CN1330749C - 产l-半胱氨酸细菌及生产l-半胱氨酸的方法 - Google Patents
产l-半胱氨酸细菌及生产l-半胱氨酸的方法 Download PDFInfo
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- CN1330749C CN1330749C CNB02105407XA CN02105407A CN1330749C CN 1330749 C CN1330749 C CN 1330749C CN B02105407X A CNB02105407X A CN B02105407XA CN 02105407 A CN02105407 A CN 02105407A CN 1330749 C CN1330749 C CN 1330749C
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Abstract
L-半胱氨酸是通过在培养基中培养棒状杆菌来生产并且在培养物中积累L-半胱氨酸并且从培养物中收集L-半胱氨酸来生产的,其中通过用编码丝氨酸乙酰转移酶的基因转化或者修饰编码丝氨酸乙酰转移酶的基因的表达调控序列等来提高丝氨酸乙酰转移酶的活性,所述修饰使得该基因在细胞中的表达应该被增强,优选地其中L-半胱氨酸降解***被进一步抑制。
Description
技术领域
本发明涉及一种生产L-半胱氨酸的方法,更进一步说涉及一种新的适合于生产L-半胱氨酸的棒状杆菌及利用该细菌生产L-半胱氨酸的方法。L-半胱氨酸和L-半胱氨酸的衍生物可以用于药品领域、化妆品领域及食品领域。
背景技术
通常地,L-半胱氨酸是通过从含角蛋白的材料如毛发、角质物、羽毛提取得到,或利用DL-2-氨基二氢噻唑-4-羧酸作为母体经微生物酶转化而得到。另外,也有利用微生物发酵来生产L-半胱氨酸的。
关于细菌的L-半胱氨酸的生物合成已被详细的研究,所述细菌例如埃希氏大肠杆菌(Kredich,NM.等,生化杂志(J.Biol.Chem.)241,4955-4965(1966);Kredich,NM.等,1987,半胱氨酸的生物合成,在Neidhardt,E.C.等编著的细胞及分子生物学中,第1卷,埃希氏大肠杆菌及鼠伤寒沙门氏菌,美国微生物学会,华盛顿特区,419-428(Neidhardt,F.C.,et al.(eds),Escherichia coli and Salmonellatyphimmium:Cellular and Molecular Biology,Vol.1,American Society forMicrobiology,Washington DC.)),已经阐明L-半胱氨酸是由L-丝氨酸经过两步反应而产生的。在埃希氏大肠杆菌中,第一步反应是乙酰辅酶A对L-丝氨酸的活化反应,该反应是用丝氨酸乙酰转移酶(EC 2.3.1.30,下文简称”SAT”)催化的,第二步反应是由上述反应生产的O-乙酰基丝氨酸生产L-半胱氨酸的反应,该反应是用O-乙酰基丝氨酸(硫羟基)裂解酶催化的。
而且,已经弄清楚,在埃希氏大肠杆菌中,L-半胱氨酸的降解会因为半胱氨酸脱巯基酶(下文简称“CD”)活性的降低而受到抑制(日本专利特开平No.11-155571)。
在埃希氏大肠杆菌中,编码SAT的基因(cysE)已经从野生菌和分泌L-半胱氨酸的突变菌中克隆出来(Denk,D.和Boeck,A.,普通微生物学(J.GeneralMicrobiol.),133,515-5250987))。而且这些cysE的核苷酸序列已经被测定,有报道表明在SAT中其第256位的甲硫氨酸残基被异亮氨酸残基所取代,L-半胱氨酸对其的反馈抑制作用是减弱的。此外,也有文章披露了用编码SAT的DNA来生产L-半胱氨酸及其类似物的方法,其中所用的SAT基因通过引入与上文不同的突变来减弱L-半胱氨酸的反馈抑制作用(国际公开号为WO97/15673)。这种SAT在第97位到第273位氨基酸区域内有一个突变,或者在第227位点的氨基酸之后有一C-末端区域的缺失。
尽管已知如上所述通过利用L-半胱氨酸对其反馈抑制作用被减弱的SAT的编码基因来生产L-半胱氨酸的技术,该申请的发明人发现,属于含有L-半胱氨酸对其的反馈抑制作用被减弱的SAT的这类埃希氏杆菌属的细菌L-半胱氨酸的产量并不稳定。但是他们还发现这种不稳定是能够被克服的,比如,通过降低细胞内CD的活性,能够使其产量变得稳定。他们还成功地获得了一种能够稳定地生产L-半胱氨酸的埃希氏大肠杆菌菌株(日本专利特开平NO.11-155571)。
进一步地,还披露了一种通过使用微生物,特别是埃希氏大肠杆菌,来生产L-半胱氨酸等物质的方法,其过量表达一种适合于从细胞中排出抗生素或其他有毒物质的某种蛋白的编码基因(***基因)(日本专利NO.29920110)。
另一方面,和对于谷氨酸棒状杆菌一样,aecD基因已经被证明为是使S-(β-氨基乙基)-半胱氨酸(AEC)具有抗性的基因(Rossol,I等,细菌杂志(J.Bacteriol.),174(9),2968-2977(1992))。已经表明,aecD基因不是生长所必需的,只有在其扩增时在AEC抗性中涉及,这种基因所编码的蛋白质,在底物为AEC、半胱氨酸或其类似物时,具有C-S裂解酶活性。Rossol等人注意到这一事实,裂解酶能够催化消除反应,还能像合成酶一样催化反向合成反应,从而提出一种含硫氨基酸的新的酶促合成途径。但是,由于利用微生物发酵来生产含硫氨基酸的成功例子非常少见,因此他们只给出了一种,可能利用C-S裂解酶的反向反应的酶促合成方法。
如上所述,培养埃希氏杆菌属的L-半胱氨酸生产菌已有了很多报道,与谷氨酸棒状杆菌的L-半胱氨酸降解有关的酶也有了相当深度的报道,但是,尚无一例是关于利用棒状杆菌发酵到一定程度可以从培养基中收集L-半胱氨酸来生产L-半胱氨酸的。
发明内容
本发明是在上文提到的现有技术基础上完成的。目的是提供一种具有L-半胱氨酸生产能力的棒状杆菌及利用该细菌生产L-半胱氨酸的方法。
本发明的发明者为了达到上文提到的目的作了艰苦的研究工作。结果他们发现,通过增加细胞内丝氨酸乙酰转移酶活性,能够给予棒状杆菌生产L-半胱氨酸的能力。他们还发现,通过抑制L-半胱氨酸降解***,L-半胱氨酸的生产能力还可以被增强,进而完成了本发明。
因此,本发明提供如下:
(1)一种具有L-半胱氨酸生产能力的棒状杆菌。
(2)根据(1)的棒状杆菌,其被修饰使得增强细胞内丝氨酸乙酰转移酶活性。
(3)根据(2)的棒状杆菌,其中通过携带L-半胱氨酸对其反馈抑制作用被减弱的丝氨酸乙酰转移酶来提高细胞内丝氨酸乙酰转移酶活性。
(4)根据(3)的棒状杆菌,其中L-半胱氨酸对其反馈抑制作用被减弱的丝氨酸乙酰转移酶包含一个突变:相应于野生型丝氨酸乙酰转移酶第256位的甲硫氨酸残基被赖氨酸和亮氨酸之外的氨基酸残基所取代,或者一个突变:缺失相应于野生型丝氨酸乙酰转移酶第256位甲硫氨酸残基处氨基酸残基开始的C-末端区。
(5)根据(2)至(4)之一的棒状杆菌,其中通过增加丝氨酸乙酰转移酶基因的表达量,来提高丝氨酸乙酰转移酶的活性。
(6)根据(5)的棒状杆菌,其中通过用编码丝氨酸乙酰转移酶的基因转化,或者通过细菌细胞内编码丝氨酸乙酰转移酶的基因的表达调控中涉及的表达调控序列或基因的修饰使得该基因的表达增强来增强细胞内的丝氨酸乙酰转移酶的活性。
(7)根据(5)的棒状杆菌,其中丝氨酸乙酰转移酶的编码基因是属于埃希氏杆菌属细菌的cysE基因。
(8)根据(1)至(6)之一的棒状杆菌,其中L-半胱氨酸降解***被进一步抑制。
(9)根据(8)的棒状杆菌,其中通过细胞内半胱氨酸脱巯基酶活性降低,而使L-半胱氨酸降解***受到抑制。
(10)根据(9)的棒状杆菌,其中通过破坏aedD基因,而使细胞内半胱氨酸脱巯基酶活性降低。
(11)一种生产L-半胱氨酸的方法,包括在培养基中培养(1)到(10)中的任一种棒状杆菌来生产并且在培养物中积累L-半胱氨酸,及从培养物中收集L-半胱氨酸。
本发明中,L-半胱氨酸生产能力指细菌在培养基中培养时,培养基中积累的L-半胱氨酸的量能达到可以从培养基中收集L-半胱氨酸的水平的本发明细菌的能力。在本发明所提到的L-半胱氨酸生产能力的范围内,在细胞的生长不降低的前提下,L-半胱氨酸的生产能力达到一定水平。
“L-半胱氨酸降解***的抑制”是指至少一种与半胱氨酸降解有关的酶被减少或被去除。“反馈抑制的减弱”是指反馈抑制被完全消除或只保留较弱的反馈抑制。在本发明中,若没有特殊说明,L-半胱氨酸是指还原L-半胱氨酸、L-胱氨酸或其混合物。
由于本发明的棒状杆菌有L-半胱氨酸的生产能力,因此可以用作发酵生产L-半胱氨酸的材料,进而培育出产L-半胱氨酸的细菌。
具体实施方式
下文,将详细解释本发明。
<1>本发明的棒状杆菌
本发明细菌是一种具有L-半胱氨酸生产能力的棒状杆菌。在本发明中,“棒状杆菌”包括那些曾经被分类到短杆菌属,现在又合并到棒状杆菌属的那些细菌(Int.J.Syst.Bacteriol.,41,255(1981)),也包括那些属于短杆菌属但和棒状杆菌亲缘关系较近的细菌,比如以下棒状杆菌:
嗜乙酰乙酸棒状杆菌(corynebacterium acetoacidophilum)
醋谷棒状杆菌(corynebacterium acetoglutamicum)
corynebacterium alkanolyticum
颈棒状杆菌(corynebacterium callumae)
谷氨酸棒状杆菌(corynebacterium glutamicum)
百合棒状杆菌(corynebacterium lillium)
corynebacterium melassecola
corynebacterium thermoaminogenes
力士棒状杆菌(corynebacterium herculis)
谷氨酸棒杆菌(Brevibacterium divaricatum)
黄色短杆菌(Brevibacterium flavum)
Brevibacterium immariophilum
乳发酵短杆菌(Brevibacterium lactofermentum)
玫瑰色短杆菌(Brevibacterium roseum)
解糖短杆菌(Brevibacterium saccharolyticum)
生硫短杆菌(Brevibacterium thiogenitalis)
产氨短杆菌(Brevibacterium ammoniagenes)
Brevibacterium album
Brevibactefium cerium
嗜氨微杆菌(Microbacterium ammoniaphilum)
特别的,还比如下列菌株:
嗜乙酰乙酸棒状杆菌(corynebacterium acetoacidophilum)ATCC 13870
醋谷棒状杆菌(corynebacterium acetoglutamicum)ATCC 15806
Corynebacterium alkanolyticum ATCC 21511
颈棒状杆菌(corynebacterium callunae)ATCC 15911
谷氨酸棒状杆菌(corynebacterium glutamicum)ATCC 13020,13032
百合棒状杆菌(corynebacterium lillium)ATCC 15990
Corvnebacterium melassecola ATCC 17965
Corvnebacterium thermoaminogenes AJ12340(FERM BP-1539)
力士棒状杆菌(corynebacterium herculis)ATCC 13868
谷氨酸棒杆菌(Brevibacterium divaricatum)ATCC 14020
黄色短杆菌(Brevibactenum flavum)ATCC 13826,ATCC 14067,AJ12418(FERMBP-2205)
Brevibacterium immariophilum ATCC 14068
乳发酵短杆菌(Brevibacterium lactofermentum)ATCC 13869
玫瑰色短杆菌(Brevibacterium roseum)ATCC 13825
解糖短杆菌(Brevibacterium saccharolyticum)ATCC 14066
生硫短杆菌(Brevibacterium thiogenitalis)ATCC 19240
产氨短杆菌(Brevibacterium anmoniagenes)ATCC 6871,ATCC6872
Brevibacterium album ATCC 15111
蜡状短杆菌(Brevibacterium cerinum)ATCC 15112
嗜氨微杆菌(Microbacterium ammoniaphilus) ATCC 15354
有些地方可以提供以上菌株,比如美国典型培养物保藏中心(10801University Boulevard,Manassas,VA 20110-2209,美国)。在那里,每一种菌株都有它的登记号,根据登记号能获得所要的菌株。菌株的登记号都列在美国典型培养物保藏中心的目录中。AJ12340菌株于1987年10月27日保藏在,通产省工业技术院国立生命科学和人体技术研究所(NIBH)(现在是,独立行政公司,国家高级科技学院,国际专利生物保藏中心,Chuo Dai-6,1-1 Higashi 1-Chome,Tsukuba-shi,Ibaraki-ken,Japan,邮编:305-8566),为布达佩斯条约规定的一个国际保藏组织,获得的登记号为FERM BP-1539。AJ12418菌株于1989年1月5日保藏在,布达佩斯条约微生物国际保藏单位,通产省工业技术院国立生命科学和人体技术研究所(NIBH),获得保藏登记号为FERM BP-2205。
本发明细菌的第一个实施方案是一种具有提高细胞内丝氨酸乙酰转移酶活性的棒状杆菌。
“修饰以使细胞内丝氨酸乙酰转移酶(以下简称为“SAT”)活性提高”的意思是指每个细胞中的SAT的活性比没有修饰过的细菌,比如野生棒状杆菌,中的SAT的活性更高。例如每个细胞中的SAT分子数增加、每一个SAT分子的SAT活性增强等。把谷氨酸棒状杆菌ATCC 13032和野生型棒状杆菌比较,由于提高了细胞内SAT的活性,棒状杆菌中L-半胱氨酸的产量也随着提高。
为了提高棒状杆菌细胞中SAT活性,可以增加细胞内SAT编码基因的拷贝数。例如,把SAT编码基因片段与细菌中的功能性载体优选多拷贝型载体相连,构建成一个重组DNA,再把该重组体导入到宿主棒状杆菌中转化。
作为SAT基因,任何来自棒状杆菌和其他生物体,比如埃希氏杆菌属细菌的基因都可以被使用。除野生型的SAT基因以外,在不丧失对L-丝氨酸和乙酰辅酶A催化活性的情况下,SAT基因可以是编码具有一个氨基酸位点被替换、缺失、***的蛋白质的DNA序列,也可以是编码具有一个或几个氨基酸位点发生倒位、异位的蛋白质的DNA序列。
在埃希氏大肠杆菌中,编码SAT的cycE基因已经从野生菌和分泌L-半胱氨酸的突变菌中被克隆出来(Denk,D.和Boeck,A.,J.GeneralMicrobiol.,133,515-525(1987))。于是,基于其核苷酸序列设计的引物,以棒状杆菌基因组DNA为模板(参考日本专利特开平NO.11-155571),利用PCR方法(聚合酶链反应,参考White,T.J.et al.,Trends Genet.,5,185(1989))而获得SAT基因。其他微生物的编码SAT的基因也可以通过同样的方法获得。
染色体DNA可以从DNA供体细菌中制备,例如运用Saito和Miura所提供的方法(参考HSaito和K.Miura,生物化学生物物理年鉴(Biochem.Biophys.Acta),72,619(1963),生物工程实验教科书(Text for BioengineeringExperiments),日本生物科学及生物工程学会编pp,97-98,Baifukan,1992)。
如果把通过PCR方法扩增得到的SAT基因与一种能在埃希氏大肠杆菌或/和棒状杆菌中自主复制的载体连接,构建出重组DNA,然后把该重组DNA导入埃希氏大肠杆菌,后面的工作变得容易了。例如能在埃希氏大肠杆菌中自主复制的载体包括pUC19、pUC18、pHSG299、pHSG399、pHSG398、RSF1010、pBR322、pACYC184、pMW219等等。
在棒状杆菌中产生作用的载体可以是在棒状杆菌中能自主复制的质粒,特别的包括如下质粒:
pAM330(参考日本专利No.58-67699)
pHM1519(参考日本专利No.58-77859)
pSFK6(参考日本专利No.2000-26288)
而且,假如把一个使质粒具有在棒状杆菌中自主复制能力的DNA片段从载体中分离出来,再***到前文提到的转化埃希氏大肠杆菌的载体中,这种载体就可以被用来作为穿梭载体,在埃希氏大肠杆菌和棒状杆菌中都能够自主复制。
下文列出了这样的一些穿梭载体及其宿主微生物,在圆括号中分别给出了这些这些载体在国际保藏单位中的登记号。
pAJ655埃希氏大肠杆菌AJ11882(FERM BP-136)
谷氨酸棒状杆菌SR8201(ATCC39135)
pAJ1844埃希氏大肠杆菌AJ11883(FERM BP-137)
谷氨酸棒状杆菌SR 8202(ATCC39 136)
pAJ611埃希氏大肠杆菌AJ11884(FERM BP-138)
pAJ3148谷氨酸棒状杆菌SR 8203(ATCC39 137)
pAJ440枯草芽孢杆菌AJ11901(FERMBP-140)
pHC4埃希氏大肠杆菌AJ12617(FERM BP-3532)
另外,在实施例中描述的质粒pVK7(参考日本专利特开平No.11-266881)也适合于作为埃希氏大肠杆菌和棒状杆菌的穿梭载体。
这些载体也可以从如下被保藏的微生物中获得。即,通过使用溶菌酶和SDS溶胞收集处于指数生长期的微生物细胞,以30000xg离心,往获得的上清液中加入聚乙二醇,然后用氯化铯-溴化乙啶等密度梯度离心分离和纯化。
为了通过SAT基因与在棒状杆菌细胞中能起作用的载体连接,构建一个重组DNA,载体借助于一个与SAT基因末端相对应的限制酶消化。连接酶一般使用T4DNA连接酶。
把上述重组DNA导入微生物细胞中可以用目前已公开的所有的转化方法。例如,用氯化钙处理感受态细胞以增强细胞对DNA的通透性,运用这种方法转化埃希氏大肠杆菌K-12取得成功(Mandel,M.和Higa,A.,分子生物学(J.MolBiol).,53,159(1970))。也可以把处于生长期的待转化的细胞与重组DNA一起培养,有报道证明用这种方法已成功转化了枯草芽孢杆菌(Duncan,C.H.,Wilson,GA.和Young,F.E.,基因(Gene),1,153(1977))。除上述方法以外,也可以把感受态细胞处理成容易吸收重组DNA的去壁细胞或原生质体,然后把重组DNA导入受体细胞,这种方法已被用于转化枯草芽孢杆菌、放线菌及酵母菌(Chang,S.和Choen,S.N.,分子遗传学(Molec.Gen,Genet.),168,111(1979);Bibb,M J.,Ward,J.M.和Hopwood,O.A.,自然(Nature),274,398(1978);Hinnen,A.,Hicks,J.B.和Fink,GR.,美国国家科学院院刊(Proc.Natl.Sci.USA),75.1929(1978))。利用电穿孔法也成功转化了棒状杆菌(Sugimoto等,日本专利特开No.2-207791)。
增加SAT基因的拷贝数可以通过向棒状杆菌染色体DNA中导入多个SAT基因拷贝来实现。为了将SAT基因的多个拷贝导入棒状杆菌染色体DNA,对作为靶物存在于染色体DNA中的多拷贝序列进行同源重组。可以使用作为存在于染色体DNA中的多拷贝,重复DNA序列,存在于转座因子末端的反向重复序列。也可以像日本专利特开No.2-109985所公开的一样,把SAT基因***转座子中,使它们一起被转移,从而将基因的多拷贝导入染色体DNA。
除了上文描述的基因扩增技术外,提高SAT的活性还可以通过更换质粒或染色体DNA中的比较强的SAT基因的表达调控序列来实现(参考日本专利特开No.1-215280))。强启动子如lac启动子、trp启动子、trc启动子等。表达调控序列的置换还可用例如热敏感性质粒基因取代,例如以棒状杆菌为宿主热敏感性质粒包括:p48K、pSFKT2、pSFKT3、pSFKT4、pSFKT5和pSFKT6(参考日本专利特开No.2000-262288),pHSC4(参考法国专利公开号为No.2667875,1992和日本专利特开No.5-7491)等等。另外,正如国际公开号为WO00/18935的专利文件公开了通过替换SAT基因启动子区的几个核苷酸而使该启动子变为强启动子。通过这样的替换或修饰启动子,SAT基因的表达能得到提高,SAT的活性也就得到提高。修饰表达调控序列可以和增加SAT基因的拷贝数联合使用。
此外当SAT基因的表达过程中存在抑制机制时,也可以通过修饰表达调控序列或与抑制有关的基因增强表达基因以消除或减弱抑制。
通过产生包含L-半胱氨酸对其反馈抑制作用被减弱的SAT的棒状杆菌(后文也称为“突变SAT”),也可以提高棒状杆菌细胞内SAT活性。突变SAT包含一个突变,即在野生型SAT点位相对应的甲硫氨酸的氨基酸残基被赖氨酸残基和亮氨酸残基之外的氨基酸残基所代替的突变,或缺失以下氨基酸残基处开始的C-末端区域的突变,该氨基酸残基是与野生型SAT点位相应的甲硫氨酸。除赖氨酸残基和亮氨酸残基外的氨基酸残基的例子包括构成常规蛋白质的氨基酸中除甲硫氨酸残基,赖氨酸残基和亮氨酸残基之外的17种氨基酸,更特别地,可以提到异亮氨酸残基。
除了前文提到的L-半胱氨酸减弱反馈抑制的突变外,本发明使用的突变SAT可以是具有包括一个或几个氨基酸残基被替换、缺失、***,增加,或倒位的基本上不降低乙酰辅酶催化L-丝氨酸激活活性的氨基酸序列的突变SAT。在具有这样的突变的SAT中,256位点处的甲硫氨酸残基位点是可以改变的。即使在这样一种情况下,L-半胱氨酸对其反馈抑制被减弱的突变SAT,可以通过用除赖氨酸残基和亮氨酸残基之外的氨基酸残基置换相应于在256位点的甲硫氨酸残基的氨基酸残基来得到。
可以通过向细胞内SAT基因导入这样一个突变来构建一个含有突变SAT的棒状杆菌,该突变使得L-半胱氨酸对编码的SAT的反馈抑制消除。导入突变的方法可以通过用紫外光照射处理、或者使用用于常规诱变处理的诱变剂象N-甲基N’-硝基N-亚硝基胍(NTG)或亚硝酸处理。
通过将突变SAT基因导入棒状杆菌也可以构建一个含有突变SAT基因的棒状杆菌。突变SAT可以通过向野生型SAT基因中导入这样的突变而获得,这样的突变使得L-半胱氨酸对编码的SAT的反馈抑制消除,比如在野生型SAT第256位点处的甲硫氨酸残基相对应的氨基酸残基用赖氨酸残基和亮氨酸残基之外的氨基酸残基替代的突变,或把野生型SAT第256位甲硫氨酸残基相对应的氨基酸残基开始的C-末端区缺失的突变。象把希望的变异导入野生型SAT基因的方法一样,定点诱变也是一种方法。作为突变SAT基因,编码埃希氏大肠杆菌的突变SAT的突变cysE已被公开(参考国际专利公开号为WO97/15673和日本专利特开No.11-155571)
本发明的细菌的第二实施方案是棒状杆菌,其中细胞内SAT活性增强的同时,L-半胱氨酸降解***受到抑制。在埃希氏大肠杆菌中,CD和胱硫醚β-裂解酶被认为是L-半胱氨酸降解有关的酶,在下文中,减弱或消除棒状杆菌中CD活性将被详细说明。
为了减弱或消除棒状杆菌的细胞内CD活性,可以用紫外光照射或用N-甲基-N’-硝基-N-亚硝基胍(NTG)或亚硝酸等诱变剂处理棒状杆菌,从中筛选出CD活性减弱的突变株。除了诱变外,还可以利用基因裂解技术获得CD活性减弱的棒状杆菌。就是,能用缺失编码CD的基因的内部序列修饰使不产生正常发挥功能的CD的包括aecD基因的DNA(缺失型CD基因)转化棒状杆菌,结果是在缺失型CD基因和染色体上CD基因之间发生重组,使染色体上的CD基因断裂。像这样的利用同源重组的基因替换的基因破坏已经被建立。有一些利用含有温度敏感性复制起点等的质粒、线性DNA的方法。
缺失型CD基因可以通过以下方法替换宿主染色体上的CD基因:***有温度敏感性复制起点、突变CD基因和一个抗药性(如抗氯霉素)标志基因的重组DNA。并用重组DNA转化棒状杆菌,并使其在复制起点不能起作用的温度下培养,然后转化菌可以在含有药物的培养基中培养以获得重组DNA被整合到染色体DNA中的转化菌株。
在上文描述的重组DNA与染色体DNA发生重组交换的转化菌中,突变CD基因与染色体自身CD基因发生了重组,染色体CD基因和突变CD基因都被***到染色体上,重组DNA的其他部分(载体片段、温度敏感性复制起点、抗药性标志)也被***到这两个这基因当中。在这个阶段,由于正常CD基因在这种状态下是优势的,转化菌株表达正常CD蛋白。
为了使染色体DNA上仅留下缺失型CD基因,通过两个CD基因的重组,从染色体DNA上消除该CD基因的一个拷贝和载体片段(包括温度敏感复制起点和抗药性标标记)。一种情况是正常的CD基因留在染色体DNA上而缺失型DNA被删除,另一种情况相反,缺失型DNA留下而正常的CD基因从染色体DNA上被删除。但是无论那一种情况,当细胞在温度敏感性复制起点能起作用的温度下培养时,被消除的DNA仍然可以作为质粒驻留在细胞中,其后如果当细胞在温度敏感性复制起点不起作用的温度下培养时,质粒中的CD基因将和质粒一起在细胞中被消除。然后用PCR、Southem杂交或其他方法筛选染色体上留有缺失型CD基因的菌株,于是就得到了含有损失型CD基因的菌株。
如下面提到的实施例中所述,通过纯化具有CD活性的蛋白质并且对该蛋白质进行氨基酸序列测定,及克隆编码该蛋白的基因并测序,证明了在黄色短杆菌和谷氨酸棒状杆菌中CD蛋白主要是由aecD基因编码的。
用Kredich等发明的方法(生物化学杂志(J.Biol.Chem.),248,6187-6196(1973))测定筛选出来的菌株的细胞抽提物的CD活性,与原始菌株中的CD活性作比较,可以证实含被破坏基因的菌株或突变菌株中的CD活性被减弱或消除。
用同样的方法,其他与L-半胱氨酸降解有关的酶活性能被减弱或消除。
已经有文章报道,谷氨酸棒状杆菌IR33的一种aecD破坏型菌株被成功构建(I.Rossol&A.Puhler,细菌杂志(J.Bacteriol.),174,2968-2977(1992))。
<2>生产L-半胱氨酸的方法
如上所述,通过培养具有L-半胱氨酸生产能力并且其细胞内SAT活性被提高的棒状杆菌,或者其中培养基中L-半胱氨酸降解***被进一步抑制以在培养基中生产和积累L-半胱氨酸的棒状杆菌,并且收集来自培养基的L-半胱氨酸,来生产L-半胱氨酸。
虽然用本发明的方法生产的L-半胱氨酸除了含有还原型半胱氨酸外还含有胱氨酸,但是本发明生产的目的是生产半胱氨酸,及还原型半胱氨酸和胱氨酸的混合物。
用本发明的棒状杆菌生产L-半胱氨酸,用常规的方法,使用含有碳源、氮源和矿物质及微生物生长必须微量营养物质如氨基酸和维生素的一般培养基培养棒状杆菌。培养基可以是合成培养基,也可以是天然培养基,只要是用于培养菌株的,可以使用任何碳源和氮源。
作为碳源,使用糖类如葡萄糖、甘油、果糖、蔗糖、麦芽糖、甘露糖、半乳糖、淀粉水解产物和糖蜜,有机酸如乙酸、柠檬酸,醇类如乙醇,它们可以单独使用,也可以与其他碳源混合使用。
作为氮源,使用氨,铵盐如硫化铵、碳酸铵、氯化铵、磷酸铵和醋酸铵,硝酸盐等。
作为有机微量营养物、氨基酸、维生素、脂肪酸、核酸,使用包含这些物质的那些,如蛋白胨、酪蛋白氨基酸、酵母抽提物、大豆蛋白降解产物等等。如果菌株是营养缺陷型,其生长需要一种氨基酸或其类似物质,优选提供所需要的营养。
作为矿物盐,使用磷酸盐、镁盐、钙盐、铁盐、锰等。
培养采用通气培养,发酵温度控制在20-45℃,PH 5-9。在培养过程中,当培养基的PH值下降,加入碳酸钙或碱如氨气中和,用所描述的上述办法在培养基中培养10-120小时后,培养基中L-半胱氨酸的量积累。
用常规方法的组合收集培养基中的L-半胱氨酸,比如利用离子交换树脂、沉降法等。
实行本发明的最佳方案
下文将用实施例更详细解释本发明:
<1>棒状杆菌中L-半胱氨酸合成酶的确定
为了达到鉴定棒状杆菌中L-半胱氨酸分解酶的目的,可以用各种色谱法从黄色短杆菌(ATCC 14067)破碎细胞的悬浮液中纯化出一种表现出CD活性的酶,用Kredich等介绍的方法测定酶的活性(生物化学杂志(J.Biol,Chem.),248,6187-6196(1973))。ATCC 14067菌株培养在基本培养基中(5g/L葡萄糖,1.5g/L尿素,15g/L硫化铵,1g/LKH2PO4,3g/LK2HPO4,0.1g/LMgSO4.7H2O,1mg/LCaCL2,30ug/L 维生素H,100ug/L 维生素B110mg/LfFeSO4.7H2O,和10mg/LMnSO4.4H2O,PH 7.0),然后制备破碎细胞的悬浮液,用DEAE-琼脂糖FF(Amercham Pharmacia Biotech)、BUTYL-Toyopearl650M(Tohso)、MonoQ(Amercham Pharmacia Biotech)、RESOURCEIOS(Amercham Pharmacia Biotech)和Superdex-200(Amercham Pharmacia Biotech)从细胞悬浮液中成功纯化出表现出CD活性的蛋白质片段,以获得在SDS-PAGE中表现出基本上单一泳带的蛋白质溶液。获得的酶蛋白质在还原条件下在SDS-PAGE中显示出分子量大约为43KDa.
通过用埃德曼降解方法测定如上所述提纯的具有CD活性的蛋白质的N-末端氨基酸序列,通过用赖氨酸内肽酶消化蛋白质获得一个肽的氨基酸序列。结果如下:
(N-末端氨基酸序列)
MRFPELEELKNRRTLKWTRFPEDVL(SEQ ID NO:1)
(内部氨基酸序列)
ILREEGK(SEQ ID NO:2)
当用FASTA法检测到一种蛋白具有以上内部序列的氨基酸序列,则证明这种蛋白与来自谷氨酸棒状杆菌的aecD基因编码的产物的一部分具有100%的同源性(I.Rossol&A.Puhler,细菌杂志(J.Bacteriol.),174,2968-2977(1992))
制备具有下列核苷酸序列的引物1和2,基于由上文提到的N-末端氨基酸序列和内部氨基酸序列推导出的DNA序列,并且以ATCC 14067菌株的染色体DNA为模板进行PCR。
(引物1)
AARTGGACNMGNTTYCCNGA(SEQ ID NO:3)
(引物2)
CTTACCCTCCTCACGAAGAA(SEQ ID NO:4)
因此发现得到的片段的核苷酸序列与报道的谷氨酸棒状杆菌aecD基因高度一致。但是如上所述测定的蛋白质的N-末端氨基酸序列位于以前报道的aecD基因的起始密码子上游129bp处。基于这个结果说明在ATCC 14067菌株中,提纯的具有CD活性的蛋白质是aecD基因产物,而且aecD基因的起始密码子ATG正确的位于以前报道的aecD基因起始密码子上游至少129bp处。
通过以上方法获得的谷氨酸棒状杆菌的aecD基因的核苷酸序列,由核苷酸序列编码的蛋白质的氨基酸序列在SEQ ID NOS:5和6中也被列出。<2>测定谷氨酸棒状杆菌aecD破坏型菌株的CD活性
已经有文章报道,谷氨酸棒状杆菌IR33的一种aecD破坏型菌株被成功构建(I.Rossol&A.Puhler,细菌杂志(J.Bacteriol.),174,2968-2977(1992)),在IR33菌株中,染色体上的aecD基因使用质粒pIR33发生同源重组而裂解,该pIR33质粒含有***了氯霉素抗性基因的失活型aecD基因。测定以上参考描述的IR33和它的母系菌株ATCC 13032的CD活性。首先破碎细胞制备细胞悬浮液,然后用Kredich等的方法测量CD的活性,结果见表1。从表中可以看出,IR33的CD活性比野生型ATCC 13032的CD活性降低了约1/3。
表1
菌株 | CD活性(mU/毫克) |
ATCC13032 | 38 |
IR33 | 12 |
如上所述,aecD基因的破坏使CD活性显著降低,则证明了在谷氨酸棒状杆菌中由aecD基因编码主要的CD活性。
<3>把来自埃希氏大肠杆菌的含有脱敏型SAT基因的质粒导入aecD破坏型菌株中
把一个来自埃希氏大肠杆菌的野生型SAT基因(cysE)或者编码脱敏型SAT蛋白的基因导入谷氨酸棒状杆菌的aecD破坏型菌株IR33和野生型菌株ATCC 13032中,其中通过野生型的SAT基因的256位点处的甲硫氨酸残基突变为异亮氨酸残基而将SAT基因脱敏(日本专利特开No.11-155571)。
分别用EcoRI消化含有野生型SAT基因的质粒pCE和含有脱敏型SAT基因的质粒pCEM256I,切下包含SAT基因的片段(日本专利特开No.11-155571),再***到质粒pVK7的EcoRI位点中,该质粒是在棒状杆菌和埃希氏大肠杆菌之间的穿梭载体(参考日本专利特开No.11-266881)。pVK7是由谷氨酸棒状杆菌中的隐蔽性质粒pAM330和埃希氏大肠杆菌中的载体pHSG299连接而构成的(Kmr,参考Takeshita,S等,基因(Gene),61,63-74,(1987),日本专利特开No.10-215883),包含由一个多克隆位点和一个来自pHSG299的lacZ’位点。
根据上文描述的方法,在pKV7的lacZ’前方***野生型SAT基因或脱敏型SAT基因而获得质粒,包含野生型SAT基因的质粒和包含脱敏型SAT基因的质粒的分别标记为pKV7-CE、pKV7-256。
把pKV7、pKV7-CE和pKV7-256分别用电穿孔技术导入ATCC13032和IR33,以卡那霉素抗性作为标记物筛选转化菌株。
<4>生产L-半胱氨酸和L-胱氨酸
每一个得到的转化体包被在M-CM2G板上(10g/L的聚胨,10g/L酵母抽提物,5g/LNaCl,5g/L葡萄糖,0.2g/L DL-甲硫氨酸,PH7.2),平板中还含有25mg/L的卡那霉素,在31.5℃中培养48小时,然后转到盛有20ml的产半胱氨酸培养基的锥形瓶中,31.5℃摇晃培养72小时。产半胱氨酸培养基含有下面所列物质:
(产半胱氨酸培养基)
葡萄糖 100g/L
(NH4)SO4 45g/L
KH2PO4 1g/L
MgSO4.7H2O 1g/L
FeSO4.7H2O 10mg/L
MnSO4.5H2O 10mg/L
盐酸硫氨素 300ug/L
生物素 100ug/L
大豆蛋白盐酸水
解产物“Aieki”
(注册商标号,
Ajinomoto Co.,Inc) 480mg/L
L-异亮氨酸 100mg/L
L-亮氨酸 100mg/L
L-甘氨酸 100mg/L
DL-甲硫氨酸 100mg/L
CaCO3 50g/L
为溶解沉淀的L-胱氨酸,加入0.5N HCl稀释液体培养基,以还原型半胱氨酸(L-Cys)和胱氨酸(L-CysH)的总量,用Leuconostoc mesenteroides生物测定法(Tsunoda T等,氨基酸(Amino acids),3,7-13(1961))测定L-半胱氨酸聚集的量。
表2
宿主菌株 | 导入的质粒 | L-Cys+L-CysH积累量 |
ATCC13032 | pVK7 | <50 |
ATCC13032 | pVK7-CE | <50 |
ATCC13032 | pVK7-256 | 140 |
IR33 | pVK7 | <50 |
IR33 | pVK7-CE | 70 |
IR33 | pVK7-256 | 250 |
以上描述,证明了通过在谷氨酸棒状杆菌中扩增来自埃希氏大肠杆菌的SAT基因引起了L-半胱氨酸的积累,而且,脱敏型SAT基因的积累效应比野生型SAT基因更具优势,另外aecD基因的损失也提高了L-半胱氨酸的积累。
序列表
<110>味之素株式会社(Ajinomoto Co.,Inc.)
<120>产L-半胱氨酸细菌及生产L-半胱氨酸的方法
<130>OP1289
<140>
<141>2002-02-09
<150>JP2001-34486
<151>2001-02-09
<160>6
<170>PatentIn Ver.2.0
<210>1
<211>25
<212>PRT
<213>黄色棒杆菌(Corynebacterium flavum)
<400>1
Met Arg Phe Pro Glu Leu Glu Glu Leu Lys Asn Arg Arg Thr Leu Lys
1 5 10 15
Trp Thr Arg Phe Pro Glu Asp Val Leu
20 25
<210>2
<211>7
<212>PRT
<213>黄色棒杆菌(Corynebacterium flavum)
<400>2
Ile Leu Arg Glu Glu Gly Lys
1 5
<210>3
<211>20
<212>DNA
<213>人工序列
<220>
<223>人工序列说明:引物
<220>
<221>misc_特征
<222>(9,11,18)
<223>n=aorgtocort
<400>3
aartggacnm gnttyccnga 20
<210>4
<211>20
<212>DNA
<213>人工序列
<220>
<223>人工序列说明:引物
<400>4
cttaccctcc tcacgaagaa 20
<210>5
<211>1620
<212>DNA
<213>谷氨酸棒杆菌(Corynebacterium glutamicum)
<220>
<221>CDS
<222>(232)..(1338)
<400>5
gatatcgccg cttgggactg tgattgggct tacctctttg gcttcgaaca ccggggttga 60
ggtagtggtt acttccatgg tttcctcagc ggaaacggct tggctatcag cactttcacc 120
cgaacagcct gcaagaagtg cgacggctaa cagggctggg attgtcctca acttcacttc 180
gggctccttc ttagtaatag gttcgtagaa aagtttacta agcctgagag t atg cga 237
Met Arg
1
ttt cct gaa ctc gaa gaa ttg aag aat cgc cgg acc ttg aaa tgg acc 285
Phe Pro Glu Leu Glu Glu Leu Lys Asn Arg Arg Thr Leu Lys Trp Thr
5 10 15
cgg ttt cca gaa gac gtg ctt cct ttg tgg gtt gcg gaa agt gat ttt 333
Arg Phe Pro Glu Asp Val Leu Pro Leu Trp Val Ala Glu Ser Asp Phe
20 25 30
ggc acc tgc ccg cag ttg aag gaa gct atg gca gat gcc gtt gag cgc 381
Gly Thr Cys Pro Gln Leu Lys Glu Ala Met Ala Asp Ala Val Glu Arg
35 40 45 50
gag gtc ttc gga tac cca cca gat gct act ggg ttg aat gat gcg ttg 429
Glu Val Phe Gly Tyr Pro Pro Asp Ala Thr Gly Leu Asn Asp Ala Leu
55 60 65
act gga ttc tac gag cgt cgc tat ggg ttt ggc cca aat ccg gaa agt 477
Thr Gly Phe Tyr Glu Arg Arg Tyr Gly Phe Gly Pro Asn Pro Glu Ser
70 75 80
gtt ttc gcc att ccg gat gtg gtt cgt ggc ctg aag ctt gcc att gag 525
Val Phe Ala Ile Pro Asp Val Val Arg Gly Leu Lys Leu Ala Ile Glu
85 90 95
cat ttc act aag cct ggt tcg gcg atc att gtg ccg ttg cct gca tac 573
His Phe Thr Lys Pro Gly Ser Ala Ile Ile Val Pro Leu Pro Ala Tyr
100 105 110
cct cct ttc att gag ttg cct aag gtg act ggt cgt cag gcg atc tac 621
Pro Pro Phe Ile Glu Leu Pro Lys Val Thr Gly Arg Gln Ala Ile Tyr
115 120 125 130
att gat gcg cat gag tac gat ttg aag gaa att gag aag gcc ttc gct 669
Ile Asp Ala His Glu Tyr Asp Leu Lys Glu Ile Glu Lys Ala Phe Ala
135 140 145
gac ggt gcg gga tca ctg ttg ttc tgc aat cca cac aac cca ctg ggc 717
Asp Gly Ala Gly Ser Leu Leu Phe Cys Asn Pro His Asn Pro Leu Gly
150 155 160
acg gtc ttt tct gaa gag tac atc cgc gag ctc acc gat att gcg gcg 765
Thr Val Phe Ser Glu Glu Tyr Ile Arg Glu Leu Thr Asp Ile Ala Ala
165 170 175
aag tac gat gcc cgc atc atc gct gat gag atc cac gcg cca ctg gtt 813
Lys Tyr Asp Ala Arg Ile Ile Ala Asp Glu Ile His Ala Pro Leu Val
180 185 190
tat gaa ggc acc cat gtg gtt gct gct ggt gtt tct gag aac gct gca 861
Tyr Glu Gly Thr His Val Val Ala Ala Gly Val Ser Glu Asn Ala Ala
195 200 205 210
aac act tgc atc acc atc acc gca act tct aag gcg tgg aac act gct 909
Asn Thr Cys Ile Thr Ile Thr Ala Thr Ser Lys Ala Trp Asn Thr Ala
215 220 225
ggt ttg aag tgt gct cag atc ttc ttc agt ggt gaa gcc gat gtg aag 957
Gly Leu Lys Cys Ala Gln Ile Phe Phe Ser Gly Glu Ala Asp Val Lys
230 235 240
gcc tgg aag aat ttg tcg gat att acc cgt gac ggt gtg tcc atc ctt 1005
Ala Trp Lys Asn Leu Ser Asp Ile Thr Arg Asp Gly Val Ser Ile Leu
245 250 255
gga ttg atc gct gcg gag aca gtg tac aac gag ggc gaa gaa ttc ctt 1053
Gly Leu Ile Ala Ala Glu Thr Val Tyr Asn Glu Gly Glu Glu Phe Leu
260 265 270
gat gag tca att cag att ctc aag gac aac cgt gac ttt gcg gct gct 1101
Asp Glu Ser Ile Gln Ile Leu Lys Asp Asn Arg Asp Phe Ala Ala Ala
275 280 285 290
gaa ctg gaa aag ctt ggc gtg aag gtc tac gca ccg gac tcc act tat 1149
Glu Leu Glu Lys Leu Gly Val Lys Val Tyr Ala Pro Asp Ser Thr Tyr
295 300 305
ttg atg tgg ttg gac ttc gct ggc acc aag ate gaa gag gcg cct tct 1197
Leu Met Trp Leu Asp Phe Ala Gly Thr Lys Ile Glu Glu Ala Pro Ser
310 315 320
aaa att ctt cgt gag gag ggt aag gtc atg ctg aat gat ggc gca gct 1245
Lys Ile Leu Arg Glu Glu Gly Lys Val Met Leu Asn Asp Gly Ala Ala
325 330 335
ttt ggt ggt ttc acc acc tgc gct cgt ctt aat ttt gcg tgt tcc aga 1293
Phe Gly Gly Phe Thr Thr Cys Ala Arg Leu Asn Phe Ala Cys Ser Arg
340 345 350
gag acc ctt gag gag ggc tgc gcc gta tcg cca gcg tgt tgt aaa 1338
Glu Th rLeu Glu Glu Gly Cys Ala Val Ser Pro Ala Cys Cys Lys
355 360 365
taatgagtaa aaagtctgtc ctgattactt ctttgatgct gttttccatg ttcttcggag 1398
ctggaaacct catcttcccg ccgatgcttg gattgtcggc aggaaccaac tatctaccag 1458
ctatcttagg atttctagca acgagtgttc tgctcccggt gctggcgatt atcgcggtgg 1518
tgttgtcggg agaaaatgtc aaggacatgg cttctcgtgg cggtaagatc tttggcctgg 1578
tgtttcctat tgctgcctat ttgtctatcg gcgcgtttta cg 1620
<210>6
<211>369
<212>PRT
<213>谷氨酸棒杆菌(Corynebacterium glutamicum)
<400>6
Met Arg Phe Pro Glu Leu Glu Glu Leu Lys Asn Arg Arg Thr Leu Lys
1 5 10 15
Trp Thr Arg Phe Pro Glu Asp Val Leu Pro Leu Trp Val Ala Glu Ser
20 25 30
Asp Phe Gly Thr Cys Pro Gln Leu Lys Glu Ala Met Ala Asp Ala Val
35 40 45
Glu Arg Glu Val Phe Gly Tyr Pro Pro Asp Ala Thr Gly Leu Asn Asp
50 55 60
Ala Leu Thr Gly Phe Tyr Glu Arg Arg Tyr Gly Phe Gly Pro Asn Pro
65 70 75 80
Glu Ser Val Phe Ala Ile Pro Asp Val Val Arg Gly Leu Lys Leu Ala
85 90 95
Ile Glu His Phe Thr Lys Pro Gly Ser Ala Ile Ile Val Pro Leu Pro
100 105 110
Ala Tyr Pro Pro Phe Ile Glu Leu Pro Lys Val Thr Gly Arg Gln Ala
115 120 125
Ile Tyr Ile Asp Ala His Glu Tyr Asp Leu Lys Glu Ile Glu Lys Ala
130 135 140
Phe Ala Asp Gly Ala Gly Ser Leu Leu Phe Cys Asn Pro His Asn Pro
145 150 155 160
Leu Gly Thr Val Phe Ser Glu Glu Tyr Ile Arg Glu Leu Thr Asp Ile
165 170 175
Ala Ala Lys Tyr Asp Ala Arg Ile Ile Ala Asp Glu Ile His Ala Pro
180 185 190
Leu Val Tyr Glu Gly Thr His Val Val Ala Ala Gly Val Ser Glu Asn
195 200 205
Ala Ala Asn Thr Cys Ile Thr Ile Thr Ala Thr Ser Lys Ala Trp Asn
210 215 220
Thr Ala Gly Leu Lys Cys Ala Gln Ile Phe Phe Ser Gly Glu Ala Asp
225 230 235 240
Val Lys Ala Trp Lys Asn Leu Ser Asp Ile Thr Arg Asp Gly Val Ser
245 250 255
Ile Leu Gly Leu Ile Ala Ala Glu Thr Val Tyr Asn Glu Gly Glu Glu
260 265 270
Phe Leu Asp Glu Ser Ile Gln Ile Leu Lys Asp Asn Arg Asp Phe Ala
275 280 285
Ala Ala Glu Leu Glu Lys Leu Gly Val Lys Val Tyr Ala Pro Asp Ser
290 295 300
Thr Tyr Leu Met Trp Leu Asp Phe Ala Gly Thr Lys Ile Glu Glu Ala
305 310 315 320
Pro Ser Lys Ile Leu Arg Glu Glu Gly Lys Val Met Leu Asn Asp Gly
325 330 335
Ala Ala Phe Gly Gly Phe Thr Thr Cys Ala Arg Leu Asn Phe Ala Cys
340 345 350
Ser Arg Glu Thr Leu Glu Glu Gly Cys Ala Val Ser Pro Ala Cys Cys
355 360 365
Lys
Claims (5)
1、具有生产L-半胱氨酸能力的棒状杆菌,其中已对所述棒状杆菌进行修饰,从而应提高细胞内丝氨酸乙酰转移酶活性,且已对所述棒状杆菌进行修饰,从而L-半胱氨酸降解***进一步被这种修饰抑制,所述修饰应降低半胱氨酸脱巯基酶活性,
其中细胞内丝氨酸乙酰转移酶活性是通过以下方法提高的:导入L-半胱氨酸对其的反馈抑制作用被减弱的丝氨酸乙酰转移酶、增加编码丝氨酸乙酰转移酶的基因的拷贝数、或修饰编码丝氨酸乙酰转移酶的基因的表达调控中所涉及的表达调控序列或基因,并且,
其中细胞内半胱氨酸脱巯基酶活性是通过破坏aecD基因而降低的。
2、权利要求1的棒状杆菌,其中L-半胱氨酸对其反馈抑制被减弱的丝氨酸乙酰转移酶包含野生型的丝氨酸乙酰转移酶256位的甲硫氨酸残基相应的氨基酸残基被赖氨酸残基和亮氨酸残基之外的氨基酸残基所取代的突变,或者包含缺失野生型的丝氨酸乙酰转移酶的第256位的甲硫氨酸残基相应的氨基酸残基开始的C-末端区的突变。
3、权利要求1的棒状杆菌,其中丝氨酸乙酰转移酶的编码基因是属于埃希氏杆菌属细菌的cysE。
4、权利要求2的棒状杆菌,其中丝氨酸乙酰转移酶的编码基因是属于埃希氏杆菌属细菌的cysE。
5、一种生产L-半胱氨酸的方法,包括在培养基中培养1到4中的任一项的棒状杆菌来生产并且在培养物中积累L-半胱氨酸,并且从培养物中收集L-半胱氨酸。
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CN112779203B (zh) * | 2021-01-19 | 2022-12-30 | 浙江工业大学 | 高产l-半胱氨酸的基因工程菌及其构建与应用 |
CN113817660A (zh) * | 2021-09-15 | 2021-12-21 | 绵阳师范学院 | 一种产丝氨酸乙酰转移酶的基因工程菌及其构建方法 |
CN114214341B (zh) * | 2021-12-30 | 2023-12-26 | 山西大学 | 番茄SlSERAT1;1基因或其片段在植物发育过程中的应用 |
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WO1997015673A1 (de) * | 1995-10-26 | 1997-05-01 | Consortium für elektrochemische Industrie GmbH | Verfahren zur herstellung von o-acetylserin, l-cystein und l-cystein-verwandten produkten |
JPH11155571A (ja) * | 1997-11-25 | 1999-06-15 | Ajinomoto Co Inc | L−システインの製造法 |
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2001
- 2001-02-09 JP JP2001034486A patent/JP4622111B2/ja not_active Expired - Fee Related
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2002
- 2002-02-01 US US10/060,218 patent/US7148047B2/en not_active Expired - Fee Related
- 2002-02-05 BR BR0200285-0A patent/BR0200285A/pt not_active Application Discontinuation
- 2002-02-08 EP EP02002066A patent/EP1234874A1/en not_active Ceased
- 2002-02-09 CN CNB02105407XA patent/CN1330749C/zh not_active Expired - Fee Related
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
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WO1997015673A1 (de) * | 1995-10-26 | 1997-05-01 | Consortium für elektrochemische Industrie GmbH | Verfahren zur herstellung von o-acetylserin, l-cystein und l-cystein-verwandten produkten |
JPH11155571A (ja) * | 1997-11-25 | 1999-06-15 | Ajinomoto Co Inc | L−システインの製造法 |
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CN1374400A (zh) | 2002-10-16 |
US20030077766A1 (en) | 2003-04-24 |
EP1234874A1 (en) | 2002-08-28 |
US7148047B2 (en) | 2006-12-12 |
JP2002233384A (ja) | 2002-08-20 |
BR0200285A (pt) | 2002-10-29 |
JP4622111B2 (ja) | 2011-02-02 |
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