JP5504608B2 - Method for producing 1,5-pentanediamine - Google Patents
Method for producing 1,5-pentanediamine Download PDFInfo
- Publication number
- JP5504608B2 JP5504608B2 JP2008272717A JP2008272717A JP5504608B2 JP 5504608 B2 JP5504608 B2 JP 5504608B2 JP 2008272717 A JP2008272717 A JP 2008272717A JP 2008272717 A JP2008272717 A JP 2008272717A JP 5504608 B2 JP5504608 B2 JP 5504608B2
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- gene
- pentanediamine
- lysine
- coli
- strain
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Images
Description
本発明は、発酵法による１，５−ペンタンジアミンの製造方法に関する。 The present invention relates to a method for producing 1,5-pentanediamine by fermentation.
１，５−ペンタンジアミンは医薬中間体などの合成原料や高分子原材料として期待され、需要が高まりつつある。発酵法による１，５−ペンタンアミンの製造方法として、これまでにリジンデカルボキシラーゼの発現を増強したエシェリヒア属細菌（特許文献１）またはコリネ型細菌（特許文献２）を用いた製造技術が開示されている。 1,5-pentanediamine is expected as a synthetic raw material such as a pharmaceutical intermediate or a polymer raw material, and the demand is increasing. As a method for producing 1,5-pentanamine by fermentation, a production technique using Escherichia bacteria (Patent Document 1) or coryneform bacteria (Patent Document 2) having enhanced expression of lysine decarboxylase has been disclosed so far. ing.
コリネ型細菌の生育上限温度はエシェリヒア属よりも１０度程度低く、また生育速度も遅いとされる。従って、コリネ型細菌を発酵法の宿主として１，５−ペンタンジアミン生産に利用する場合には、培養温度を下げて培養する必要があると考えられる。すなわち、発酵に用いられている通常の微生物は、発酵中に自らが発生する発酵熱により培地の温度が上昇し、発酵に必要な酵素が失活したり生産菌が死滅したりするために、発酵中に培地を冷却することが必要となっている。従って、発酵におけるコストとしては、培養中に発生する発酵熱の冷却エネルギーが余分にかかってしまう。 The upper limit temperature for growth of coryneform bacteria is about 10 degrees lower than that of the genus Escherichia, and the growth rate is also slow. Therefore, when coryneform bacteria are used for the production of 1,5-pentanediamine as a host for fermentation, it is considered necessary to culture at a lower culture temperature. In other words, normal microorganisms used in fermentation increase the temperature of the medium due to the heat of fermentation generated by itself during fermentation, in order to inactivate enzymes necessary for fermentation or kill production bacteria, It is necessary to cool the medium during fermentation. Therefore, as the cost in fermentation, the cooling energy of the fermentation heat generated during the culture is excessive.
一方、１，５−ペンタンジアミンの前駆体であるＬ−リジンを工業規模で発酵生産しているエシェリヒア属細菌（特に大腸菌）は分子生物学的に解析が進んでおり、培養温度も３７℃前後とコリネ型細菌と比較して高く、生育速度も速いことから、効率のよい１，５−ペンタンジアミン製造が可能になることが期待される。しかしながら、エシェリヒア属細菌はアスパラギン酸をβ−ホスホアスパラギン酸に変える反応を触媒する酵素であるアスパルトキナーゼ（以下、「ＡＫ」と略すことがある）のうち、ＡＫIIIがＬ−リジンによるフィードバック阻害を受けることが知られている。 On the other hand, Escherichia bacteria (especially Escherichia coli) that fermentably produce L-lysine, which is a precursor of 1,5-pentanediamine, on an industrial scale has been analyzed molecularly, and the culture temperature is also around 37 ° C. Therefore, it is expected that efficient production of 1,5-pentanediamine will be possible. However, among Escherichia bacteria, among the aspartokinase (hereinafter sometimes abbreviated as “AK”) that catalyzes the reaction of converting aspartate to β-phosphoaspartate, AKIII inhibits feedback inhibition by L-lysine. It is known to receive.
大腸菌のＡＫは３種あり（ＡＫＩ、ＡＫII、ＡＫIII）、うちＡＫＩとＡＫIIはホモセリンデヒドロゲナーゼ（以下、「ＨＤ」と略すことがある）との複合酵素である。複合酵素の内のひとつはｔｈｒＡ遺伝子にコードされるＡＫＩ−ＨＤＩであり、もう一方はｍｅｔＬＭ遺伝子にコードされるＡＫII−ＨＤIIである。ＡＫIはスレオニンとイソロイシンによる協奏的抑制及びスレオニンによる阻害を受け、ＡＫIIはメチオニンによる抑制を受けるとされている。これらに対し、ＡＫIIIのみは単機能酵素であり、ｌｙｓＣと名付けられた遺伝子の産物であって、Ｌ−リジンによるフィードバック阻害を受けることが知られている。ＡＫIIIがＬ−リジンによるフィードバック阻害を受けると、ＡＫIIIの酵素活性がＬ−リジンによって抑制され、その結果、生産物であるＬ−リジン並びにその代謝産物である１，５−ペンタンジアミンの生産量も抑制される。 There are three types of AK of E. coli (AKI, AKII, AKIII), and AKI and AKII are complex enzymes of homoserine dehydrogenase (hereinafter sometimes abbreviated as “HD”). One of the complex enzymes is AKI-HDI encoded by the thrA gene, and the other is AKII-HDII encoded by the metLM gene. AKI is said to be subject to concerted suppression by threonine and isoleucine and inhibition by threonine, and AKII is suppressed by methionine. On the other hand, only AKIII is a monofunctional enzyme and is a product of a gene named lysC, and is known to undergo feedback inhibition by L-lysine. When AKIII is subjected to feedback inhibition by L-lysine, the enzyme activity of AKIII is suppressed by L-lysine. As a result, the production amount of L-lysine as a product and 1,5-pentanediamine as its metabolite is also increased. It is suppressed.
そこで、Ｌ−リジンによるフィードバック阻害を受けない変異型ＡＫIIIの変異酵素を取得することができれば、エシェリヒア属細菌（特に大腸菌）を用いて効率のよい１，５−ペンタンジアミンの発酵生産を行うことが期待できるが、ＡＫIIIの変異酵素についての報告はあるものの（特許文献３および４）、同変異酵素が１，５−ペンタンジアミンの生産性を改善した例は知られていない。
本発明は上記事情に鑑みてなされたものであり、その課題は、簡便かつ効率的な１，５−ペンタンジアミンの製造方法を開発することである。 This invention is made | formed in view of the said situation, The subject is developing a simple and efficient manufacturing method of 1, 5-pentanediamine.
上記課題を解決するために、本発明者らが鋭意検討した結果、Ｌ−リジンによるフィードバック阻害を解除する変異を有するアスパルトキナーゼIIIを発現し、かつリジンデカルボキシラーゼ（ｃａｄＡ）の発現が増強された大腸菌（Ｅ．ｃｏｌｉ）を培養することにより、高い対糖収率で１，５−ペンタンジアミンを製造できることを見出し、本発明を完成させるに至った。 As a result of intensive studies by the present inventors in order to solve the above problems, aspartokinase III having a mutation that cancels feedback inhibition by L-lysine is expressed, and expression of lysine decarboxylase (cadA) is enhanced. By culturing Escherichia coli (E. coli), it was found that 1,5-pentanediamine can be produced with a high sugar yield, and the present invention has been completed.
すなわち、本発明は、１，５−ペンタンジアミンを生産する能力を有する大腸菌（Ｅ.ｃｏｌｉ）を培養することによる１，５−ペンタンジアミンの製造法であって、前記細菌が１００ｍＭのＬ−リジン存在化におけるＬ−リジンによる阻害解除度が８０％以上のアスパルトキナーゼIIIを発現し、かつリジンデカルボキシラーゼの発現が増強されていることを特徴とする１，５−ペンタンジアミンの製造方法を提供するものである。 That is, the present invention relates to a method for producing 1,5-pentanediamine by culturing E. coli having the ability to produce 1,5-pentanediamine, wherein the bacterium is 100 mM L-lysine. Provided is a method for producing 1,5-pentanediamine, which expresses aspartokinase III having a degree of inhibition release by L-lysine of 80% or more in the presence and enhanced expression of lysine decarboxylase To do.
また、本発明の好ましい態様によれば、Ｌ−リジンによるフィードバック阻害を解除する変異を有するアスパルトキナーゼIIIが、大腸菌（Ｅ.ｃｏｌｉ）由来のアスパルトキナーゼIIIの変異体であることを特徴とする１，５−ペンタンジアミンの製造方法である。 According to a preferred embodiment of the present invention, the aspartokinase III having a mutation that cancels feedback inhibition by L-lysine is a mutant of aspartokinase III derived from E. coli. Is a process for producing 1,5-pentanediamine.
さらに、本発明は、リジンデカルボキシラーゼが、大腸菌（Ｅ.ｃｏｌｉ）由来であることを特徴とする１，５−ペンタンジアミンの製造方法を提供するものである。 Furthermore, the present invention provides a method for producing 1,5-pentanediamine, wherein lysine decarboxylase is derived from E. coli.
本発明によれば、既存の方法に比較してより生産性の高い１，５−ペンタンジアミンの製造方法が提供される。 ADVANTAGE OF THE INVENTION According to this invention, the manufacturing method of 1,5-pentanediamine with higher productivity is provided compared with the existing method.
本発明は、１，５−ペンタンジアミンを生産する能力を有する大腸菌（以下、１，５−ペンタンジアミン生産菌ともいう）に１００ｍＭのＬ−リジン存在化におけるＬ−リジンによる阻害解除度が８０％以上のアスパルトキナーゼIII（以下、変異型ＡＫIIIともいう）を発現させることを特徴とする。 In the present invention, the degree of inhibition release by L-lysine in the presence of 100 mM L-lysine in E. coli having the ability to produce 1,5-pentanediamine (hereinafter also referred to as 1,5-pentanediamine-producing bacterium) is 80%. The above aspartokinase III (hereinafter also referred to as mutant AKIII) is expressed.
１，５−ペンタンジアミン生産菌に変異型ＡＫIIIを発現させる方法としては、変異型ＡＫIIIをコードする遺伝子（以下、変異型ＬｙｓＣともいう）を１，５−ペンタンジアミン生産菌に導入する方法が好ましく適用される。 As a method for expressing mutant AKIII in 1,5-pentanediamine-producing bacteria, a method in which a gene encoding mutant AKIII (hereinafter also referred to as mutant LysC) is introduced into 1,5-pentanediamine-producing bacteria is preferable. Applied.
変異型ＬｙｓＣを供与する生物としては、特に限定はないが、エシェリヒア属に属する微生物であることが好ましく、大腸菌（Ｅ．ｃｏｌｉ）であることがより好ましい。具体的にはナイトハルトらの著書（Neidhardt F.C. et.al., Escherichia coli and Salmonella Typhimurium, American Society for Microbiology, Washington D.C., 1208, table 1）にあげられる大腸菌が利用でき、たとえば、大腸菌のＪＭ１０９株やＭＣ１０６１株等があげられる。ここで、大腸菌の野生型ＬｙｓＣの遺伝子配列は配列番号１、大腸菌の野生型ＡＫIIIのアミノ酸配列は配列番号２で表され、変異型は該野生型の塩基配列もしくはアミノ酸配列が欠失、置換または付加したものである。 The organism that provides the mutant LysC is not particularly limited, but is preferably a microorganism belonging to the genus Escherichia, more preferably E. coli. Specifically, Escherichia coli listed in the book of Neidhardt et al. (Neidhardt FC et.al., Escherichia coli and Salmonella Typhimurium, American Society for Microbiology, Washington DC, 1208, table 1) can be used. MC1061 strain and the like. Here, the wild-type LysC gene sequence of Escherichia coli is represented by SEQ ID NO: 1, the amino acid sequence of wild-type AKIII of E. coli is represented by SEQ ID NO: 2, and the mutant type is a deletion, substitution or substitution of the wild-type base sequence or amino acid sequence. It is added.
大腸菌由来の変異型ＬｙｓＣを取得する方法としては、特開平１１−２８５３８１に開示される方法により取得することができる。変異型ＡＫIIIの具体例としては、特開平１１−２８５３８１に開示される配列番号２のアミノ酸配列中、Ｎ−末端から（１）３２３番目のグリシン残基がアスパラギン酸残基に置換する変異、（２）３２３番目のグリシン残基がアスパラギン酸残基に置換しかつ４０８番目のグリシン残基がアスパラギン酸残基に置換する変異、（３）３４番目のアルギニン残基がシステイン残基に置換しかつ３２３番目のグリシン残基がアスパラギン酸残基に置換する変異、（４）３２５番目のロイシン残基がフェニルアラニン残基に置換する変異、（６）３１８番目のメチオニン残基がイソロイシン残基に置換する変異、（７）３１８番目のメチオニン残基がイソロイシン残基に置換しかつ３４９番目のバリン残基がメチオニン残基に置換する変異、（８）３４５番目のセリン残基がロイシン残基に置換する変異、（９）３４７番目のバリン残基がメチオニン残基に置換する変異、（１０）３５２番目のスレオニン残基がイソロイシン残基に置換する変異、（１１）３５２番目のスレオニン残基がイソロイシン残基に置換しかつ３６９番目のセリン残基がフェニルアラニン残基に置換する変異、（１２）１６４番目のグルタミン酸残基がリジン残基に置換する変異または（１３）４１７番目のメチオニン残基がイソロイシン残基に置換しかつ４１９番目のシステイン残基がチロシン残基に置換する変異、菊地慶実著「大腸菌における新規リジンデカルボキシラーゼの発見及びその分子生物学的解析」（東北大学学位論文、１９９９年）に開示される（１４）２５０番目のグルタミン酸残基がリジン残基に置換する変異または（１５）３４４番目のスレオニン残基がメチオニン残基に置換する変異、（１６）３５４番目のアスパラギン酸残基がアスパラギンに置換する変異、あるいは本明細書の実施例にある（１７）３５４番目のアスパラギン酸残基がバリン残基に置換する変異、（１８）３８１番目のグルタミン酸残基がバリン残基に置換する変異、（１９）３５４番目のアスパラギン酸残基がバリン残基に置換しかつ３８１番目のグルタミン酸残基がバリン残基に置換する変異、（２０）３２１番目のセリン残基がフェニルアラニン残基に置換する変異または（２１）４２１番目のグリシン残基がアスパラギン酸残基に置換する変異が挙げられるが、これらの中でも（１６）〜（２１）に挙げられる変異型ＡＫIIIが１，５−ペンタンジアミン生産菌における１，５−ペンタンジアミンの生産効率に向上する変異として好ましく用いられる。 As a method of obtaining E. coli-derived mutant LysC, it can be obtained by the method disclosed in JP-A-11-285381. As a specific example of mutant AKIII, in the amino acid sequence of SEQ ID NO: 2 disclosed in JP-A-11-285381, (1) a mutation in which the 323rd glycine residue from the N-terminus is substituted with an aspartic acid residue ( 2) a mutation in which the 323rd glycine residue is substituted with an aspartic acid residue and the 408th glycine residue is substituted with an aspartic acid residue; (3) the 34th arginine residue is substituted with a cysteine residue; 323th glycine residue substitution with aspartic acid residue, (4) 325th leucine residue substitution with phenylalanine residue, (6) 318th methionine residue substitution with isoleucine residue (7) a mutation in which the 318th methionine residue is replaced with an isoleucine residue and the 349th valine residue is replaced with a methionine residue, (8 Mutation in which the 345th serine residue is substituted with a leucine residue, (9) Mutation in which the 347th valine residue is substituted with a methionine residue, (10) Mutation in which the 352nd threonine residue is substituted with an isoleucine residue (11) Mutation in which the 352nd threonine residue is substituted with an isoleucine residue and the 369th serine residue is substituted with a phenylalanine residue, (12) Mutation in which the 164th glutamic acid residue is substituted with a lysine residue Or (13) Mutation in which the 417th methionine residue is replaced with an isoleucine residue and the 419th cysteine residue is replaced with a tyrosine residue, “Discovery of a novel lysine decarboxylase in Escherichia coli and its molecular organism (14) The 250th glutamic acid residue is rigid. A mutation that substitutes a residue or (15) a mutation that substitutes a threonine residue at position 344 with a methionine residue, (16) a mutation that substitutes an aspartic acid residue at position 354, or an example of the present specification. (17) Mutation in which the 354th aspartic acid residue is substituted with a valine residue, (18) Mutation in which the 381st glutamic acid residue is substituted with a valine residue, (19) The 354th aspartic acid residue is in valine. A mutation in which the 381st glutamic acid residue is substituted with a valine residue, (20) a mutation in which the 321st serine residue is substituted with a phenylalanine residue, or (21) the 421st glycine residue is asparagine. Examples include mutations that substitute for acid residues, and among these, mutant AKIII listed in (16) to (21) is 1,5-pentanedi. It is preferably used as a mutation that improves the production efficiency of 1,5-pentanediamine in amine-producing bacteria.
これらの配列において、上記アミノ酸残基の置換を起こすような塩基配列の変異を有するものが、本発明の変異型ＡＫIIIをコードする核酸である。なお、置換されたアミノ酸残基に対応するコドンは、そのアミノ酸残基をコードするものであれば種類は特に問わない。また、菌種や菌株の違いにより保持する野生型ＡＫIIIのアミノ酸配列がわずかに相異するものがある。このような酵素の活性に関与しない位置でのアミノ酸残基の置換、欠失あるいは挿入を有するものも本発明の変異型ＬｙｓＣに含まれる。こうして得られた該変異型ＬｙｓＣを、組換えＤＮＡとして大腸菌（宿主）に導入し、発現させることによりフィードバック阻害が解除されたＡＫIIIを保有する大腸菌を取得できる。 Among these sequences, a nucleic acid encoding the mutant AKIII of the present invention has a nucleotide sequence mutation that causes substitution of the amino acid residue. The codon corresponding to the substituted amino acid residue is not particularly limited as long as it encodes the amino acid residue. In addition, there are those in which the amino acid sequences of wild-type AKIII retained are slightly different due to differences in bacterial species and strains. Such mutant LysCs of the present invention also include amino acid residue substitutions, deletions or insertions at positions not involved in enzyme activity. The mutant LysC thus obtained is introduced into E. coli (host) as a recombinant DNA and expressed to obtain E. coli having AKIII in which feedback inhibition is released.
変異型ＬｙｓＣを組換えＤＮＡとして大腸菌に導入する方法としては、該遺伝子断片を取り出し、他のベクターに挿入する方法が挙げられる。本発明において用いることのできることのできるベクターＤＮＡとしては、プラスミドベクターＤＮＡが好ましく、例えばｐＵＣ１９、ｐＵＣ１８、ｐＢＲ３２２、ｐＨＳＧ２９９、ｐＨＳＧ２９８、ｐＨＳＧ３９９、ｐＨＳＧ３９８、ｐＲＳＦ１０１０、ｐＭＷ１１９、ｐＭＷ１１８、ｐＭＷ２１９、ｐＭＷ２１８等が挙げられる。他にもファージＤＮＡのベクターも利用できる。その際、変異型ＬｙｓＣの発現を効率的に実施するために、変異型ＬｙｓＣの上流に、ｌａｃ、ｔｒｐ、ＰＬ等の微生物内で働く他のプロモーターを連結してもよく、野生型ＬｙｓＣに含まれるプロモーターをそのまま、あるいは増幅して用いてもよい。 Examples of the method for introducing mutant LysC into E. coli as a recombinant DNA include a method of taking out the gene fragment and inserting it into another vector. The vector DNA that can be used in the present invention is preferably a plasmid vector DNA, and examples thereof include pUC19, pUC18, pBR322, pHSG299, pHSG298, pHSG399, pHSG398, pRSF1010, pMW119, pMW118, pMW219, pMW218 and the like. In addition, phage DNA vectors can also be used. At that time, in order to efficiently carry out the expression of the mutant LysC, other promoters working in the microorganism such as lac, trp, and PL may be linked upstream of the mutant LysC, and are included in the wild-type LysC. The promoter may be used as it is or after amplification.
また、上記のように、変異型ＬｙｓＣを自律複製可能なベクターＤＮＡに挿入したものを宿主に導入し、プラスミドのように染色体外ＤＮＡとして宿主に保持させてもよいが、変異型ＬｙｓＣをトランスダクション、トランスポゾン（Berg D.E. and Berg C.M., Bio/Technol., 1, 417 (1983)）、Ｍｕファージ（特開平２−１０９９８５）または相同性組換え（Experiments in Molecular Genetics, ColdSpring Harbor Lab. (1972)）を用いた方法で宿主微生物の染色体に組み込んでもよい。 In addition, as described above, mutant LysC inserted into a vector DNA capable of autonomous replication may be introduced into the host and retained in the host as extrachromosomal DNA like a plasmid, but mutant LysC may be transduced. , Transposon (Berg DE and Berg CM, Bio / Technol., 1, 417 (1983)), Mu phage (JP-A-2-109985) or homologous recombination (Experiments in Molecular Genetics, ColdSpring Harbor Lab. (1972)) It may be incorporated into the chromosome of the host microorganism by a method using
次に、本発明で用いられるリジンデカルボキシラーゼについて説明する。リジンデカルボキシラーゼとは、アミノ酸の一種であるリジンから二酸化炭素を除去することにより１，５−ペンタンジアミンを生成し得る酵素である、このような活性を有していれば起源は問わないが、好ましくは微生物由来のものが使用できる。 Next, lysine decarboxylase used in the present invention will be described. Lysine decarboxylase is an enzyme that can produce 1,5-pentanediamine by removing carbon dioxide from lysine, which is a kind of amino acid. Preferably, those derived from microorganisms can be used.
このような微生物由来のリジンデカルボキシラーゼとしては、バシラス・ハロドゥランス（Bacillus halodurans）、バシラス・サブチリス（Bacillus subtilis）、大腸菌（Escherichia coli） 、セレノモナス・ルミナンチウム（Selenomonas tuminantium）、ビブリオ・コレラ（Vibrio cholerae）、ビブリオ・パラヘモリティカス（Vibrio parahaemolyticus）、ストレプトマイセス・コエリカーラ（Streptomyces coelicolor）、ストレプトマイセス・ピロサス（Sterptomyces pilosus）、エイケネラ・コロデンス（Eikenella corrodens） 、イユバクテリウム・アシダミノフィルム（Eubacterium acidaminophilum） 、サルモネラ・ティフィムリウム（Salmonella typhimurium） 、ハフニア・アルベイ（Hafnia alvei）、ナイセリア・メニンギチデス（Neisseria meningitidis） 、テルモプラズマ・アシドフィルム（Thermoplasma acidophilum） またはピロコッカス・アビシ（Pyrococcus abyssi）等由来が挙げられるが、好ましくは大腸菌由来のものである。 Examples of lysine decarboxylase derived from such microorganisms include Bacillus halodurans, Bacillus subtilis, Escherichia coli, Selenomonas tuminantium, Vibrio cholerae , Vibrio parahaemolyticus, Streptomyces coelicolor, Sterptomyces pilosus, Eikenella corrodens, Eubacterium acidum phil, um Salmonella typhimurium, Hafnia alvei, Neisseria meningitidis, Thermoplasma acidoph (Thermoplasma acidoph ilum) or Pyrococcus abyssi, and the like, preferably from E. coli.
大腸菌由来のリジンデカルボキシラーゼをコードする遺伝子（以下、リジンデカルボキシラーゼ遺伝子ともいう）として、ｃａｄＡまたはｌｄｃＣが好ましく、このうちｃａｄＡがより好適に用いられる。すなわち、Ｌ−リジンによるフィードバック阻害が解除された変異を有する変異型ＬｙｓＣを保持する大腸菌のリジンデカルボキシラーゼをコードするｃａｄＡまたはｌｄｃＣ遺伝子のコピー数を高め、または細菌細胞内のｃａｄＡまたはｌｄｃＣ遺伝子の発現が増強されるようにｃａｄＡまたはｌｄｃＣ遺伝子のプロモーターを改変することにより、リジンデカルボキシラーゼの活性が増強され、１，５−ペンタンジアミン生産性を一層向上させることができる。 As a gene encoding lysine decarboxylase derived from E. coli (hereinafter also referred to as lysine decarboxylase gene), cadA or ldcC is preferable, and cadA is more preferably used. That is, increasing the copy number of cadA or ldcC gene encoding lysine decarboxylase of Escherichia coli carrying mutant LysC having a mutation in which feedback inhibition by L-lysine is released, or expressing cadA or ldcC gene in bacterial cells By modifying the promoter of the cadA or ldcC gene so as to be enhanced, the activity of lysine decarboxylase is enhanced, and 1,5-pentanediamine productivity can be further improved.
リジンデカルボキシラーゼ遺伝子をクローニングする方法に特に制限はない。既知の遺伝子情報に基づき、ＰＣＲ（polymerase chain reaction）法を用いて必要な遺伝領域を増幅取得する方法、既知の遺伝子情報に基づきゲノムライブラリーやｃＤＮＡライブラリーより相同性や酵素活性を指標としてクローニングする方法などが挙げられる。本発明においては、これらの遺伝子は、遺伝的多形性などによる変異型も含む。なお、遺伝的多形性とは、遺伝子上の自然突然変異により遺伝子の塩基配列が一部変化しているものをいう。例えばＥ．ｃｏｌｉ Ｋ１２株の染色体ＤＮＡよりＰＣＲ法を用いて、リジンデカルボキシラーゼ遺伝子であるｃａｄＡまたはｌｄｃＣ遺伝子をクローニングすることができる。この際使用する染色体ＤＮＡはＥ．ｃｏｌｉ由来であればどの菌株由来でもよい。特に好ましくは、配列番号３に示したｃａｄＡ遺伝子または配列番号４に示したｌｄｃＣ遺伝子のＤＮＡ配列である。 There is no particular limitation on the method for cloning the lysine decarboxylase gene. Based on known gene information, PCR (polymerase chain reaction) method is used to amplify and acquire necessary genetic regions, cloning based on known gene information from genomic and cDNA libraries using homology and enzyme activity as indicators The method of doing is mentioned. In the present invention, these genes include mutants due to genetic polymorphism and the like. Genetic polymorphism means that the base sequence of a gene is partially changed due to a natural mutation on the gene. For example, E.I. The lysine decarboxylase gene cadA or ldcC gene can be cloned from the chromosomal DNA of E. coli strain K12 using PCR. The chromosomal DNA used here is E. coli. Any strain may be used so long as it is derived from E. coli. Particularly preferred is the DNA sequence of the cadA gene shown in SEQ ID NO: 3 or the ldcC gene shown in SEQ ID NO: 4.
リジンデカルボキシラーゼ遺伝子を保持するベクターＤＮＡは、通常大腸菌で利用するベクターＤＮＡにリジンデカルボキシラーゼ遺伝子を導入することにより得ることができる。上記ベクターＤＮＡの好適な例として、例えば、ｐＢＲ３２２、ｐＵＣ１９、ｐＢｌｕｅｓｃｒｉｐｔＫＳ+、ｐＡＣＹＣ１７７、ｐＡＣＹＣ１８４、ｐＡＹＣ３２、ｐＭＷ１１９、ｐＥＴ２２ｂ等が挙げられる。他にもファージＤＮＡのベクターも利用できる。 A vector DNA carrying a lysine decarboxylase gene can be obtained by introducing a lysine decarboxylase gene into a vector DNA usually used in E. coli. Preferable examples of the vector DNA include pBR322, pUC19, pBluescriptKS +, pACYC177, pACYC184, pAYC32, pMW119, pET22b and the like. In addition, phage DNA vectors can also be used.
ベクターＤＮＡが導入された大腸菌は、選択マーカー（ 例えばアンピシリン）で選択される。リジンデカルボキシラーゼ（ｃａｄＡまたはｌｄｃＣ）遺伝子発現を増強させる方法としては、遺伝子コピー数の増加が含まれるため、大腸菌で機能し得るベクター中にリジンデカルボキシラーゼ遺伝子を導入すると、遺伝子のコピー数が増加する。好ましくはマルチコピーベクターが使用され、マルチコピーベクターとしては、ｐＢＲ３２２、ｐＵＣ１９、ｐＢｌｕｅｓｃｒｉｐｔＫＳ+、ｐＡＣＹＣ１７７、ｐＡＣＹＣ１８４、ｐＡＹＣ３２、ｐＭＷ１１９、ｐＥＴ２２ｂなどが挙げられる。また、遺伝子発現の増強は、例えば、相同組換えなどの方法により、遺伝子の複数コピーを細菌染色体中に導入することにより達成され得る。 E. coli into which the vector DNA has been introduced is selected with a selection marker (for example, ampicillin). Methods to enhance lysine decarboxylase (cadA or ldcC) gene expression include increased gene copy number, so introducing the lysine decarboxylase gene into a vector that can function in E. coli increases the gene copy number . Preferably, a multicopy vector is used, and examples of the multicopy vector include pBR322, pUC19, pBluescriptKS +, pACYC177, pACYC184, pAYC32, pMW119, pET22b, and the like. In addition, enhancement of gene expression can be achieved by introducing multiple copies of the gene into bacterial chromosomes by methods such as homologous recombination, for example.
リジンデカルボキシラーゼ遺伝子の発現増強は、本来の天然プロモーターの代わりに、より強力なプロモーターの制御下に、本発明のＤＮＡを配置することによっても達成され得る。好ましくは、上記リジンデカルボキシラーゼ遺伝子が、該遺伝子の発現を可能とするプロモーターの下流に導入されることによって達成され得る。用語「本来のプロモーター」は、野生型生物に存在するＤＮＡ領域であって、遺伝子のオープンリーディングフレーム（ＯＲＦ）の上流に位置し、同遺伝子の発現を促進する領域を意味する。プロモーターの強度の評価法は、例えば、Deuschle U., Kammerer W., Gents R., Bujard H.（エシェリヒア・コリにおけるプロモーター：ｉｎ ｖｉｖｏ強度の階層は代替的構造を示す。 EMBO J. 1986, 5, 2987-2994）により記載されている。 Enhanced expression of the lysine decarboxylase gene can also be achieved by placing the DNA of the present invention under the control of a stronger promoter instead of the native promoter. Preferably, the lysine decarboxylase gene can be achieved by being introduced downstream of a promoter that enables expression of the gene. The term “native promoter” refers to a region of DNA present in a wild-type organism that is located upstream of the open reading frame (ORF) of the gene and promotes expression of the gene. For example, Deuschle U., Kammerer W., Gents R., Bujard H. (Promoter in Escherichia coli: Hierarchy of in vivo strength indicates an alternative structure. EMBO J. 1986, 5 , 2987-2994).
翻訳の向上は、本来のシャイン−ダルガーノ（Shine-Dalgarno）配列（ＳＤ配列）の代わりに、より効果的なＳＤ配列を本発明のＤＮＡ中に導入することにより達成され得る。ＳＤ配列は、リボソームの１６ＳＲＮＡと相互作用するｍＲＮＡの開始コドンの上流領域である（Shine J. and Dalgarno L., Proc. Natl. Acad. Sci. U S A, 1974, 71, 4, 1342-6）。用語「本来のＳＤ配列」とは、野生型に存在するＳＤ配列を意味する。効果的なＳＤ配列としては、Ｔ７ファージ由来φ１０遺伝子のＳＤ配列が挙げられる（Olins P.O. et al, Gene, 1988, 73, 227-235）。 Improved translation can be achieved by introducing a more effective SD sequence into the DNA of the present invention instead of the original Shine-Dalgarno sequence (SD sequence). The SD sequence is the upstream region of the start codon of mRNA that interacts with ribosomal 16S RNA (Shine J. and Dalgarno L., Proc. Natl. Acad. Sci. USA, 1974, 71, 4, 1342-6). The term “original SD sequence” means an SD sequence present in the wild type. An effective SD sequence includes the SD sequence of T10 phage-derived φ10 gene (Olins P.O. et al, Gene, 1988, 73, 227-235).
上記のようにして得られるＬ−リジンによるフィードバック阻害が解除されたＡＫIIIを保持し、かつリジンデカルボキシラーゼの発現が増強された大腸菌を、好適な培地中で培養し、該培養物中に１，５−ペンタンジアミンを生産蓄積させ、該培養物から１，５−ペンタンジアミンを採取することにより、１，５−ペンタンジアミンを効率よく製造することができる。 Escherichia coli that retains AKIII that has been desensitized to feedback inhibition by L-lysine and is enhanced in expression of lysine decarboxylase obtained as described above is cultured in a suitable medium, By producing and accumulating 5-pentanediamine and collecting 1,5-pentanediamine from the culture, 1,5-pentanediamine can be efficiently produced.
本発明は、上記形質転換大腸菌を培養することを含む１，５−ペンタンジアミンの製造方法に関するものである。本発明における培養方法について説明する。 The present invention relates to a method for producing 1,5-pentanediamine, which comprises culturing the transformed E. coli. The culture method in the present invention will be described.
培養には、バッチ培養、流加培養（フェドバッチ培養）、連続培養のいずれも採用することができる。これら本発明における大腸菌の培養には、分離膜を用いることも可能である。 For the culture, any of batch culture, fed-batch culture (fed batch culture), and continuous culture can be employed. A separation membrane can also be used for culturing E. coli according to the present invention.
培地は炭素源、窒素源、無機イオンおよび必要に応じてその他の有機微量成分を含有する通常の培地が使用可能であり、１，５−ペンタンジアミンが産生される限り特に制限はない。 As the medium, a normal medium containing a carbon source, a nitrogen source, inorganic ions and other organic trace components as required can be used, and there is no particular limitation as long as 1,5-pentanediamine is produced.
炭素源としてはグルコース、フラクトース、糖蜜などの糖類、フマール酸、クエン酸、コハク酸などの有機酸、メタノール、エタノール、グリセロールなどのアルコール類などを１〜１５％、窒素源として酢酸アンモニウムなどの有機アンモニウム塩、硫酸アンモニウム、塩化アンモニウム、リン酸アンモニウム、硝酸アンモニウムなどの無機アンモニウム塩、アンモニアガス、アンモニア水、尿素などを０．１％〜４．０％、有機微量成分としてはピリドキサルリン酸やビオチンなどの被要求性物質が０．００００００１％〜０．１％、それぞれ適当量含有する培地が用いられる。 Carbon sources include sugars such as glucose, fructose and molasses, organic acids such as fumaric acid, citric acid and succinic acid, alcohols such as methanol, ethanol and glycerol, etc., and organic substances such as ammonium acetate as a nitrogen source. Inorganic ammonium salts such as ammonium salt, ammonium sulfate, ammonium chloride, ammonium phosphate, and ammonium nitrate, ammonia gas, aqueous ammonia, urea, etc. are 0.1% to 4.0%. Organic trace components such as pyridoxal phosphate, biotin, etc. A medium containing 0.0000001% to 0.1% of the required substance, each in an appropriate amount, is used.
その他、培地にはリン酸カルシウム、硫酸マグネシウム、塩化カルシウム、塩化ナトリウム、硫酸亜鉛、硫酸銅、硫酸第１鉄などが微量物質として必要に応じて添加されうる。さらにチアミン、ナイアシンなどの要求ビタミン、又はこれらを含有する酵母エキス、コーンスティープリカー、その他天然物を適当量含有した培地を用いることもできる。 In addition, calcium phosphate, magnesium sulfate, calcium chloride, sodium chloride, zinc sulfate, copper sulfate, ferrous sulfate and the like can be added to the medium as trace substances as necessary. Furthermore, required vitamins such as thiamine and niacin, or yeast extract, corn steep liquor and other natural products containing these may be used.
次に、培養条件について説明する。培養は通常、好気的条件下で行うが、１，５−ペンタンジアミンの生成に応じて嫌気的条件下で行うことも可能である。 Next, culture conditions will be described. Cultivation is usually performed under aerobic conditions, but can also be performed under anaerobic conditions depending on the production of 1,5-pentanediamine.
この１，５−ペンタンジアミン発酵における培養ｐＨ調整には水酸化ナトリウムまたはアンモニアおよび塩酸またはジカルボン酸を使用することが好ましい。これら中和剤を用い培養ｐＨを５〜８に、特に好ましくはｐＨ ６．５〜７．５に制御するのがよい。なお中和剤の状態に制限はなく、気体、液体、固体または水溶液で使用される。特に好ましくは水溶液である。 It is preferable to use sodium hydroxide or ammonia and hydrochloric acid or dicarboxylic acid for adjusting the culture pH in this 1,5-pentanediamine fermentation. Using these neutralizing agents, the culture pH is preferably controlled to 5 to 8, particularly preferably pH 6.5 to 7.5. In addition, there is no restriction | limiting in the state of a neutralizer, It uses by gas, a liquid, solid, or aqueous solution. Particularly preferred is an aqueous solution.
培養温度は一般に２５℃〜４５℃、特に好ましくは３０℃〜３７℃が用いられる。また、培養時間に特に制限はなく、一般に４〜１２０時間振とうまたは撹拌培養することで好ましい結果が得られる。 The culture temperature is generally 25 ° C to 45 ° C, particularly preferably 30 ° C to 37 ° C. Moreover, there is no restriction | limiting in particular in culture | cultivation time, Generally a favorable result is obtained by shaking or stirring culture for 4 to 120 hours.
培養液中に生成した１，５−ペンタンジアミンは、そのまま単離精製することなく利用することもできるし、菌体を遠心分離などで除去した後、濃縮、蒸留および晶析などの従来知られている方法を組み合わせて分離・精製することもできる。例えば、特開２００４−２２２５６９号公報に示される晶析を用いた精製法を用いることができる。 The 1,5-pentanediamine produced in the culture solution can be used without isolation and purification as it is, and after the cells have been removed by centrifugation or the like, conventional methods such as concentration, distillation and crystallization are known. It is also possible to separate and purify by combining these methods. For example, a purification method using crystallization disclosed in Japanese Patent Application Laid-Open No. 2004-222569 can be used.
以下、本発明の１，５−ペンタンジアミンの製造方法をさらに詳細に説明するために、実施例を挙げて説明する。本発明はこれらの実施例に限定されない。 Hereinafter, in order to explain the manufacturing method of 1,5-pentanediamine of the present invention in more detail, examples will be described. The present invention is not limited to these examples.
下記実施例においては、リジンによるフィードバック阻害が解除された変異を有する変異型ＬｙｓＣの例として大腸菌由来の遺伝子、発現が増強されたリジンデカルボシキラーゼ遺伝子の例として、大腸菌由来のｇａｐＡ遺伝子プロモーターと連結した大腸菌由来のｃａｄＡ遺伝子（配列番号３）を用いた。 In the following examples, an Escherichia coli-derived gene as an example of a mutant LysC having a mutation in which feedback inhibition by lysine is released, an example of a lysine decarboxylase gene with enhanced expression, an E. coli-derived gapA gene promoter, The ligated E. coli-derived cadA gene (SEQ ID NO: 3) was used.
（実施例１）変異型ＬｙｓＣ遺伝子の取得
＜１＞野生型ｌｙｓＣのクローニング
大腸菌（Ｅ．ｃｏｌｉ）のＡＫIIIをコードする遺伝子（ｌｙｓＣ）の塩基配列は既に報告されており（Cassan M., Parsot C., Cohen G.N. and Patte J.C., J. Biol. Chem., 261, 1052 (1986)）、オープンリーディングフレーム（ＯＲＦ）は１３４７塩基対よりなり、４４９アミノ酸残基からなる蛋白質をコードしていることがわかっている。この遺伝子にはオペレーターが存在しＬ−リジンによる抑制を受けるため、このオペレーター領域を除くために、ＳＤ配列とＯＲＦのみを含む領域を、ＰＣＲ法を用いて増幅し、クローニングすることにした。
Example 1 Acquisition of Mutant LysC Gene <1> Cloning of wild-type lysC The base sequence of the gene (lysC) encoding AKIII of E. coli has already been reported (Cassan M., Parsot C Cohen GN and Patte JC, J. Biol. Chem., 261, 1052 (1986)), the open reading frame (ORF) consists of 1347 base pairs and encodes a protein consisting of 449 amino acid residues. know. Since this gene has an operator and is suppressed by L-lysine, in order to remove this operator region, it was decided to amplify and clone a region containing only the SD sequence and the ORF using the PCR method.
Ｅ．ｃｏｌｉ Ｋ−１２ Ｗ３１１０株の全ゲノムＤＮＡを斎藤、三浦の方法（Biochem. Biophys. Acta., 72, 619(1963)）により調製し、配列番号５及び６に示す配列を有する２種のプライマーを用いてエルリッチらの方法（ＰＣＲ Technology, Stocktonpress (1989)）に従ってＰＣＲ反応を行い、ｌｙｓＣの増幅を行った。得られたＤＮＡを制限酵素ＥｃｏＲＩとＸｂａＩで消化した後、低コピーベクターｐＭＷ１１９（宝酒造株式会社）のＥｃｏＲＩ、ＸｂａＩ部位に挿入したプラスミドとしてｐＳＹ１を得た。 E. The total genomic DNA of E. coli K-12 W3110 strain was prepared by the method of Saito and Miura (Biochem. Biophys. Acta., 72, 619 (1963)), and two primers having the sequences shown in SEQ ID NOs: 5 and 6 were prepared. The PCR reaction was performed according to the method of Elrich et al. (PCR Technology, Stocktonpress (1989)) to amplify lysC. After digesting the obtained DNA with restriction enzymes EcoRI and XbaI, pSY1 was obtained as a plasmid inserted into the EcoRI and XbaI sites of the low copy vector pMW119 (Takara Shuzo).
ＡＫI、ＡＫIIおよびＡＫIIIの完全欠損株であるＥ．ｃｏｌｉ Ｇｉｆ１０６Ｍ１株（thrA1101、ilvA296、metL1000、lysC1001、arg-1000）をｐＳＹ１で形質転換したところ、Ｇｉｆ１０６Ｍ１のホモセリンおよびジアミノピメリン酸の要求性が相補されたので、プラスミドに挿入されたＤＮＡ断片は、活性のある野生型ＡＫIIIをコードする遺伝子であるｌｙｓＣを含有すると確認した。また、ＡＫI、ＡＫIIおよびＡＫIII完全欠損株Ｅ．ｃｏｌｉ Ｇｉｆ１０６Ｍ１株にｐＳＹ１を導入して得られる形質転換株をＧｉｆ１０６Ｍ１／ｐＳＹ１株と命名し、最少培地Ｍ９に５０ｍＭのＬ−リジンを加えた培地で培養した結果、Ｇｉｆ１０６Ｍ１／ｐＳＹ１株は野生型のｌｙｓＣを含有するプラスミドを保持し、同プラスミド上のｌｙｓＣにコードされる野生型ＡＫIIIが唯一のＡＫであるため、野生型ＡＫIIIがＬ−リジンにより阻害され、Ｇｉｆ１０６Ｍ１／ｐＳＹ１株はＬ−スレオニン、Ｌ−イソロイシン、Ｌ−メチオニン及びジアミノピメリン酸（ＤＡＰ）の合成ができなくなり生育が抑えられた。 E. coli, which is a completely deficient strain of AKI, AKII and AKIII. When E. coli Gif106M1 strain (thrA1101, ilvA296, metL1000, lysC1001, arg-1000) was transformed with pSY1, the requirement for homoserine and diaminopimelate of Gif106M1 was complemented, so that the DNA fragment inserted into the plasmid It was confirmed to contain lysC, a gene encoding a certain wild type AKIII. In addition, AKI, AKII and AKIII complete deletion strains E. coli. The transformant obtained by introducing pSY1 into E. coli Gif106M1 strain was named Gif106M1 / pSY1 strain and cultured in a medium containing 50 mM L-lysine in the minimal medium M9. As a result, the Gif106M1 / pSY1 strain Since the wild type AKIII encoded by lysC on the plasmid is the only AK, the wild type AKIII is inhibited by L-lysine, and the Gif106M1 / pSY1 strain is L-threonine, L- Isoleucine, L-methionine and diaminopimelic acid (DAP) could not be synthesized and growth was suppressed.
＜２＞変異型ｌｙｓＣの取得
Ｌ−リジンによる阻害の解除されたＡＫIIIをコードする変異型ｌｙｓＣを含有するプラスミド保持株は、著量のＬ−リジンが添加された最少培地Ｍ９上での生育が可能になると予想し、生育がＬ−リジンに耐性となっている株を選択することによって、変異型ｌｙｓＣを含有するプラスミド保持株を選択することにした。まず、変異型ｌｙｓＣを効率よく取得するために、＜１＞で作製したｐＳＹ１上のｌｙｓＣに変異処理を行なうことにした。
<2> Acquisition of mutant lysC Plasmid-bearing strains containing mutant lysC encoding AKIII that has been desensitized by L-lysine can grow on minimal medium M9 supplemented with a significant amount of L-lysine. It was decided that a plasmid-carrying strain containing a mutant lysC was selected by selecting a strain that was expected to be capable of growing and was resistant to L-lysine. First, in order to efficiently obtain mutant lysC, it was decided to perform mutation treatment on lysC on pSY1 prepared in <1>.
（１−２−１）変異型ｌｙｓＣを含有するプラスミド保持株の選択条件の検討
Ｇｉｆ１０６Ｍ１／ｐＳＹ１株をそれぞれ種々の濃度のＬ−リジンを含有するＭ９寒天平板培地上で培養を行なった。そしてＬ−リジンによる生育阻止濃度を調べ、変異型ｌｙｓＣを含有するプラスミド保持株の選択条件の検討を行なった。各種濃度でＬ−リジンを含むＭ９培地での形質転換体の生育を表１に示す。なお、表中の＋は形質転換株が生育したことを示し、±はやや生育したことを示し、−は生育しなかったことを示す。
(1-2-1) Examination of selection conditions of plasmid-bearing strain containing mutant lysC Gif106M1 / pSY1 strain was cultured on M9 agar plate medium each containing various concentrations of L-lysine. Then, the growth inhibitory concentration by L-lysine was examined, and the selection conditions for the plasmid-carrying strain containing the mutant lysC were examined. Table 1 shows the growth of transformants on M9 medium containing L-lysine at various concentrations. In the table, + indicates that the transformed strain has grown, ± indicates that it has grown slightly, and − indicates that it has not grown.
変異導入実験にはｐＳＹ１を用い、最少培地Ｍ９にＬ−リジン５０ｍＭを添加したものを、変異型ｌｙｓＣを含有するプラスミド保持株の選択に用いた。以下、実施例１においてこの培地を選択培地という。 In the mutagenesis experiment, pSY1 was used, and L-lysine 50 mM added to a minimal medium M9 was used for selection of a plasmid-carrying strain containing a mutant lysC. Hereinafter, this medium is referred to as a selective medium in Example 1.
（１−２−２）ヒドロキシルアミンによるｐＳＹ１のインビトロ変異処理
ｐＳＹ１プラスミドへの変異の導入には、プラスミドを直接ヒドロキシルアミンで処理するインビトロ変異処理法を用いた。
(1-2-2) In Vitro Mutation Treatment of pSY1 with Hydroxylamine An in vitro mutation treatment method in which the plasmid was directly treated with hydroxylamine was used to introduce the mutation into the pSY1 plasmid.
（ヒドロキシルアミンによるインビトロ変異処理）
２μｇのｐＳＹ１を０．４Ｍ ヒドロキシルアミン中（５００ｍＭ リン酸ナトリウム緩衝液（ｐＨ６．０） ２０μｌ、４Ｍ ヒドロキシルアミン塩酸塩 ２０μｌ、１０ｍＭ ＥＤＴＡ（ｐＨ６．０）２０μｌ、水を加えて計１００μｌとする）で、６０℃及び６２．５℃の条件下で、３０分処理した。処理後のＤＮＡをGFX PCR DNA and Gel Band Purification Kit（ＧＥ Ｈｅａｌｔｈｃａｒｅ製)で精製後、高効率コンピテントセルＥ．ｃｏｌｉ ＸＬ１０Ｇｏｌｄ（Ｔａｋａｒａ社製）に導入し、完全培地（Ｌbroth：1％ Bacto trypton, 0.5％ Yeast extract, 0.5％ NaCl, 1.5％寒天）に撒きコロニーを形成させた。プレート上に出現したコロニーをすべて掻き集め、QIAprep Spin Miniprep Kit （キアゲン社）によりプラスミド抽出し、ＡＫI、ＡＫIIおよびＡＫIII完全欠損株であるＧｉｆ１０６Ｍ１株に導入し、これを（１−２−１）で設定した選択培地に撒き、生育可能な株を選択し候補株とした。
(In vitro mutation treatment with hydroxylamine)
2 μg of pSY1 in 0.4 M hydroxylamine (500 μM sodium phosphate buffer (pH 6.0) 20 μl, 4 M hydroxylamine hydrochloride 20 μl, 10 mM EDTA (pH 6.0) 20 μl, water added to make a total of 100 μl) , At 60 ° C. and 62.5 ° C. for 30 minutes. The treated DNA is purified by GFX PCR DNA and Gel Band Purification Kit (manufactured by GE Healthcare). The cells were introduced into E. coli XL10 Gold (manufactured by Takara) and seeded in complete medium (Lbroth: 1% Bacto trypton, 0.5% Yeast extract, 0.5% NaCl, 1.5% agar) to form colonies. All the colonies that appeared on the plate were scraped and extracted with the QIAprep Spin Miniprep Kit (Qiagen), and introduced into the Gif106M1 strain, which is completely deficient in AKI, AKII, and AKIII. This was set in (1-2-1). The selected strain was seeded and a viable strain was selected as a candidate strain.
上記で得られた候補株合計１６株を再度選択培地にスポットし、Ｌ−リジン耐性が認められた１６株を得た。これら１６株がどれだけリジン耐性を有するかを調べるために、Ｌ−リジン存在下での液体培養によって増殖を調べた。１００または２００ｍＭのＬ−リジンを含むＭ９培地において、培養時間４８時間、温度３７℃の条件下、攪拌１８０ｒｐｍで上記１６株の試験管培養を行い、菌体濁度を測定した結果を表２に示す。 A total of 16 candidate strains obtained above were spotted on the selective medium again, and 16 strains with L-lysine resistance were obtained. To examine how resistant these 16 strains are to lysine, growth was examined by liquid culture in the presence of L-lysine. Table 2 shows the results of measuring the cell turbidity in M9 medium containing 100 or 200 mM L-lysine and culturing the above 16 strains at 180 rpm with stirring at a temperature of 37 ° C. for 48 hours. Show.
２００ｍＭのリジン濃度でも耐性のあったＮｏ．１、Ｎｏ．４、Ｎｏ．６、Ｎｏ．１０、Ｎｏ．１１株の５株の有する変異型ｌｙｓＣをそれぞれｌｙｓＣＭ１、ｌｙｓＣＭ４、ｌｙｓＣＭ６、ｌｙｓＣＭ１０、ｌｙｓＣＭ１１と命名した。 No. which was resistant even at a lysine concentration of 200 mM. 1, no. 4, no. 6, no. 10, no. The mutated lysC of 11 strains was named lysCM1, lysCM4, lysCM6, lysCM10, and lysCM11, respectively.
（１−２−３）ポイントミューテーションによるｐＳＹ１の１アミノ酸置換
ｐＳＹ１のプラスミドＤＮＡを鋳型とし、配列番号１８に示す合成ＤＮＡプライマーと、プロメガ（Ｐｒｏｍｅｇａ）社のGeneEditor in vitro Site-Directed Mutagenesis Systemを用いて、配列番号２に示すアミノ酸配列を有するＡＫIII遺伝子に、Ｎ−末端から３５２番目のスレオニン残基がイソロイシン残基に置換された変異を導入した。この変異を有するアミノ酸配列を有する遺伝子は、上記に示したとおりリジンによるフィードバック阻害が解除されることが既に分かっており、これをｌｙｓＣＭＰと命名した。そして、ｐＳＹ１と同様に、図１に示すようにｌｙｓＣＭＰをｐＭＷ１１９のＥｃｏＲＩ、ＸｂａＩ部位に挿入したプラスミドｐＳＹＭＰを得た。
(1-2-3) One amino acid substitution of pSY1 by point mutation Using the plasmid DNA of pSY1 as a template, using a synthetic DNA primer shown in SEQ ID NO: 18 and GeneEditor in vitro Site-Directed Mutagenesis System of Promega Thus, a mutation in which the threonine residue at position 352 from the N-terminal was replaced with an isoleucine residue was introduced into the AKIII gene having the amino acid sequence shown in SEQ ID NO: 2. A gene having an amino acid sequence having this mutation has already been found to release feedback inhibition by lysine as shown above, and this was named lysCMP. Then, similarly to pSY1, a plasmid pSYMP was obtained in which lysCMP was inserted into the EcoRI and XbaI sites of pMW119 as shown in FIG.
（１−２−４）野生型ｌｙｓＣおよび変異型ｌｙｓＣの塩基配列の決定
常法に従い、今回取得したｌｙｓＣＭ１、ｌｙｓＣＭ４、ｌｙｓＣＭ６、ｌｙｓＣＭ１０、ｌｙｓＣＭ１１、ｌｙｓＣＭＰそれぞれについて塩基配列を決定し、野生型ｌｙｓＣとの変異点を明らかにした。結果を表３に示す。（１−２−２）で取得した変異型ｌｙｓＣの塩基配列の変異の種類は５種類であった。ｌｙｓＣＭ６に関しては、野生型ｌｙｓＣ遺伝子と塩基配列は同一であり、変異は確認されなかった。（１−２−３）で合成したｌｙｓＣＭＰにおいては、配列番号２に示すアミノ酸配列のＮ−末端から３５２番目のスレオニン残基がイソロイシン残基に置換された変異を確認した。
(1-2-4) Determination of nucleotide sequences of wild-type lysC and mutant-type lysC According to a conventional method, the nucleotide sequences of lysCM1, lysCM4, lysCM6, lysCM10, lysCM11, and lysCMP obtained this time were determined, The mutation point was clarified. The results are shown in Table 3. There were five types of mutations in the base sequence of the mutant lysC obtained in (1-2-2). Regarding lysCM6, the nucleotide sequence was the same as that of the wild-type lysC gene, and no mutation was confirmed. In lysCMP synthesized in (1-2-3), a mutation was confirmed in which the threonine residue at position 352 from the N-terminus of the amino acid sequence shown in SEQ ID NO: 2 was replaced with an isoleucine residue.
＜３＞変異型ｌｙｓＣの単離及び変異型ｌｙｓＣ遺伝子産物の検討
上記変異型ｌｙｓＣであることが確認できたｌｙｓＣＭ１、ｌｙｓＣＭ４、ｌｙｓＣＭ１０、ｌｙｓＣＭ１１をｐＳＹ１同様ｐＭＷ１１９に挿入したプラスミドを得、それぞれ図１に示した通り、ｐＳＹＭ１、ｐＳＹＭ４、ｐＳＹＭ１０、ｐＳＹＭ１１と命名した。そして、ＡＫI、ＡＫIIおよびＡＫIII完全欠損株Ｇｉｆ１０６Ｍ１をｐＳＹ１、ｐＳＹＭ１、ｐＳＹＭ４、ｐＳＹＭ１０、ｐＳＹＭ１１またはｐＳＹＭＰで形質転換し、各形質転換株から無細胞抽出液を調製し、ＡＫIIIの酵素活性を測定した。
<3> Isolation of mutant lysC and examination of mutant lysC gene product Plasmids were obtained by inserting lysCM1, lysCM4, lysCM10, and lysCM11, which were confirmed to be the above mutant lysC, into pMW119, similar to pSY1, respectively. As shown, they were named pSYM1, pSYM4, pSYM10, and pSYM11. Then, AKI, AKII, and AKIII completely deficient strain Gif106M1 were transformed with pSY1, pSYM1, pSYM4, pSYM10, pSYM11, or pSYMP, and cell-free extracts were prepared from each transformed strain, and the enzyme activity of AKIII was measured.
無細胞抽出液（粗酵素液）は次のようにして調製した。形質転換株をトリプトン１０ｇ／Ｌ、酵母エキス５ｇ／Ｌ、ＮａＣｌ ５ｇ／Ｌからなる培地で培養し、ＯＤ６００が約０．５になったところで集菌した。菌体を菌体破砕液（５０ｍＭ リン酸カリウム緩衝液ｐＨ７．５、３００ｍＭ 塩化ナトリウム、５ｍＭ βメルカプトエタノール、１０ｍＭ イミダゾール）にて懸濁後、超音波処理で菌体を破砕した。遠心分離後、可溶性画分をＮｉ−ＮＴＡ ａｇａｒｏｓｅカラム（キアゲン社）に通し、洗浄緩衝液（５０ｍＭ リン酸カリウム緩衝液ｐＨ７．５、３００ｍＭ 塩化ナトリウム、５ｍＭ βメルカプトエタノール、２０ｍＭ イミダゾール）にてカラムを洗浄した。次いで、溶出用緩衝液（５０ｍＭ リン酸カリウム緩衝液ｐＨ７．５、３００ｍＭ 塩化ナトリウム、５ｍＭ βメルカプトエタノール、２５０ｍＭ イミダゾール）をカラムに通し、その上清を粗酵素液とした。 A cell-free extract (crude enzyme solution) was prepared as follows. The transformed strain was cultured in a medium consisting of tryptone 10 g / L, yeast extract 5 g / L, NaCl 5 g / L, and collected when OD 600 reached about 0.5. The cells were suspended in a cell disruption solution (50 mM potassium phosphate buffer pH 7.5, 300 mM sodium chloride, 5 mM β-mercaptoethanol, 10 mM imidazole), and then disrupted by sonication. After centrifugation, the soluble fraction is passed through a Ni-NTA agarose column (Qiagen), and the column is washed with a washing buffer (50 mM potassium phosphate buffer pH 7.5, 300 mM sodium chloride, 5 mM β-mercaptoethanol, 20 mM imidazole). Washed. Next, an elution buffer (50 mM potassium phosphate buffer pH 7.5, 300 mM sodium chloride, 5 mM β-mercaptoethanol, 250 mM imidazole) was passed through the column, and the supernatant was used as a crude enzyme solution.
ＡＫIII活性の測定はブラックの方法（Methods in Enzymology, Vol. 5, p820-827, Academic Press Inc., New York (1962)）に従った。すなわち、最終液量１ｍＬ中に１００ｍＭ トリス−硫酸緩衝液（ｐＨ７．５）、１０ｍＭ ＡＴＰ、１０ｍＭ 硫酸マグネシウム、６００ｍＭ ヒドロキシルアミン・塩酸塩、６００ｍＭ 硫酸アンモニウム、５０ｍＭ Ｌ−アスパラギン酸及び前記粗酵素液を加え、３０℃、１５分間反応させた後、１０％ 塩化鉄（ＩＩ）６水和物−３．３％トリクロロ酢酸−０．７Ｎ塩酸液１．５ｍＬを加え、遠心分離で沈殿物を除去した液を、５４０ｎｍの吸光度を測定した。活性は１分間に生成するヒドロキサム酸の量で表示（１μｍｏｌ／ｍｉｎ）した。 The AKIII activity was measured according to the black method (Methods in Enzymology, Vol. 5, p820-827, Academic Press Inc., New York (1962)). That is, 100 mM Tris-sulfate buffer (pH 7.5), 10 mM ATP, 10 mM magnesium sulfate, 600 mM hydroxylamine / hydrochloride, 600 mM ammonium sulfate, 50 mM L-aspartic acid and the crude enzyme solution were added to 1 mL of the final solution volume, After reacting at 30 ° C. for 15 minutes, 1.5 mL of 10% iron (II) chloride hexahydrate-3.3% trichloroacetic acid-0.7N hydrochloric acid solution was added, and the liquid from which the precipitate was removed by centrifugation was added. Absorbance at 540 nm was measured. The activity was expressed as the amount of hydroxamic acid generated per minute (1 μmol / min).
ＡＫIIIの酵素活性を測定する際、酵素反応液中に１００ｍＭの濃度のＬ−リジンを加え、Ｌ−リジンによる阻害の具合を調べた。なお、表４中、阻害解除度（％）は、Ｌ−リジン非存在下での酵素活性に対する１００ｍＭ Ｌ−リジン存在下での酵素活性で表示した。結果を表４に示した。 When measuring the enzyme activity of AKIII, L-lysine at a concentration of 100 mM was added to the enzyme reaction solution, and the degree of inhibition by L-lysine was examined. In Table 4, the degree of inhibition release (%) was expressed as the enzyme activity in the presence of 100 mM L-lysine relative to the enzyme activity in the absence of L-lysine. The results are shown in Table 4.
この結果が示すように、野生型ＡＫIIIはＬ−リジンによる阻害を強く受け、１００ｍＭ Ｌ−リジン存在下になると、１００％酵素活性が阻害された。それに対し、今回得られた変異型ＡＫIIIはいずれも１００ｍＭ Ｌ−リジン存在下での阻害はほとんどみられなかった。また総蛋白当りの比活性は、いずれもほとんどが野生型と同等もしくはそれ以上のものであり、変異導入による活性低下の問題はほとんどなかった。特に、ｌｙｓＣＭ１１は、比活性、阻害解除度共にもっとも向上していた。なお、表４中、阻害解除度（％）とは、反応液中Ｌ−リジン非存在下でのＡＫ活性に対する１００ｍＭ Ｌ−リジンの存在下でのＡＫ活性である。 As this result shows, wild type AKIII was strongly inhibited by L-lysine, and when it was in the presence of 100 mM L-lysine, 100% enzyme activity was inhibited. In contrast, none of the mutant AKIII obtained this time showed any inhibition in the presence of 100 mM L-lysine. In addition, the specific activity per total protein was almost equal to or higher than that of the wild type, and there was almost no problem of decreased activity due to mutagenesis. In particular, lysCM11 was most improved in both specific activity and inhibition release. In Table 4, the degree of inhibition release (%) is the AK activity in the presence of 100 mM L-lysine relative to the AK activity in the absence of L-lysine in the reaction solution.
（実施例２）変異型ＬｙｓＣを用いた１，５−ペンタンジアミンの発酵生産
実施例１において取得したＬ−リジンによるフィードバック阻害を解除する変異を有する変異型ＬｙｓＣを用いて、１，５−ペンタンジアミン生産を行った。
(Example 2) Fermentative production of 1,5-pentanediamine using mutant LysC 1,5-pentane using mutant LysC having a mutation that cancels feedback inhibition by L-lysine obtained in Example 1 Diamine production was performed.
変異型ＬｙｓＣを有するｐＳＹＭ１、ｐＳＹＭ４、ｐＳＹＭ１０、ｐＳＹＭ１１またはｐＳＹＭＰを、Ｅ．ｃｏｌｉ Ｗ３１１０株に導入し、１，５−ペンタンジアミン発酵試験を行った。培養は、以下の１，５−ペンタンジアミン生産培地を用い、培養温度３７℃、攪拌８００〜１０００ｒｐｍの条件下で１２時間行った。ＨＰＬＣの分析条件は以下に示す。培養結果を表５に示す。 PSYM1, pSYM4, pSYM10, pSYM11 or pSYMP having a mutated LysC were transformed into E. coli. E. coli strain W3110 was introduced to conduct a 1,5-pentanediamine fermentation test. The following 1,5-pentanediamine production medium was used for culturing for 12 hours under conditions of a culture temperature of 37 ° C. and stirring of 800 to 1000 rpm. The analysis conditions of HPLC are shown below. The culture results are shown in Table 5.
この結果が示すように、Ｌ−リジンによるフィードバック阻害解除型遺伝子保持株を用いた試験では、いずれにおいても１，５−ペンタンジアミンが生産された。特に、Ｗ３１１０／ｐＳＹＭ１１を用いた時に、もっとも１，５−ペンタンジアミンを生産することがわかった。 As this result shows, 1,5-pentanediamine was produced in any of the tests using the feedback inhibition-cancelled gene-bearing strain with L-lysine. In particular, it was found that 1,5-pentanediamine was produced most when W3110 / pSYM11 was used.
（１，５−ペンタンジアミン生産培地）
Ａ：（ＮＨ４）２ＳＯ４ １６ｇ／Ｌ
ＫＨ２ＰＯ４ １ｇ／Ｌ
ＭｇＳＯ４・７Ｈ２Ｏ １ｇ／Ｌ
ＦｅＳＯ４・７Ｈ２Ｏ ０．０１ｇ／Ｌ
ＭｎＳＯ４・５Ｈ２Ｏ ０．０１ｇ／Ｌ
Yeast Extract (Difco) ２ｇ／Ｌ
ＫＯＨでｐＨ６．５に調整し、１２１℃で２０分オートクレーブ
Ｂ：２５％グルコース（１１５℃で１０分オートクレーブ）
Ａ：Ｂを４：１で混合し、抗生物質（アンピシリン ５０μｇ／ｍｌ）を加える。
(1,5-pentanediamine production medium)
A: (NH 4 ) 2 SO 4 16 g / L
KH 2 PO 4 1 g / L
MgSO 4 · 7H 2 O 1g / L
FeSO 4 · 7H 2 O 0.01g / L
MnSO 4 · 5H 2 O 0.01g / L
Yeast Extract (Difco) 2g / L
Adjust pH to 6.5 with KOH and autoclave B: 25% glucose at 121 ° C for 20 minutes (autoclave at 115 ° C for 10 minutes)
A: B is mixed 4: 1 and antibiotics (ampicillin 50 μg / ml) are added.
（１，５−ペンタンジアミン濃度のＨＰＬＣによる分析方法）
使用カラム：ＣＡＰＣＥＬＬ ＰＡＫ Ｃ１８（資生堂）
移動相：０．１％（ｗ／ｗ）リン酸水溶液：アセトニトリル＝４．５：５．５
検出：ＵＶ３６０ｎｍ
サンプル前処理：分析サンプル２５μｌに内標として、１，４−ジアミノブタン（０．０３Ｍ）を２５μｌ、炭酸水素ナトリウム（０．０７５Ｍ）を１５０μｌおよび２，４−ジニトロフルオロベンゼン（０．２Ｍ）のエタノール溶液を添加混合し、３７℃の温度で１時間保温する。上記の反応溶液５０μｌを１ｍｌアセトニトリルに溶解後、１０，０００ｒｐｍで５分間遠心した後の上清１０μｌをＨＰＬＣ分析した。
(Analysis method by HPLC of 1,5-pentanediamine concentration)
Column used: CAPCELL PAK C18 (Shiseido)
Mobile phase: 0.1% (w / w) phosphoric acid aqueous solution: acetonitrile = 4.5: 5.5
Detection: UV360nm
Sample pretreatment: 25 μl of analytical sample was 25 μl of 1,4-diaminobutane (0.03 M), 150 μl of sodium hydrogen carbonate (0.075 M) and 2,4-dinitrofluorobenzene (0.2 M) as internal standards. The ethanol solution is added and mixed, and kept at a temperature of 37 ° C. for 1 hour. After dissolving 50 μl of the above reaction solution in 1 ml acetonitrile, 10 μl of the supernatant after centrifugation at 10,000 rpm for 5 minutes was analyzed by HPLC.
（実施例３）リジンデカルボキシラーゼ遺伝子発現増強株の作製
染色体中に存在するリジンデカルボキシラーゼ遺伝子の発現量を増強させるために、リジンデカルボキシラーゼ遺伝子のプロモーターを大腸菌のｇａｐＡ遺伝子（グリセルアルデヒドデヒドロゲナーゼ遺伝子）プロモーターと置換した株の作製を試みた。プロモーターの置換は、ＦＬＰレコンビナーゼを用いた相同組換えによる遺伝子破壊方法を改変して行った。以下に、作製方法を示す。
(Example 3) Preparation of a lysine decarboxylase gene expression enhancement strain In order to enhance the expression level of a lysine decarboxylase gene present in a chromosome, the lysine decarboxylase gene promoter was changed to the E. coli gapA gene (glyceraldehyde dehydrogenase gene). An attempt was made to create a strain that replaced the promoter. The replacement of the promoter was performed by modifying the gene disruption method by homologous recombination using FLP recombinase. A manufacturing method will be described below.
＜１＞ｇａｐＡ遺伝子プロモーターのクローニング
大腸菌Ｗ３１１０株を培養し遠心回収後、UltraClean Microbial DNA Isolation Kit（ＭＯ ＢＩＯ社製）を用いてゲノムＤＮＡの抽出を行った。詳細な操作方法は、付属のプロトコールに従った。
<1> Cloning of gapA Gene Promoter After Escherichia coli W3110 was cultured and collected by centrifugation, genomic DNA was extracted using UltraClean Microbial DNA Isolation Kit (manufactured by MO BIO). The detailed operation method followed the attached protocol.
上記のようにして得られたゲノムＤＮＡを鋳型、オリゴヌクレオチド（配列番号７（ＫＳ０２９）、配列番号８（ＫＳ０３０））をプライマーセットとして、ＰＣＲによりｇａｐＡ遺伝子プロモーター（ｇａｐＡ遺伝子の上流５００ｂｐ、以下ｇａｐＡプロモーターと略す。）の増幅を行った。ＰＣＲ増幅反応には、KOD-Plus polymerase（東洋紡社製）を用い、反応バッファー、ｄＮＴＰｍｉｘなどは付属のものを使用した。上記のように付属のプロトコールに従い調整したゲノムＤＮＡを５０ｎｇ／サンプル、プライマーを５０ｐｍｏｌ／サンプル、及びKOD-Plus polymeraseを１ユニット／サンプルになるように５０μlの反応系に調製した。反応溶液をＰＣＲ増幅装置ｉＣｙｃｌｅｒ（ＢＩＯ−ＲＡＤ社製）により９４℃の温度で５分熱変成させた後、９４℃（熱変成）：３０秒、５５℃（プライマーのアニール）：３０秒、６８℃（相補鎖の伸張）：３０秒を１サイクルとして３０サイクル行い、その後４℃の温度に冷却した。なお、遺伝子増幅用プライマー（配列番号７（ＫＳ０２９）、８（ＫＳ０３０））には、５末端側及び3末端側にＨｉｎｄＩＩＩ認識配列が付加されるようにして作製した。 Using the genomic DNA obtained as described above as a template and oligonucleotides (SEQ ID NO: 7 (KS029), SEQ ID NO: 8 (KS030)) as a primer set, a gapA gene promoter (500 bp upstream of the gapA gene, hereinafter the gapA promoter) is obtained by PCR. Amplification). For the PCR amplification reaction, KOD-Plus polymerase (manufactured by Toyobo Co., Ltd.) was used, and the attached reaction buffer, dNTPmix, etc. were used. A 50 μl reaction system was prepared so that the genomic DNA prepared as described above was 50 ng / sample, the primer was 50 pmol / sample, and the KOD-Plus polymerase was 1 unit / sample. The reaction solution was thermally denatured at 94 ° C. for 5 minutes with a PCR amplification apparatus iCycler (manufactured by BIO-RAD), then 94 ° C. (thermal denature): 30 seconds, 55 ° C. (primer annealing): 30 seconds, 68 ° C (extension of complementary strand): 30 cycles of 30 seconds, followed by cooling to a temperature of 4 ° C. The gene amplification primers (SEQ ID NOs: 7 (KS029) and 8 (KS030)) were prepared such that HindIII recognition sequences were added to the 5 and 3 terminal sides.
各ＰＣＲ増幅断片を精製し末端をT4 Polynucleotide Kinase（タカラバイオ社製）によりリン酸化後、ｐＵＣ１１８ベクター（制限酵素ＨｉｎｃＩＩで切断し、切断面を脱リン酸化処理したもの）にライゲーションした。ライゲーションは、DNA Ligation Kit Ver.2（タカラバイオ社製）を用いて行った。ライゲーション溶液を大腸菌ＤＨ５αのコンピテント細胞（タカラバイオ社製）に形質転換し、抗生物質アンピシリンを５０μｇ／ｍＬを含むＬＢプレートに蒔いて一晩培養した。生育したコロニーについて、ミニプレップでプラスミドＤＮＡを回収し、制限酵素ＨｉｎｄＩＩＩで切断し、ｇａｐＡプロモーターが挿入されているプラスミドを選抜した。これら一連の操作は、全て付属のプロトコールに従い行った。 Each PCR amplified fragment was purified and the end was phosphorylated with T4 Polynucleotide Kinase (manufactured by Takara Bio Inc.), and then ligated to pUC118 vector (cleaved with restriction enzyme HincII and subjected to dephosphorylation of the cut surface). Ligation was performed using DNA Ligation Kit Ver.2 (Takara Bio Inc.). The ligation solution was transformed into competent cells of Escherichia coli DH5α (manufactured by Takara Bio Inc.) and plated on an LB plate containing 50 μg / mL of the antibiotic ampicillin and cultured overnight. For the grown colonies, the plasmid DNA was collected with a miniprep, cut with the restriction enzyme HindIII, and a plasmid in which the gapA promoter was inserted was selected. All of these series of operations were performed according to the attached protocol.
＜２＞リジンデカルボキシラーゼ遺伝子のクローニング
次に、＜１＞で得られた大腸菌Ｗ３１１０のゲノムＤＮＡを鋳型として、オリゴヌクレオチド（配列番号９（ＣａｄＡＦ２）、配列番号１０（ＣａｄＡＲ２））をプライマーセットとしてＰＣＲを行い、リジンデカルボキシラーゼをコードしているｃａｄＡ遺伝子のクローニングを行った。ＰＣＲ増幅反応は、伸張反応のみ２分に変えた以外は＜１＞と同様な条件で行った。なお、遺伝子増幅用プライマー（配列番号９（ＣａｄＡＦ２）、配列番号１０（ＣａｄＡＲ２））には、５末端側にＨｉｎｄＩＩＩ、３末端側にＸｂａＩ認識配列が付加されるようにして作製した。得られたＤＮＡ断片を＜１＞と同様な方法でｐＵＣ１１８ベクターにライゲーションし、ｃａｄＡ遺伝子が挿入されているｐＵＣ１１８ベクターを得た。得られたベクターを制限酵素ＨｉｎｄＩＩＩおよびＸｂａＩで切断して、ｃａｄＡ遺伝子が挿入されているプラスミドを確認した。
<2> Cloning of lysine decarboxylase gene Next, PCR was performed using E. coli W3110 genomic DNA obtained in <1> as a template and oligonucleotides (SEQ ID NO: 9 (CadAF2), SEQ ID NO: 10 (CadAR2)) as primer sets. The cadA gene encoding lysine decarboxylase was cloned. The PCR amplification reaction was performed under the same conditions as in <1> except that only the extension reaction was changed to 2 minutes. The gene amplification primers (SEQ ID NO: 9 (CadAF2), SEQ ID NO: 10 (CadAR2)) were prepared such that HindIII was added to the 5 terminal side and an XbaI recognition sequence was added to the 3 terminal side. The obtained DNA fragment was ligated to the pUC118 vector in the same manner as in <1> to obtain a pUC118 vector in which the cadA gene was inserted. The obtained vector was cleaved with restriction enzymes HindIII and XbaI to confirm a plasmid into which the cadA gene was inserted.
次に、このｃａｄＡ遺伝子が挿入されたｐＵＣ１１８ベクターを制限酵素ＨｉｎｄＩＩＩおよびＸｂａＩで切断し、得られたｃａｄＡ遺伝子を含むＤＮＡ断片をｐＵＣ１９のＨｉｎｄＩＩＩ／ＸｂａＩ切断部位にライゲーションし、得られたプラスミドＤＮＡを回収し、制限酵素ＨｉｎｄＩＩＩおよびＸｂａＩで切断することにより、ｃａｄＡ遺伝子が挿入された発現ベクターを選抜した。得られたプラスミドをｐＨＳ７とした。 Next, the pUC118 vector into which the cadA gene has been inserted is cleaved with restriction enzymes HindIII and XbaI, and the resulting DNA fragment containing the cadA gene is ligated to the HindIII / XbaI cleavage site of pUC19, and the resulting plasmid DNA is recovered. Then, an expression vector into which the cadA gene was inserted was selected by cutting with restriction enzymes HindIII and XbaI. The obtained plasmid was designated as pHS7.
＜３＞クロラムフェニコール耐性遺伝子のクローニング
次に、クロラムフェニコール耐性遺伝子（ｃａｔ遺伝子）およびその上下流にＦＬＰ認識サイト（ＦＲＴ）を有するベクターｐＫＤ３を鋳型、オリゴヌクレオチド（配列番号１１、配列番号１２）をプライマーセットとして、ＰＣＲによりｃａｔ遺伝子のクローニングを行った。ＰＣＲ増幅反応は、伸張反応のみ１分に変えた以外は＜１＞と同様な条件で行った。なお、遺伝子増幅用プライマー（配列番号１１、配列番号１２）には、５末端側にＢａｍＨＩ、３末端側にＳａｃＩ認識配列が付加されるようにして作製した。得られたＤＮＡ断片を＜１＞と同様な方法でｐＵＣ１１８ベクターにライゲーションし、ｃａｔ遺伝子が挿入されているｐＵＣ１１８ベクターを得た。得られたベクターを制限酵素ＢａｍＨＩおよびＳａｃＩで切断して、ｃａｔ遺伝子が挿入されているプラスミドを確認した。
<3> Cloning of Chloramphenicol Resistance Gene Next, a vector pKD3 having a chloramphenicol resistance gene (cat gene) and an FLP recognition site (FRT) upstream and downstream thereof as a template, oligonucleotide (SEQ ID NO: 11, sequence) The cat gene was cloned by PCR using No. 12) as a primer set. The PCR amplification reaction was performed under the same conditions as in <1> except that only the extension reaction was changed to 1 minute. The gene amplification primers (SEQ ID NO: 11, SEQ ID NO: 12) were prepared such that BamHI was added to the 5 terminal side and a SacI recognition sequence was added to the 3 terminal side. The obtained DNA fragment was ligated to the pUC118 vector in the same manner as in <1> to obtain a pUC118 vector into which the cat gene was inserted. The obtained vector was cleaved with restriction enzymes BamHI and SacI to confirm the plasmid into which the cat gene was inserted.
＜４＞ｃａｔ遺伝子およびｇａｐＡプロモーターのｐＨＳ７への挿入
次に、ｃａｔ遺伝子が挿入されているｐＵＣ１１８ベクターを制限酵素ＢａｍＨＩで切断し、得られたＤＮＡ断片を上記ｐＨＳ７のＢａｍＨＩ／ＳａｃＩ切断部位に導入したプラスミドを作製した。得られたベクターを制限酵素ＢａｍＨＩおよびＳａｃＩで切断して、ｃａｔ遺伝子が挿入されていることを確認した。このようにして得られたプラスミドをｐＫＳ５とした。
<4> Insertion of cat gene and gapA promoter into pHS7 Next, the pUC118 vector into which the cat gene was inserted was cleaved with the restriction enzyme BamHI, and the resulting DNA fragment was introduced into the BamHI / SacI cleavage site of pHS7. A plasmid was prepared. The obtained vector was cleaved with restriction enzymes BamHI and SacI to confirm that the cat gene was inserted. The plasmid thus obtained was designated as pKS5.
＜５＞ｇａｐＡプロモーター−ｃａｄＡ遺伝子カセットの染色体への導入
次に、ｇａｐＡプロモーターが挿入されたｐＵＣ１１８ベクターを制限酵素ＨｉｎｄＩＩＩで切断し、得られたDNA断片を上記ｐＫＳ５のＨｉｎｄＩＩＩ切断部位に導入したプラスミドを作製した。このプラスミドＤＮＡを鋳型、オリゴヌクレオチド（配列番号１３（Ｍ１３ ＲＶ）、配列番号８（ＫＳ０３０））をプライマーセットとしてＰＣＲを行った。ＰＣＲにはＰｒｅｍｉｘＴａｑ ＥｘＴａｑバージョン Ｖｅｒ（タカラバイオ社製）を用いた。このＰＣＲにより、約５００ｂｐの増幅断片が得られるプラスミドを目的のプラスミドとして選抜した。このようにして得られたプラスミドをｐＫＳ８とした。
<5> Introduction of gapA Promoter-cadA Gene Cassette into Chromosome Next, a pUC118 vector into which the gapA promoter has been inserted is cleaved with the restriction enzyme HindIII, and the resulting DNA fragment is introduced into the HindIII cleavage site of pKS5. Produced. PCR was performed using this plasmid DNA as a template and oligonucleotides (SEQ ID NO: 13 (M13 RV), SEQ ID NO: 8 (KS030)) as a primer set. For PCR, PremixTaq ExTaq version Ver (manufactured by Takara Bio Inc.) was used. A plasmid from which an amplified fragment of about 500 bp was obtained by this PCR was selected as a target plasmid. The plasmid thus obtained was designated as pKS8.
＜４＞のようにして得られたｐＫＳ８を鋳型、オリゴヌクレオチド（配列番号１４（ＫＳ０３２）、配列番号１５（ＫＳ０３３））をプライマーセットとして、ＰＣＲによりｇａｐＡプロモーター、ｃａｄＡ遺伝子、ｃａｔ遺伝子を含むＤＮＡ断片を増幅した。ＰＣＲ増幅反応にはKOD-Plus polymerase（東洋紡社製）を用い、反応バッファー、ｄＮＴＰｍｉｘなどは付属のものを使用した。プラスミドＤＮＡを５０ｎｇ／サンプル、プライマーを５０ｐｍｏｌ／サンプル、及びKOD-Plus polymeraseを１ユニット／サンプルになるように５０μlの反応系に調製した。反応溶液をＰＣＲ増幅装置ｉＣｙｃｌｅｒ（ＢＩＯ−ＲＡＤ社製）により９４℃の温度で５分熱変成させた後、９４℃（熱変成）：３０秒、６５℃（プライマーのアニール）：３０秒、６８℃（相補鎖の伸張）：３分３０秒を１サイクルとして３０サイクル行い、その後４℃の温度に冷却した。得られた約３．５ｋｂの増幅断片を定法に従って電気泳動後のアガロースゲルから抽出し、５００ｎｇ／μｌとなるように調整した。 DNA fragment containing gapA promoter, cadA gene, and cat gene by PCR using pKS8 obtained as described in <4> as a template and oligonucleotides (SEQ ID NO: 14 (KS032), SEQ ID NO: 15 (KS033)) as a primer set Was amplified. For the PCR amplification reaction, KOD-Plus polymerase (manufactured by Toyobo Co., Ltd.) was used, and the attached reaction buffer, dNTPmix, etc. were used. A 50 μl reaction system was prepared so that the plasmid DNA was 50 ng / sample, the primer was 50 pmol / sample, and the KOD-Plus polymerase was 1 unit / sample. The reaction solution was thermally denatured at 94 ° C. for 5 minutes with a PCR amplification apparatus iCycler (manufactured by BIO-RAD), then 94 ° C. (thermal denature): 30 seconds, 65 ° C. (primer annealing): 30 seconds, 68 ° C. (Extension of complementary strand): 30 cycles of 3 minutes 30 seconds were performed, and then cooled to a temperature of 4 ° C. The obtained amplified fragment of about 3.5 kb was extracted from the agarose gel after electrophoresis according to a conventional method and adjusted to 500 ng / μl.
Ｗ３１１０株にＦＬＰレコンビナーゼを有するプラスミドｐＫＤ４６を導入した株（Ｗ３１１０／ｐＫＤ４６と表記する。）を５ｍｌのＬＢ培地に植菌し、一晩３０℃で培養した（前培養）。得られた前培養液を５ｍｌのＳＯＢ培地（１ｍＭアラビノース含有）に１％植菌し、ＯＤ６００が０．６になるまで３０℃で培養した（本培養）。本培養液を遠心して集菌した後、氷冷した１０％グリセロールで３回洗浄し、最終的に５０μｌの１０％グリセロールに菌体を懸濁した。この菌体懸濁液に上記のように精製したＰＣＲ増幅断片を２μｌ添加し、３０分氷冷した。この懸濁液をエレクトロポレーション用キュベット（０．２ｃｍ）に移し、ＧｅｎｅＰｕｌｓｅｒ Ｘｃｅｌｌ（ＢＩＯ−ＲＡＤ社製）を用いてエレクトロポレーションを行った（２５００Ｖ、２５μＦ、２００Ω）。電気パルスを与えた後、キュベットに１ｍｌのＳＯＣ培地を投入し菌体懸濁液を回収して、３７℃で２．５時間培養した。培養液を２５μｇ／Ｌのクロラムフェニコールを添加したＬＢ寒天培地に塗布し、３７℃で一晩培養した。 A strain obtained by introducing the plasmid pKD46 having FLP recombinase into the W3110 strain (denoted as W3110 / pKD46) was inoculated into 5 ml of LB medium and cultured overnight at 30 ° C. (preculture). 1% of the obtained preculture was inoculated into 5 ml of SOB medium (containing 1 mM arabinose), and cultured at 30 ° C. until OD600 reached 0.6 (main culture). The culture was collected by centrifugation, washed 3 times with ice-cold 10% glycerol, and finally the cells were suspended in 50 μl of 10% glycerol. 2 μl of the PCR amplified fragment purified as described above was added to this cell suspension, and the mixture was ice-cooled for 30 minutes. This suspension was transferred to an electroporation cuvette (0.2 cm), and electroporation was performed using GenePulser Xcell (manufactured by BIO-RAD) (2500 V, 25 μF, 200Ω). After applying the electric pulse, 1 ml of SOC medium was put into the cuvette, and the cell suspension was collected and cultured at 37 ° C. for 2.5 hours. The culture solution was applied to an LB agar medium supplemented with 25 μg / L chloramphenicol and cultured at 37 ° C. overnight.
得られたコロニーがアンピシリンを添加したＬＢ培地で生育しないことを確認した後、目的とするｃａｄＡ遺伝子のプロモーターがｇａｐＡプロモーターと置換されている株であることを、抽出したゲノムを鋳型、オリゴヌクレオチド（配列番号１６（ＫＳ００７）、配列番号１７（ＫＳ００８））をプライマーとしたＰＣＲにより行った。目的とする相同組換え株では約３．８ｋｂの増幅断片が得られるのに対し、目的の位置に相同組換えで挿入されていない株では約２．３ｋｂの増幅断片が得られる。その結果、約３．８ｋｂの増幅断片が確認できた。このｃａｄＡ遺伝子のプロモーターがｇａｐＡプロモーターと置換されている株をＷ３１１０（ｇａｐＡ−ｃａｄＡ）株とした。 After confirming that the obtained colonies did not grow on the LB medium supplemented with ampicillin, it was confirmed that the target cadA gene promoter was replaced with the gapA promoter. This was performed by PCR using SEQ ID NO: 16 (KS007) and SEQ ID NO: 17 (KS008)) as primers. In the target homologous recombination strain, an amplified fragment of about 3.8 kb is obtained, while in a strain not inserted by homologous recombination at the target position, an amplified fragment of about 2.3 kb is obtained. As a result, an amplified fragment of about 3.8 kb was confirmed. A strain in which the promoter of the cadA gene was replaced with the gapA promoter was designated as W3110 (gapA-cadA) strain.
（実施例４）リジンデカルボキシラーゼ遺伝子発現増強株を用いた１，５−ペンタンジアミンの発酵生産
大腸菌で１，５−ペンタンジアミンを大量生産させるためには、リジンデカルボキシラーゼ遺伝子の発現を増強した宿主を用いることが好ましい。そこで、変異型ｌｙｓＣが大腸菌で１，５−ペンタンジアミンを大量する際に及ぼす影響を評価するに当たり、実施例３で作製したＷ３１１０（ｇａｐ−ｃａｄＡ）株を使用した。
(Example 4) Fermentative production of 1,5-pentanediamine using a lysine decarboxylase gene expression-enhanced strain In order to mass-produce 1,5-pentanediamine in E. coli, a host with enhanced expression of lysine decarboxylase gene Is preferably used. Therefore, the W3110 (gap-cadA) strain prepared in Example 3 was used to evaluate the effect of the mutant lysC on the large amount of 1,5-pentanediamine in Escherichia coli.
Ｗ３１１０（ｇａｐＡ−ｃａｄＡ）株をｐＳＹＭ１、ｐＳＹＭ４、ｐＳＹＭ１０またはｐＳＹＭ１１で形質転換し、得られた形質転換体について実施例２と同様にして１，５−ペンタンジアミン生産性を調べた。結果を表６に示す。 The W3110 (gapA-cadA) strain was transformed with pSYM1, pSYM4, pSYM10 or pSYM11, and the resulting transformants were examined for 1,5-pentanediamine productivity in the same manner as in Example 2. The results are shown in Table 6.
この結果が示すように、いずれの株を用いても１，５−ペンタンジアミンが生産されたが、特に、Ｗ３１１０（ｇａｐ−ｃａｄＡ）／ｐＳＹＭ１株、Ｗ３１１０（ｇａｐ−ｃａｄＡ）／ｐＳＹＭ４株、Ｗ３１１０（ｇａｐ−ｃａｄＡ）／ｐＳＹＭ１０株、Ｗ３１１０（ｇａｐ−ｃａｄＡ）／ｐＳＹＭ１１株は１．０ｇ／Ｌ以上の１，５−ペンタンジアミンを生産し、その中でもＷ３１１０（ｇａｐ−ｃａｄＡ）／ｐＳＹＭ１１を用いた時に、もっとも１，５−ペンタンジアミンを生産することがわかった。 As can be seen from the results, 1,5-pentanediamine was produced using any strain, and in particular, W3110 (gap-cadA) / pSYM1, W3110 (gap-cadA) / pSYM4 strain, W3110 ( gap-cadA) / pSYM10 strain, W3110 (gap-cadA) / pSYM11 strain produced 1.0 g / L or more of 1,5-pentanediamine, and among them, when using W3110 (gap-cadA) / pSYM11, However, it was found that 1,5-pentanediamine was produced.
（比較例１）野生型ｌｙｓＣを導入した株による１，５−ペンタンジアミンの発酵生産
Ｗ３１１０株をｐＳＹ１で形質転換し、得られた形質転換体について実施例２と同様にして、１，５−ペンタンジアミン生産性を調べた。結果を表７に示す。
(Comparative Example 1) Fermentative production of 1,5-pentanediamine by a strain into which wild-type lysC was introduced W3110 strain was transformed with pSY1, and the obtained transformant was subjected to 1,5- The pentanediamine productivity was investigated. The results are shown in Table 7.
（比較例２）野生型ｌｙｓＣを導入したリジンデカルボキシラーゼ遺伝子発現増強株による１，５−ペンタンジアミンの発酵生産
実施例２で得られたＷ３１１０（ｇａｐＡ−ｃａｄＡ）株をｐＳＹ１で形質転換し、得られた形質転換体について実施例２と同様に１，５−ペンタンジアミン生産性を調べた。結果を表８に示す。
(Comparative Example 2) Fermentative production of 1,5-pentanediamine using a lysine decarboxylase gene expression-enhanced strain into which wild-type lysC was introduced. The W3110 (gapA-cadA) strain obtained in Example 2 was transformed with pSY1 and obtained. The resulting transformant was examined for 1,5-pentanediamine productivity in the same manner as in Example 2. The results are shown in Table 8.
これらを比較した結果、本発明によって開示されたリジンによるフィードバック阻害が解除された変異型ｌｙｓＣをリジンデカルボキシラーゼ遺伝子の発現を増強した宿主株に導入することによって、１，５−ペンタンジアミンの生産効率が向上することが明らかとなり、特にｌｙｓＣＭ１、ｌｙｓＣＭ４、ｌｙｓＣＭ１０またはｌｙｓＣＭ１１をリジンデカルボキシラーゼ遺伝子の発現を増強した宿主株に導入することによって、特に１，５−ペンタンジアミンの生産効率が向上することが明らかとなった。 As a result of comparing these, the introduction of the mutant lysC released from feedback inhibition by lysine disclosed by the present invention into a host strain with enhanced expression of lysine decarboxylase gene, the production efficiency of 1,5-pentanediamine It is clear that the production efficiency of 1,5-pentanediamine is particularly improved by introducing lysCM1, lysCM4, lysCM10 or lysCM11 into a host strain with enhanced expression of the lysine decarboxylase gene. It became.
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