host chromosome also can be used as carrier.This integration can realize that (140 roll up, 1995:973-987) for Steiner etc., Genetics but preferably integrate by homologous recombination like that as mentioned by allos or homologous recombination.Among the application, gene rib1, rib2, rib4 and rib7 can be present in genomic different positions respectively or be present in and maybe can be co-located on the different carriers on genome or the carrier.

Be used for the inventive method comprise rib gene 1,2,7 and 4 or their biology of combination of function equivalent demonstrate the raising of riboflavin production.

In the methods of the invention, be used for producing the biological culture of riboflavin at the substratum that allows its growth.These substratum can be synthetic substratum or natural substratum.Can use and select to be fit to the substratum that well known to a person skilled in the art of this biological growth.Used substratum comprise carbon source, nitrogenous source, inorganic salt and comprise a small amount of VITAMIN as required and trace element to support microorganism growth.

Favourable carbon source example is that sugar is (as monose, disaccharides or polysaccharide are (as glucose, fructose, seminose, wood sugar, semi-lactosi, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or Mierocrystalline cellulose), compounding sugar source (as molasses), sugar phosphoric acid salt is (as 1, the 6-hexose diphosphate), sugar alcohol (as N.F,USP MANNITOL), polyol (as glycerine), alcohols (as methyl alcohol or ethanol), carboxylic acid is (as citric acid, lactic acid or acetate), fat (soybean oil or Canola oil), amino acid such as aminoacid mixture (as casamino acids) or single amino acids (as glycine or aspartic acid) or aminosugar, these that mention also can be simultaneously as nitrogenous source later.

Favourable nitrogenous source is organic or inorganic nitrogen-containing compound or comprise the material of these compounds.Example has ammonium salt such as NH ₄Cl or (NH ₄) ₂SO ₄, nitrate, urea, perhaps compound nitrogen source, as corn steep liquor, beer yeast autolysate, soyflour, gluten, yeast extract, meat extract, casein hydrolysate, yeast or Rhizoma Solani tuber osi protein, all these can be often simultaneously as nitrogenous source.

The example of inorganic salt has calcium salt, magnesium salts, sodium salt, cobalt salt, molybdenum salt, manganese salt, sylvite, zinc salt, mantoquita and molysite.And as the negatively charged ion of these salt, muriate, vitriol, phosphoric acid salt should be mentioned especially.An important factor of boosting productivity in the inventive method is to producing Fe in the substratum ²⁺Or Fe ³⁺The control of ionic concn.

As required, answer some somatomedins of restock such as VITAMIN or growth stimulant (as vitamin H, riboflavin, VITMAIN B1, folic acid, nicotinic acid, pantothenate or vitamin B6, amino acid (as L-Ala, halfcystine, proline(Pro), aspartic acid, glutamine, Serine, phenylalanine, ornithine or Xie Ansuan), carboxylic acid (as citric acid, formic acid, pimelic acid or lactic acid), or the material as dithiothreitol (DTT) in the nutritional medium.

The blending ratio of above-mentioned nutritive substance is decided according to fermented type is different, and will adjust according to case.All medium components can be ready to when the fermentation beginning, carry out as required before this separately or common sterilization, also can add continuously during the fermentation as required or add in batches.

Growth conditions is set at and makes the well-grown of described biology, thereby obtains the highest output as far as possible.Preferred growth temperature is 15-40 ℃, preferred 25-37 ℃ especially.The preferred 3-9 of pH value scope, preferred especially 5-8.Usually, incubation time can be from some hrs by some days, and preferred 8 hours to 21 days, preferred especially 4 hours to 14 days, described growth time was normally enough.At this moment the phase, the product in the substratum can farthest accumulate.

Those skilled in the art can find Optimum of culture medium from following textbook for example: using microbe physiology, " practical approach " (Applied Microbial Physiology, " A PracticalApproach ") (P.M.Rhodes, P.F.Stanbury edits, IRL Press, 1997, the 53-73 page or leaf, ISBN 0 19 963,577 3).And the substratum and the growth conditions that are of value to genus bacillus and other biological can particularly be known in the embodiment 9 at the open text of patent EP A 0405370, the substratum and the growth conditions that are applicable to the candiyeast growth can particularly be known in its table 3 at the open text of patent WO 88/09822, the substratum and the growth conditions that are applicable to the AshbyaCif growth can be at (Microbiology such as Schmidt, 142, know in the paper of 1996:419-426) delivering.

Method of the present invention can be implemented with batch culture or fed batch cultivation continuously or discontinuously.

Depend on the productivity that used biology is initial, can access more or less raising by method riboflavin productivity of the present invention.In general productivity can advantageously improve at least 5% than original strain, and preferably at least 10%, preferred especially 20%, especially especially preferably improve 100% at least than original strain productivity.

Embodiment:

Separate rib gene 1,2,3,4 from ashbya gossypii and yeast saccharomyces cerevisiae, 5 and 7 at patent WO 95/26406 and WO 93/03183 and describe to some extent especially in an embodiment, and its operation is similar.These open texts are mentioned herein especially.

Sequence 1 has shown to comprise and has transformed the DNA construct that also carries rib1, rib2, rib4 and rib7 gene fragment outside the required selected marker.

Ordinary method is the sequential analysis of connection, microbial transformation, microbial culture and the recombinant DNA of clone, restriction enzyme digestion, agarose gel electrophoresis, dna fragmentation for example, these methods can be implemented with reference to the method that (1989) such as Sambrook (Cold Spring Harbor Laboratory Press:ISBN 0-87969-309-6) describes unless otherwise indicated.

(Sanger etc., (1977) Proc.Natl.Acad.Sci.USA74 5463-5467) adopt the order-checking of ABI laser fluorescence dna sequencing instrument to recombinant DNA molecules according to the Sanger method.The fragment of polymerase chain reaction is checked order and is revised to avoid the mistake that polysaccharase causes in the construct to be expressed.

Embodiment 1

Comprise rib1, rib2, the clone of the DNA construct of rib4 and rib7 gene copy (carrier Tef-G418-Tef rib1,2,7,4).

Rib expression of gene construct: carrier TefG418Tefrib3,4,5 describe in WO 99/61623.This carrier is cut with the KpnI enzyme, precipitation, and dissolving also partly digests with NheI subsequently again.To from sepharose, separate with the big fragment that NheI and KpnI enzyme are cut once.The rib7 gene (is described in WO 95/26406) from the pJR765 carrier with PCR (primer: TCGAGGTACCGGGCCCCCCCTCGA; TCGAACTAGTAGACCAGTCAT) clone comes out.This specific PCR product is cut with the KpnI/SpeI enzyme and is cut carrier with above-mentioned KpnI/NheI enzyme and is connected.Produce TefG418Tefrib7 thus, 4 carriers.

The rib2 gene passes through pcr amplification from carrier pJR758 (WO 95/26406), and product is cut (primer: CCCAACTAGTCTGCAGGACAATTTAAA with SpeI and NheI enzyme; AGTGCTAGCCTACAATTCGCAGCAAAAT).The carrier TefG418Tefrib7 that this dna fragmentation and NheI enzyme are cut and handled through Phosphoric acid esterase, 4 connect.Produce TefG418Tefrib7 thus, 4,2. carrier.

The Rib1 gene by PCR from carrier pJR765 (WO 95/26406) amplification (primer: GTAGTCTAGAACTAGCTCGAAACGTG; GATTCTAGAACTAGAACTAGTGGATCCG) and use XbaI enzyme cutting.The carrier TefG418Tefrib7 that this dna fragmentation and NheI enzyme are cut and handled through Phosphoric acid esterase, 4,2 connect.

The DNA construct that obtains has constituted Tef-G418-rib1,2,7,4 carriers.

Embodiment 2

DNA construct is transformed into the fungi ashbya gossypii.

Cut DNA construct described in the embodiment 1 (carrier Tef-G418-rib1,2,4,7) with the thorough enzyme of Restriction Enzyme XbaI, the insertion fragment of carrying the rib gene order is by sepharose separation carrying out purifying.

MA2 substratum (10g/l bactopeptone, 1g/l yeast extract, 0.3g/l inositol and 10g/l D-glucose) is cultivated with the mould spore of A Shu capsule.Culture was cultivated 12 hours down in 4 ℃, rocked under 28 ℃ subsequently and cultivated 13 hours.Cell suspension centrifugation, cell precipitation is resuspended with the 50mM potassium phosphate buffer (containing 25mM DTT) of 5ml pH7.5.After 30 minutes, cell recentrifuge precipitation is in 25ml STM damping fluid (270mM sucrose, 10mM TRIS-HCl pH7.5,1mM MgCl 28 ℃ of following thermal treatments ₂) resuspended.0.5ml this suspension insert fragment with the above-mentioned purifying of about 3 μ g and 40U SpeI enzyme is handled, and with Biorad gene pulse producer carry out electroporation (100 Ω, 20 μ F, 1.5kV).Behind the electroporation, these cells are handled and are applied on the MA2 agar culture plate with the MA2 substratum of 1ml.In order to use antibiotic-screening, these flat boards comprise the 5ml low melting-point agarose covering of microbiotic G418 (200 μ g/ml) with one deck after cultivating 5 hours under 28 ℃.Transformant carries out clone purification (Steiner and Philipsen (1995) Genetics, 140 by micromanipulative technique; 973-987).

By the transformant gene DNA being carried out the successful integration that pcr analysis confirms construct.Genomic dna by Carle and Olson (Proc.Natl.Acad.Sci, 1985,82,3756-3760) and Wright and Philipsen (Gene, 1991,109,99-105) described method is separated.PCR R.Saiki (PCR method, 1990, Academic Press, method 13-20) is carried out with the special primer of construct.The segmental analysis of PCR is undertaken by separating in sepharose.

Concerning all transformants, all can verify by PCR method to genomic successfully the integration.

Embodiment 3

The mensuration of riboflavin among the reorganization ashbya gossypii clone:

Construct described in ashbya gossypii LU21 (wild type strain, ATCC 10895) and the embodiment 1 was cultivated 4 days in 28 ℃ on nutrient agar through bacterial strain LU21#1 and the #2 that conversion obtains.Comprise 10ml substratum (the from then on dull and stereotyped inoculation culture that goes up of 27.5g/l yeast extract, 0.5g/l sal epsom, 50ml/l soya-bean oil, 100ml triangular flask pH7.0) with 3.After cultivating 40 hours with 28 ℃ and 180 rev/mins on the shaking table, the 1ml nutrient solution is transferred in the 250ml triangular flask that comprises 20mlYPD substratum (10g/l yeast extract, 20g/l bactopeptone, 20g/l glucose).Cultivate down at 28 ℃ and 300 rev/mins.After 190 hours, from each bottle, get the 1ml sample, and handle with the 1M perchloric acid of 1ml.Sample is used HPLC assay determination riboflavin content after filtration.Calibrate with riboflavin standard substance (10mg/l, 20mg/l, 30mg/l, 40mg/l, 50mg/l).

Measure the parameter of the used HPLC method of riboflavin:

Post ODS Hypersil 5mm 200 * 2.1mm (HP)

Elutriant A is with 340ml H ₃PO ₄(89%) adds in the water to pH2.3

Elutriant B 100% acetonitrile

Gradient

Residence time 0-6 minute .:2%B-50%B

6-6.5 minute: 50%B-2%B

Flow velocity 0.5ml/ minute

Detect 280nm

40 ℃ of temperature

Sample introduction 2-10ml

With initial strain relatively, include the clone #1 of extra rib gene 1,2,4 and 7 copies and #2 batch and show riboflavin productivity be significantly improved (Fig. 1).

Fig. 1 shows different clones' riboflavin production.Compare with the bacterial strain of unmodified, by importing rib1,2,4 and 7 genes, riboflavin production can bring up to 135%.

Sequence table

＜110〉BASF Aktiengesellchaft

＜120〉be used to improve the optimization of the hereditary bacterial strain of riboflavin production

<130>NAE?176/01

<140>

<141>

<160>8

＜170〉PatentIn version 2 .0

<210>1

<211>1052

<212>DNA

＜213〉ashbya gossypii (Ashbya gossypii)

<220>

<221>CDS

<222>(137)..(1042)

<400>1

aaaggctttt?ccgtaggtgc?tttgtcattc?aacaatccac?gtcggaattg?gcgactatat?60

agtgtagggc?ccataaagca?gtagtcggtg?ttgatagctg?tgtcagacca?actctttgtt?120

aattactgaa?gctgat?atg?act?gaa?tac?aca?gtg?cca?gaa?gtg?acc?tgt?gtc?172

Met?Thr?Glu?Tyr?Thr?Val?Pro?Glu?Val?Thr?Cys?Val

1 5 10

gca?cgc?gcg?cgc?ata?ccg?acg?gta?cag?ggc?acc?gat?gtc?ttc?ctc?cat 220

Ala?Arg?Ala?Arg?Ile?Pro?Thr?Val?Gln?Gly?Thr?Asp?Val?Phe?Leu?His

15 20 25

cta?tac?cac?aac?tcg?atc?gac?agc?aag?gaa?cac?cta?gcg?att?gtc?ttc 268

Leu?Tyr?His?Asn?Ser?Ile?Asp?Ser?Lys?Glu?His?Leu?Ala?Ile?Val?Phe

30 35 40

ggc?gag?aac?ata?cgc?tcg?cgg?agt?ctg?ttc?cgg?tac?cgg?aaa?gac?gac 316

Gly?Glu?Asn?Ile?Arg?Ser?Arg?Ser?Leu?Phe?Arg?Tyr?Arg?Lys?Asp?Asp

45 50 55 60

acg?cag?cag?gcg?cgg?atg?gtg?cgg?ggc?gcc?tac?gtg?ggc?cag?ctg?tac 364

Thr?Gln?Gln?Ala?Arg?Met?Val?Arg?Gly?Ala?Tyr?Val?Gly?Gln?Leu?Tyr

65 70 75

ccc?ggg?cgg?acc?gag?gca?gac?gcg?gat?cgg?cgt?cag?ggc?ctg?gag?ctg 412

Pro?Gly?Arg?Thr?Glu?Ala?Asp?Ala?Asp?Arg?Arg?Gln?Gly?Leu?Glu?Leu

80 85 90

cgg?ttt?gat?gag?aca?ggg?cag?ctg?gtg?gtg?gag?cgg?gcg?acg?acg?tgg 460

Arg?Phe?Asp?Glu?Thr?Gly?Gln?Leu?Val?Val?Glu?Arg?Ala?Thr?Thr?Trp

95 100 105

acc?agg?gag?ccg?aca?ctg?gtg?cgg?ctg?cac?tcg?gag?tgt?tac?acg?ggc 508

Thr?Arg?Glu?Pro?Thr?Leu?Val?Arg?Leu?His?Ser?Glu?Cys?Tyr?Thr?Gly

110 115 120

gag?acg?gcg?tgg?agc?gcg?cgg?tgc?gac?tgc?ggg?gag?cag?ttc?gac?cag 556

Glu?Thr?Ala?Trp?Ser?Ala?Arg?Cys?Asp?Cys?Gly?Glu?Gln?Phe?Asp?Gln

125 130 135 140

gcg?ggt?aag?ctg?atg?gct?gcg?gcg?aca?gag?ggc?gag?gtg?gtt?ggc?ggt 604

Ala?Gly?Lys?Leu?Met?Ala?Ala?Ala?Thr?Glu?Gly?Glu?Val?Val?Gly?Gly

145 150 155

gcg?ggg?cac?ggc?gtg?atc?gtg?tac?ctg?cgg?cag?gag?ggc?cgc?ggc?atc 652

Ala?Gly?His?Gly?Val?Ile?Val?Tyr?Leu?Arg?Gln?Glu?Gly?Arg?Gly?Ile

160 165 170

ggg?cta?ggc?gag?aag?ctg?aag?gcg?tac?aac?ctg?cag?gac?ctg?ggc?gcg 700

Gly?Leu?Gly?Glu?Lys?Leu?Lys?Ala?Tyr?Asn?Leu?Gln?Asp?Leu?Gly?Ala

175 180 185

gac?acg?gtg?cag?gcg?aac?gag?ctg?ctc?aac?cac?cct?gcg?gac?gcg?cgc 748

Asp?Thr?Val?Gln?Ala?Asn?Glu?Leu?Leu?Asn?His?Pro?Ala?Asp?Ala?Arg

190 195 200

gac?ttc?tcg?ttg?ggg?cgc?gca?atc?cta?ctg?gac?ctc?ggt?atc?gag?gac 796

Asp?Phe?Ser?Leu?Gly?Arg?Ala?Ile?Leu?Leu?Asp?Leu?Gly?Ile?Glu?Asp

205 210 215 220

atc?cgg?ttg?ctc?acg?aat?aac?ccc?gac?aag?gtg?cag?cag?gtg?cac?tgt 844

Ile?Arg?Leu?Leu?Thr?Asn?Asn?Pro?Asp?Lys?Val?Gln?Gln?Val?His?Cys

225 230 235

ccg?ccg?gcg?cta?cgc?tgc?atc?gag?cgg?gtg?ccc?atg?gtg?ccg?ctt?tca 892

Pro?Pro?Ala?Leu?Arg?Cys?Ile?Glu?Arg?Val?Pro?Met?Val?Pro?Leu?Ser

240 245 250

tgg?act?cag?ccc?aca?cag?ggc?gtg?cgc?tcg?cgc?gag?ctg?gac?ggc?tac 940

Trp?Thr?Gln?Pro?Thr?Gln?Gly?Val?Arg?Ser?Arg?Glu?Leu?Asp?Gly?Tyr

255 260 265

ctg?cgc?gcc?aag?gtc?gag?cgc?atg?ggg?cac?atg?ctg?cag?cgg?ccg?ctg 988

Leu?Arg?Ala?Lys?Val?Glu?Arg?Met?Gly?His?Met?Leu?Gln?Arg?Pro?Leu

270 275 280

gtg?ctg?cac?acg?tct?gcg?gcg?gcc?gag?ctc?ccc?cgc?gcc?aac?aca?cac 1036

Val?Leu?His?Thr?Ser?Ala?Ala?Ala?Glu?Leu?Pro?Arg?Ala?Asn?Thr?His

285 290 295 300

ata?taa?tctttgctat 1052

Ile

<210>2

<211>301

<212>PRT

＜213〉ashbya gossypii

<400>2

Met?Thr?Glu?Tyr?Thr?Val?Pro?Glu?Val?Thr?Cys?Val?Ala?Arg?Ala?Arg

1 5 10 15

Ile?Pro?Thr?Val?Gln?Gly?Thr?Asp?Val?Phe?Leu?His?Leu?Tyr?His?Asn

20 25 30

Ser?Ile?Asp?Ser?Lys?Glu?His?Leu?Ala?Ile?Val?Phe?Gly?Glu?Asn?Ile

35 40 45

Arg?Ser?Arg?Ser?Leu?Phe?Arg?Tyr?Arg?Lys?Asp?Asp?Thr?Gln?Gln?Ala

50 55 60

Arg?Met?Val?Arg?Gly?Ala?Tyr?Val?Gly?Gln?Leu?Tyr?Pro?Gly?Arg?Thr

65 70 75 80

Glu?Ala?Asp?Ala?Asp?Arg?Arg?Gln?Gly?Leu?Glu?Leu?Arg?Phe?Asp?Glu

85 90 95

Thr?Gly?Gln?Leu?Val?Val?Glu?Arg?Ala?Thr?Thr?Trp?Thr?Arg?Glu?Pro

100 105 110

Thr?Leu?Val?Arg?Leu?His?Ser?Glu?Cys?Tyr?Thr?Gly?Glu?Thr?Ala?Trp

115 120 125

Ser?Ala?Arg?Cys?Asp?Cys?Gly?Glu?Gln?Phe?Asp?Gln?Ala?Gly?Lys?Leu

130 135 140

Met?Ala?Ala?Ala?Thr?Glu?Gly?Glu?Val?Val?Gly?Gly?Ala?Gly?His?Gly

145 150 155 160

Val?Ile?Val?Tyr?Leu?Arg?Gln?Glu?Gly?Arg?Gly?Ile?Gly?Leu?Gly?Glu

165 170 175

Lys?Leu?Lys?Ala?Tyr?Asn?Leu?Gln?Asp?Leu?Gly?Ala?Asp?Thr?Val?Gln

180 185 190

Ala?Asn?Glu?Leu?Leu?Asn?His?Pro?Ala?Asp?Ala?Arg?Asp?Phe?Ser?Leu

195 200 205

Gly?Arg?Ala?Ile?Leu?Leu?Asp?Leu?Gly?Ile?Glu?Asp?Ile?Arg?Leu?Leu

210 215 220

Thr?Asn?Asn?Pro?Asp?Lys?Val?Gln?Gln?Val?His?Cys?Pro?Pro?Ala?Leu

225 230 235 240

Arg?Cys?Ile?Glu?Arg?Val?Pro?Met?Val?Pro?Leu?Ser?Trp?Thr?Gln?Pro

245 250 255

Thr?Gln?Gly?Val?Arg?Ser?Arg?Glu?Leu?Asp?Gly?Tyr?Leu?Arg?Ala?Lys

260 265 270

Val?Glu?Arg?Met?Gly?His?Met?Leu?Gln?Arg?Pro?Leu?Val?Leu?His?Thr

275 280 285

Ser?Ala?Ala?Ala?Glu?Leu?Pro?Arg?Ala?Asn?Thr?His?Ile

290 295 300

<210>3

<211>2050

<212>DNA

＜213〉ashbya gossypii

<220>

<221>CDS

<222>(209)..(2038)

<400>3

agacgtcaca?gatatactac?tgatgttgtt?ctccagagta?tactacgccc?ctaccatatt?60

cgatcttgtg?gtattgacga?tattcctctg?tttggtttta?ctggcactat?tccgtttgac?120

ggtatagcgc?tattcgttca?tagtgacaca?tgcggcacta?gctattcagc?gaatccttta?180

taaactgcta?cttaacgttc?gtaacacc?atg?ctc?aaa?ggc?gtt?cct?ggc?ctt 232

Met?Leu?Lys?Gly?Val?Pro?Gly?Leu

1 5

ctt?ttt?aag?gag?acg?caa?cgt?cat?ctg?aaa?ccc?agg?ctg?gtt?agg?att 280

Leu?Phe?Lys?Glu?Thr?Gln?Arg?His?Leu?Lys?Pro?Arg?Leu?Val?Arg?Ile

10 15 20

atg?gaa?aac?aca?tcg?cag?gat?gag?agt?cgc?aaa?aga?cag?gtc?gct?tcg 328

Met?Glu?Asn?Thr?Ser?Gln?Asp?Glu?Ser?Arg?Lys?Arg?Gln?Val?Ala?Ser

25 30 35 40

aac?ttg?agc?agc?gat?gcc?gat?gag?ggc?tcg?ccg?gca?gtt?acg?agg?ccg 376

Asn?Leu?Ser?Ser?Asp?Ala?Asp?Glu?Gly?Ser?Pro?Ala?Val?Thr?Arg?Pro

45 50 55

gtt?aaa?atc?acc?aaa?cgc?ctc?agg?aag?aag?aac?ctc?ggg?aca?ggc?gag 424

Val?Lys?Ile?Thr?Lys?Arg?Leu?Arg?Lys?Lys?Asn?Leu?Gly?Thr?Gly?Glu

60 65 70

cta?cgg?gac?aaa?gca?gga?ttc?aag?ttg?aag?gtg?caa?gac?gtg?agc?aaa 472

Leu?Arg?Asp?Lys?Ala?Gly?Phe?Lys?Leu?Lys?Val?Gln?Asp?Val?Ser?Lys

75 80 85

aac?cgt?cac?aga?cag?gtc?gat?ccg?gaa?tac?gaa?gtc?gtg?gta?gat?ggc 520

Asn?Arg?His?Arg?Gln?Val?Asp?Pro?Glu?Tyr?Glu?Val?Val?Val?Asp?Gly

90 95 100

ccg?atg?cgc?aag?atc?aaa?ccg?tat?ttc?ttc?aca?tac?aag?act?ttc?tgc 568

Pro?Met?Arg?Lys?Ile?Lys?Pro?Tyr?Phe?Phe?Thr?Tyr?Lys?Thr?Phe?Cys

105 110 115 120

aag?gag?cgc?tgg?aga?gat?cgg?aag?ttg?ctt?gat?gtg?ttt?gtg?gat?gaa 616

Lys?Glu?Arg?Trp?Arg?Asp?Arg?Lys?Leu?Leu?Asp?Val?Phe?Val?Asp?Glu

125 130 135

ttt?cgg?gac?cgc?gat?agg?cct?tac?tac?gag?aaa?gtc?atc?ggt?tcg?ggt 664

Phe?Arg?Asp?Arg?Asp?Arg?Pro?Tyr?Tyr?Glu?Lys?Val?Ile?Gly?Ser?Gly

140 145 150

ggt?gtg?ctc?ctg?aac?ggt?aag?tca?tcg?acg?tta?gat?agc?gta?ttg?cgt 712

Gly?Val?Leu?Leu?Ash?Gly?Lys?Ser?Ser?Thr?Leu?Asp?Ser?Val?Leu?Arg

155 160 165

aat?gga?gac?ctc?att?tcg?cac?gag?ctg?cac?cgt?cat?gag?cca?ccg?gtc 760

Ash?Gly?Asp?Leu?Ile?Ser?His?Glu?Leu?His?Arg?His?Glu?Pro?Pro?Val

170 175 180

tcc?tct?agg?ccg?att?agg?acg?gtg?tac?gaa?gat?gat?gac?atc?ctg?gtg 808

Ser?Ser?Arg?Pro?Ile?Arg?Thr?Val?Tyr?Glu?Asp?Asp?Asp?Ile?Leu?Val

185 190 195 200

att?gac?aag?ccc?agc?ggg?att?cca?gcc?cat?ccc?acc?ggg?cgt?tac?cgc 856

Ile?Asp?Lys?Pro?Ser?Gly?Ile?Pro?Ala?His?Pro?Thr?Gly?Arg?Tyr?Arg

205 210 215

ttc?aac?tcc?att?acg?aaa?ata?ctt?gaa?aaa?cag?ctt?gga?tac?act?gtt 904

Phe?Asn?Ser?Ile?Thr?Lys?Ile?Leu?Glu?Lys?Gln?Leu?Gly?Tyr?Thr?Val

220 225 230

cat?cca?tgt?aac?cga?ctg?gac?cgc?cta?acc?agt?ggc?cta?atg?ttc?ttg 952

His?Pro?Cys?Asn?Arg?Leu?Asp?Arg?Leu?Thr?Ser?Gly?Leu?Met?Phe?Leu

235 240 245

gca?aaa?act?cca?aag?gga?gcc?gat?gag?atg?ggt?gat?cag?atg?aag?gcg 1000

Ala?Lys?Thr?Pro?Lys?Gly?Ala?Asp?Glu?Met?Gly?Asp?Gln?Met?Lys?Ala

250 255 260

cgc?gaa?gtg?aag?aaa?gaa?tat?gtt?gcc?cgg?gtt?gtt?ggg?gaa?ttt?cct 1048

Arg?Glu?Val?Lys?Lys?Glu?Tyr?Val?Ala?Arg?Val?Val?Gly?Glu?Phe?Pro

265 270 275 280

ata?ggt?gag?ata?gtt?gtg?gat?atg?cca?ctg?aag?act?ata?gag?ccg?aag 1096

Ile?Gly?Glu?Ile?Val?Val?Asp?Met?Pro?Leu?Lys?Thr?Ile?Glu?Pro?Lys

285 290 295

ctt?gcc?cta?aac?atg?gtt?tgc?gac?ccg?gaa?gac?gaa?gcg?ggc?aag?ggc 1144

Leu?Ala?Leu?Asn?Met?Val?Cys?Asp?Pro?Glu?Asp?Glu?Ala?Gly?Lys?Gly

300 305 310

gct?aag?acg?cag?ttc?aaa?aga?atc?agc?tac?gat?gga?caa?acg?agc?ata 1192

Ala?Lys?Thr?Gln?Phe?Lys?Arg?Ile?Ser?Tyr?Asp?Gly?Gln?Thr?Ser?Ile

315 320 325

gtc?aag?tgc?caa?ccg?tac?acg?ggc?cgg?acg?cat?cag?atc?cgt?gtt?cac 1240

Val?Lys?Cys?Gln?Pro?Tyr?Thr?Gly?Arg?Thr?His?Gln?Ile?Arg?Val?His

330 335 340

ttg?caa?tac?ctg?ggc?ttc?cca?att?gcc?aac?gat?ccg?att?tat?tcc?aat 1288

Leu?Gln?Tyr?Leu?Gly?Phe?Pro?Ile?Ala?Asn?Asp?Pro?Ile?Tyr?Ser?Asn

345 350 355 360

ccg?cac?ata?tgg?ggc?cca?agt?ctg?ggc?aag?gaa?tgc?aaa?gca?gac?tac 1336

Pro?His?Ile?Trp?Gly?Pro?Ser?Leu?Gly?Lys?Glu?Cys?Lys?Ala?Asp?Tyr

365 370 375

aag?gag?gtc?atc?caa?aaa?cta?aac?gaa?att?ggt?aag?act?aaa?tct?gcg 1384

Lys?Glu?Val?Ile?Gln?Lys?Leu?Asn?Glu?Ile?Gly?Lys?Thr?Lys?Ser?Ala

380 385 390

gaa?agt?tgg?tac?cat?tct?gat?tcc?caa?ggt?gaa?gtt?ttg?aaa?ggg?gaa 1432

Glu?Ser?Trp?Tyr?His?Ser?Asp?Ser?Gln?Gly?Glu?Val?Leu?Lys?Gly?Glu

395 400 405

caa?tgc?gat?gaa?tgt?ggc?acc?gaa?ctg?tac?act?gac?ccg?ggc?ccg?aat 1480

Gln?Cys?Asp?Glu?Cys?Gly?Thr?Glu?Leu?Tyr?Thr?Asp?Pro?Gly?Pro?Asn

410 415 420

gat?ctt?gac?tta?tgg?ttg?cat?gca?tat?cgg?tat?gaa?tcc?act?gaa?ctg 1528

Asp?Leu?Asp?Leu?Trp?Leu?His?Ala?Tyr?Arg?Tyr?Glu?Ser?Thr?Glu?Leu

425 430 435 440

gat?gag?aac?ggt?gct?aaa?aag?tgg?agt?tac?tct?act?gcg?ttt?cct?gag 1576

Asp?Glu?Asn?Gly?Ala?Lys?Lys?Trp?Ser?Tyr?Ser?Thr?Ala?Phe?Pro?Glu

445 450 455

tgg?gct?ctt?gag?cag?cac?ggc?gac?ttc?atg?cgg?ctt?gcc?atc?gaa?cag 1624

Trp?Ala?Leu?Glu?Gln?His?Gly?Asp?Phe?Met?Arg?Leu?Ala?Ile?Glu?Gln

460 465 470

gct?aag?aaa?tgc?cca?ccc?gcg?aag?aca?tca?ttt?agc?gtt?ggt?gcc?gtg 1672

Ala?Lys?Lys?Cys?Pro?Pro?Ala?Lys?Thr?Ser?Phe?Ser?Val?Gly?Ala?Val

475 480 485

tta?gtt?aat?ggg?acc?gag?att?ttg?gcc?act?ggt?tac?tca?cgg?gag?ctg 1720

Leu?Val?Asn?Gly?Thr?Glu?Ile?Leu?Ala?Thr?Gly?Tyr?Ser?Arg?Glu?Leu

490 495 500

gaa?ggc?aac?acg?cac?gct?gaa?caa?tgt?gca?ctt?caa?aaa?tat?ttt?gaa 1768

Glu?Gly?Asn?Thr?His?Ala?Glu?Gln?Cys?Ala?Leu?Gln?Lys?Tyr?Phe?Glu

505 510 515 520

caa?cat?aaa?acc?gac?aag?gtt?cct?att?ggt?aca?gta?ata?tac?acg?act 1816

Gln?His?Lys?Thr?Asp?Lys?Val?Pro?Ile?Gly?Thr?Val?Ile?Tyr?Thr?Thr

525 530 535

atg?gag?cct?tgt?tct?ctc?cgt?ctc?agt?ggt?aat?aaa?ccg?tgt?gtt?gag 1864

Met?Glu?Pro?Cys?Ser?Leu?Arg?Leu?Ser?Gly?Asn?Lys?Pro?Cys?Val?Glu

540 545 550

cgt?ata?atc?tgc?cag?cag?ggt?aat?att?act?gct?gtt?ttt?gtt?ggc?gta 1912

Arg?Ile?Ile?Cys?Gln?Gln?Gly?Asn?Ile?Thr?Ala?Val?Phe?Val?Gly?Val

555 560 565

ctt?gag?cca?gac?aac?ttc?gtg?aag?aac?aat?aca?agt?cgt?gcg?cta?ttg 1960

Leu?Glu?Pro?Asp?Asn?Phe?Val?Lys?Asn?Asn?Thr?Ser?Arg?Ala?Leu?Leu

570 575 580

gaa?caa?cat?ggt?ata?gac?tat?att?ctt?gtc?cct?ggg?ttt?caa?gaa?gaa 2008

Glu?Gln?His?Gly?Ile?Asp?Tyr?Ile?Leu?Val?Pro?Gly?Phe?Gln?Glu?Glu

585 590 595 600

tgt?act?gaa?gcc?gca?ttg?aag?ggt?cat?tga?ttttgctgcg?aa 2050

Cys?Thr?Glu?Ala?Ala?Leu?Lys?Gly?His

605 610

<210>4

<211>609

<212>PRT

＜213〉ashbya gossypii

<400>4

Met?Leu?Lys?Gly?Val?Pro?Gly?Leu?Leu?Phe?Lys?Glu?Thr?Gln?Arg?His

1 5 10 15

Leu?Lys?Pro?Arg?Leu?Val?Arg?Ile?Met?Glu?Asn?Thr?Ser?Gln?Asp?Glu

20 25 30

Ser?Arg?Lys?Arg?Gln?Val?Ala?Ser?Asn?Leu?Ser?Ser?Asp?Ala?Asp?Glu

35 40 45

Gly?Ser?Pro?Ala?Val?Thr?Arg?Pro?Val?Lys?Ile?Thr?Lys?Arg?Leu?Arg

50 55 60

Lys?Lys?Asn?Leu?Gly?Thr?Gly?Glu?Leu?Arg?Asp?Lys?Ala?Gly?Phe?Lys

65 70 75 80

Leu?Lys?Val?Gln?Asp?Val?Ser?Lys?Ash?Arg?His?Arg?Gln?Val?Asp?Pro

85 90 95

Glu?Tyr?Glu?Val?Val?Val?Asp?Gly?Pro?Met?Arg?Lys?Ile?Lys?Pro?Tyr

100 105 110

Phe?Phe?Thr?Tyr?Lys?Thr?Phe?Cys?Lys?Glu?Arg?Trp?Arg?Asp?Arg?Lys

115 120 125

Leu?Leu?Asp?Val?Phe?Val?Asp?Glu?Phe?Arg?Asp?Arg?Asp?Arg?Pro?Tyr

130 135 140

Tyr?Glu?Lys?Val?Ile?Gly?Ser?Gly?Gly?Val?Leu?Leu?Asn?Gly?Lys?Ser

145 150 155 160

Ser?Thr?Leu?Asp?Ser?Val?Leu?Arg?Ash?Gly?Asp?Leu?Ile?Ser?His?Glu

165 170 175

Leu?His?Arg?His?Glu?Pro?Pro?Val?Ser?Ser?Arg?Pro?Ile?Arg?Thr?Val

180 185 190

Tyr?Glu?Asp?Asp?Asp?Ile?Leu?Val?Ile?Asp?Lys?Pro?Ser?Gly?Ile?Pro

195 200 205

Ala?His?Pro?Thr?Gly?Arg?Tyr?Arg?Phe?Asn?Ser?Ile?Thr?Lys?Ile?Leu

210 215 220

Glu?Lys?Gln?Leu?Gly?Tyr?Thr?Val?His?Pro?Cys?Asn?Arg?Leu?Asp?Arg

225 230 235 240

Leu?Thr?Ser?Gly?Leu?Met?Phe?Leu?Ala?Lys?Thr?Pro?Lys?Gly?Ala?Asp

245 250 255

Glu?Met?Gly?Asp?Gln?Met?Lys?Ala?Arg?Glu?Val?Lys?Lys?Glu?Tyr?Val

260 265 270

Ala?Arg?Val?Val?Gly?Glu?Phe?Pro?Ile?Gly?Glu?Ile?Val?Val?Asp?Met

275 280 285

Pro?Leu?Lys?Thr?Ile?Glu?Pro?Lys?Leu?Ala?Leu?Asn?Met?Val?Cys?Asp

290 295 300

Pro?Glu?Asp?Glu?Ala?Gly?Lys?Gly?Ala?Lys?Thr?Gln?Phe?Lys?Arg?Ile

305 310 315 320

Ser?Tyr?Asp?Gly?Gln?Thr?Ser?Ile?Val?Lys?Cys?Gln?Pro?Tyr?Thr?Gly

325 330 335

Arg?Thr?His?Gln?Ile?Arg?Val?His?Leu?Gln?Tyr?Leu?Gly?Phe?Pro?Ile

340 345 350

Ala?Asn?Asp?Pro?Ile?Tyr?Ser?Asn?Pro?His?Ile?Trp?Gly?Pro?Ser?Leu

355 360 365

Gly?Lys?Glu?Cys?Lys?Ala?Asp?Tyr?Lys?Glu?Val?Ile?Gln?Lys?Leu?Asn

370 375 380

Glu?Ile?Gly?Lys?Thr?Lys?Ser?Ala?Glu?Ser?Trp?Tyr?His?Ser?Asp?Ser

385 390 395 400

Gln?Gly?Glu?Val?Leu?Lys?Gly?Glu?Gln?Cys?Asp?Glu?Cys?Gly?Thr?Glu

405 410 415

Leu?Tyr?Thr?Asp?Pro?Gly?Pro?Asn?Asp?Leu?Asp?Leu?Trp?Leu?His?Ala

420 425 430

Tyr?Arg?Tyr?Glu?Ser?Thr?Glu?Leu?Asp?Glu?Asn?Gly?Ala?Lys?Lys?Trp

435 440 445

Ser?Tyr?Ser?Thr?Ala?Phe?Pro?Glu?Trp?Ala?Leu?Glu?Gln?His?Gly?Asp

450 455 460

Phe?Met?Arg?Leu?Ala?Ile?Glu?Gln?Ala?Lys?Lys?Cys?Pro?Pro?Ala?Lys

465 470 475 480

Thr?Ser?Phe?Ser?Val?Gly?Ala?Val?Leu?Val?Asn?Gly?Thr?Glu?Ile?Leu

485 490 495

Ala?Thr?Gly?Tyr?Ser?Arg?Glu?Leu?Glu?Gly?Asn?Thr?His?Ala?Glu?Gln

500 505 510

Cys?Ala?Leu?Gln?Lys?Tyr?Phe?Glu?Gln?His?Lys?Thr?Asp?Lys?Val?Pro

515 520 525

Ile?Gly?Thr?Val?Ile?Tyr?Thr?Thr?Met?Glu?Pro?Cys?Ser?Leu?Arg?Leu

530 535 540

Ser?Gly?Asn?Lys?Pro?Cys?Val?Glu?Arg?Ile?Ile?Cys?Gln?Gln?Gly?Asn

545 550 555 560

Ile?Thr?Ala?Val?Phe?Val?Gly?Val?Leu?Glu?Pro?Asp?Asn?Phe?Val?Lys

565 570 575

Asn?Asn?Thr?Ser?Arg?Ala?Leu?Leu?Glu?Gln?His?Gly?Ile?Asp?Tyr?Ile

580 585 590

Leu?Val?Pro?Gly?Phe?Gln?Glu?Glu?Cys?Thr?Glu?Ala?Ala?Leu?Lys?Gly

595 600 605

His

<210>5

<211>730

<212>DNA

＜213〉second ashbya gossypii

<220>

<221>CDS

<222>(195)..(713)

<400>5

ttgagctata?tgtaagtcta?ttaattgatt?actaatagca?atttatggta?tcctctgttc?60

tgcatatcga?cggttactca?cgtgatgatc?agcttgaggc?ttcgcggata?aagttccatc?120

gattactata?aaaccatcac?attaaacgtt?cactataggc?atacacacag?actaagttca?180

agttagcagt?gaca?atg?att?aag?gga?tta?ggc?gaa?gtt?gat?caa?acc?tac 230

Met?Ile?Lys?Gly?Leu?Gly?Glu?Val?Asp?Gln?Thr?Tyr

1 5 10

gat?gcg?agc?tct?gtc?aag?gtt?ggc?att?gtc?cac?gcg?aga?tgg?aac?aag 278

Asp?Ala?Ser?Ser?Val?Lys?Val?Gly?Ile?Val?His?Ala?Arg?Trp?Asn?Lys

15 20 25

act?gtc?att?gac?gct?ctc?gtc?caa?ggt?gca?att?gag?aaa?ctg?ctt?gct 326

Thr?Val?Ile?Asp?Ala?Leu?Val?Gln?Gly?Ala?Ile?Glu?Lys?Leu?Leu?Ala

30 35 40

atg?gga?gtg?aag?gag?aag?aat?atc?act?gta?agc?acc?gtt?cca?ggt?gcg 374

Met?Gly?Val?Lys?Glu?Lys?Asn?Ile?Thr?Val?Ser?Thr?Val?Pro?Gly?Ala

45 50 55 60

ttt?gaa?cta?cca?ttt?ggc?act?cag?cgg?ttt?gcc?gag?ctg?acc?aag?gca 422

Phe?Glu?Leu?Pro?Phe?Gly?Thr?Gln?Arg?Phe?Ala?Glu?Leu?Thr?Lys?Ala

65 70 75

agt?ggc?aag?cat?ttg?gac?gtg?gtc?atc?cca?att?gga?gtc?ctg?atc?aaa 470

Ser?Gly?Lys?His?Leu?Asp?Val?Val?Ile?Pro?Ile?Gly?Val?Leu?Ile?Lys

80 85 90

ggc?gac?tca?atg?cac?ttt?gaa?tat?ata?tca?gac?tct?gtg?act?cat?gcc 518

Gly?Asp?Ser?Met?His?Phe?Glu?Tyr?Ile?Ser?Asp?Ser?Val?Thr?His?Ala

95 100 105

tta?atg?aac?cta?cag?aag?aag?att?cgt?ctt?cct?gtc?att?ttt?ggt?ttg 566

Leu?Met?Asn?Leu?Gln?Lys?Lys?Ile?Arg?Leu?Pro?Val?Ile?Phe?Gly?Leu

110 115 120

cta?acg?tgt?cta?aca?gag?gaa?caa?gcg?ttg?aca?cgt?gca?ggc?ctc?ggt 614

Leu?Thr?Cys?Leu?Thr?Glu?Glu?Gln?Ala?Leu?Thr?Arg?Ala?Gly?Leu?Gly

125 130 135 140

gaa?tct?gaa?ggc?aag?cac?aac?cac?ggt?gaa?gac?tgg?ggt?gct?gct?gcc 662

Glu?Ser?Glu?Gly?Lys?His?Asn?His?Gly?Glu?Asp?Trp?Gly?Ala?Ala?Ala

145 150 155

gtg?gag?atg?gct?gta?aag?ttt?ggc?cca?cgc?gcc?gaa?caa?atg?aag?aag 710

Val?Glu?Met?Ala?Val?Lys?Phe?Gly?Pro?Arg?Ala?Glu?Gln?Met?Lys?Lys

160 165 170

tga?atattaaaaa?atcacta 730

<210>6

<211>172

<212>PRT

＜213〉ashbya gossypii

<400>6

Met?Ile?Lys?Gly?Leu?Gly?Glu?Val?Asp?Gln?Thr?Tyr?Asp?Ala?Ser?Ser

1 5 10 15

Val?Lys?Val?Gly?Ile?Val?His?Ala?Arg?Trp?Asn?Lys?Thr?Val?Ile?Asp

20 25 30

Ala?Leu?Val?Gln?Gly?Ala?Ile?Glu?Lys?Leu?Leu?Ala?Met?Gly?Val?Lys

35 40 45

Glu?Lys?Asn?Ile?Thr?Val?Ser?Thr?Val?Pro?Gly?Ala?Phe?6lu?Leu?Pro

50 55 60

Phe?Gly?Thr?Gln?Arg?Phe?Ala?Glu?Leu?Thr?Lys?Ala?Ser?Gly?Lys?His

65 70 75 80

Leu?Asp?Val?Val?Ile?Pro?Ile?Gly?Val?Leu?Ile?Lys?Gly?Asp?Ser?Met

85 90 95

His?Phe?Glu?Tyr?Ile?Ser?Asp?Ser?Val?Thr?His?Ala?Leu?Met?Asn?Leu

100 105 110

Gln?Lys?Lys?Ile?Arg?Leu?Pro?Val?Ile?Phe?Gly?Leu?Leu?Thr?Cys?Leu

115 120 125

Thr?Glu?Glu?Gln?Ala?Leu?Thr?Arg?Ala?Gly?Leu?Gly?Glu?Ser?Glu?Gly

130 135 140

Lys?His?Asn?His?Gly?Glu?Asp?Trp?Gly?Ala?Ala?Ala?Val?Glu?Met?Ala

145 150 155 160

Val?Lys?Phe?Gly?Pro?Arg?Ala?Glu?Gln?Met?Lys?Lys

165 170

<210>7

<211>1109

<212>DNA

＜213〉ashbya gossypii

<220>

<221>CDS

<222>(352)..(1092)

<400>7

gaagaagcgc?aggcgccagt?ccgagctgga?ggagaacgag?gcggcgcggt?tgacgaacag?60

cgcgctgccc?atggacgatg?cgggtataca?gacggcgggt?atacagacgg?cgggtggtgc?120

cgagagaggc?accaggccgg?cttcctccag?cgatgcaagg?aagagaaggg?gaccagaggc?180

gaagttcaag?ccatctaagg?tacagaagcc?ccaattgaag?cgaactgcat?cgtcccgggc?240

ggatgagaac?gagttctcga?tattatagag?gcccccgttt?cgagtgattg?gcgtcaaaaa?300

cggctatctg?ccttcgtccg?cccccaccac?cctcgggaac?actggcaaac?c?atg?gcg 357

Met?Ala

1

cta?ata?cca?ctt?tct?caa?gat?ctg?gct?gat?ata?cta?gca?ccg?tac?tta 405

Leu?Ile?Pro?Leu?Ser?Gln?Asp?Leu?Ala?Asp?Ile?Leu?Ala?Pro?Tyr?Leu

5 10 15

ccg?aca?cca?ccg?gac?tca?tcc?gca?cgc?ctg?ccg?ttt?gtc?acg?ctg?acg 453

Pro?Thr?Pro?Pro?Asp?Ser?Ser?Ala?Arg?Leu?Pro?Phe?Val?Thr?Leu?Thr

20 25 30

tat?gcg?cag?tcc?cta?gat?gct?cgt?atc?gcg?aag?caa?aag?ggt?gaa?agg 501

Tyr?Ala?Gln?Ser?Leu?Asp?Ala?Arg?Ile?Ala?Lys?Gln?Lys?Gly?Glu?Arg

35 40 45 50

acg?gtt?att?tcg?cat?gag?gag?acc?aag?aca?atg?acg?cat?tat?cta?cgc 549

Thr?Val?Ile?Ser?His?Glu?Glu?Thr?Lys?Thr?Met?Thr?His?Tyr?Leu?Arg

55 60 65

tac?cat?cat?agc?ggc?atc?ctg?att?ggc?tcg?ggc?aca?gcc?ctt?gcg?gac 597

Tyr?His?His?Ser?Gly?Ile?Leu?Ile?Gly?Ser?Gly?Thr?Ala?Leu?Ala?Asp

70 75 80

gac?ccg?gat?ctc?aat?tgc?cgg?tgg?aca?cct?gca?gcg?gac?ggg?gcg?gat 645

Asp?Pro?Asp?Leu?Asn?Cys?Arg?Trp?Thr?Pro?Ala?Ala?Asp?Gly?Ala?Asp

85 90 95

tgc?acc?gaa?cag?tct?tca?cca?cga?ccc?att?atc?ttg?gat?gtt?cgg?ggc 693

Cys?Thr?Glu?Gln?Ser?Ser?Pro?Arg?Pro?Ile?Ile?Leu?Asp?Val?Arg?Gly

100 105 110

aga?tgg?aga?tac?cgc?ggg?tcc?aaa?ata?gag?tat?ctg?cat?aac?ctt?ggc 741

Arg?Trp?Arg?Tyr?Arg?Gly?Ser?Lys?Ile?Glu?Tyr?Leu?His?Asn?Leu?Gly

115 120 125 130

aag?ggg?aag?gcg?ccc?ata?gtg?gtc?acg?ggg?ggt?gag?ccg?gag?gtc?cgc 789

Lys?Gly?Lys?Ala?Pro?Ile?Val?Val?Thr?Gly?Gly?Glu?Pro?Glu?Val?Arg

135 140 145

gaa?cta?ggc?gtc?agt?tac?ctg?cag?ctg?ggt?gtc?gac?gag?ggt?ggc?cgc 837

Glu?Leu?Gly?Val?Ser?Tyr?Leu?Gln?Leu?Gly?Val?Asp?Glu?Gly?Gly?Arg

150 155 160

ttg?aat?tgg?ggc?gag?ttg?ttt?gag?cga?ctc?tat?tct?gag?cac?cac?ctg 885

Leu?Asn?Trp?Gly?Glu?Leu?Phe?Glu?Arg?Leu?Tyr?Ser?Glu?His?His?Leu

165 170 175

gaa?agt?gtc?atg?gtc?gaa?ggc?ggc?gcg?gag?gtg?ctc?aac?cag?ctg?ctg 933

Glu?Ser?Val?Met?Val?Glu?Gly?Gly?Ala?Glu?Val?Leu?Asn?Gln?Leu?Leu

180 185 190

ctg?cgc?cca?gat?att?gtg?gac?agt?ctg?gtg?atc?acg?ata?gga?tcc?aag 981

Leu?Arg?Pro?Asp?Ile?Val?Asp?Ser?Leu?Val?Ile?Thr?Ile?Gly?Ser?Lys

195 200 205 210

ttc?ctg?ggc?tca?cta?ggt?gtt?gcg?gtc?tca?cca?gct?gag?gag?gtg?aac 1029

Phe?Leu?Gly?Ser?Leu?Gly?Val?Ala?Val?Ser?Pro?Ala?Glu?Glu?Val?Asn

215 220 225

cta?gag?cat?gtg?aac?tgg?tgg?cac?gga?aca?agt?gac?agt?gtt?ttg?tgc 1077

Leu?Glu?His?Val?Asn?Trp?Trp?His?Gly?Thr?Ser?Asp?Ser?Val?Leu?Cys

230 235 240

ggc?cgg?ctc?gca?tag?cggttatgac?tggtcta 1109

Gly?Arg?Leu?Ala

245

<210>8

<211>246

<212>PRT

＜213〉ashbya gossypii

<400>8

Met?Ala?Leu?Ile?Pro?Leu?Ser?Gln?Asp?Leu?Ala?Asp?Ile?Leu?Ala?Pro

1 5 10 15

Tyr?Leu?Pro?Thr?Pro?Pro?Asp?Ser?Ser?Ala?Arg?Leu?Pro?Phe?Val?Thr

20 25 30

Leu?Thr?Tyr?Ala?Gln?Ser?Leu?Asp?Ala?Arg?Ile?Ala?Lys?Gln?Lys?Gly

35 40 45

Glu?Arg?Thr?Val?Ile?Ser?His?Glu?Glu?Thr?Lys?Thr?Met?Thr?His?Tyr

50 55 60

Leu?Arg?Tyr?His?His?Ser?Gly?Ile?Leu?Ile?Gly?Ser?Gly?Thr?Ala?Leu

65 70 75 80

Ala?Asp?Asp?Pro?Asp?Leu?Asn?Cys?Arg?Trp?Thr?Pro?Ala?Ala?Asp?Gly

85 90 95

Ala?Asp?Cys?Thr?Glu?Gln?Ser?Ser?Pro?Arg?Pro?Ile?Ile?Leu?Asp?Val

100 105 110

Arg?Gly?Arg?Trp?Arg?Tyr?Arg?Gly?Ser?Lys?Ile?Glu?Tyr?Leu?His?Asn

115 120 125

Leu?Gly?Lys?Gly?Lys?Ala?Pro?Ile?Val?Val?Thr?Gly?Gly?Glu?Pro?Glu

130 135 140

Val?Arg?Glu?Leu?Gly?Val?Ser?Tyr?Leu?Gln?Leu?Gly?Val?Asp?Glu?Gly

145 150 155 160

Gly?Arg?Leu?Asn?Trp?Gly?Glu?Leu?Phe?Glu?Arg?Leu?Tyr?Ser?Glu?His

165 170 175

His?Leu?Glu?Ser?Val?Met?Val?Glu?Gly?Gly?Ala?Glu?Val?Leu?Asn?Gln

180 185 190

Leu?Leu?Leu?Arg?Pro?Asp?Ile?Val?Asp?Ser?Leu?Val?Ile?Thr?Ile?Gly

195 200 205

Ser?Lys?Phe?Leu?Gly?Ser?Leu?Gly?Val?Ala?Val?Ser?Pro?Ala?Glu?Glu

210 215 220

Val?Asn?Leu?Glu?His?Val?Asn?Trp?Trp?His?Gly?Thr?Ser?Asp?Ser?Val

225 230 235 240

Leu?Cys?Gly?Arg?Leu?Ala

245

Claims (6)

1. the method that is used for microorganisms riboflavin, described method demonstrates more highly active AshbyaCif (Ashbya) microorganism by cultivating can to produce riboflavin and to compare with wild-type ATCC 10895 at least two kinds of gene products that are selected from rib1, rib2, rib4 and rib7, separates the riboflavin that is produced from substratum subsequently.

2. the method for claim 1, three kinds of gene products that wherein are selected from rib1, rib2, rib4 and rib7 demonstrate higher activity.

3. the method for claim 1, four kinds of gene products that wherein are selected from rib1, rib2, rib4 and rib7 demonstrate higher activity.

4. as any described method among the claim 1-3, wherein the higher gene activity of rib gene product is that raising by genetic expression is caused.

5. method as claimed in claim 4, the raising of wherein said genetic expression are that the increase by gene copy number is caused.

6. any described method in the claim as described above, wherein the rib gene product has the peptide sequence shown in SEQ ID NO:2,4,6,8, or passes through to replace, inserts or delete the sub peptide sequence that obtains of 5% amino acid code at the most by these sequences.