CN108546697A - 酶法制备beta丙氨酸 - Google Patents
酶法制备beta丙氨酸 Download PDFInfo
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- CN108546697A CN108546697A CN201810306203.1A CN201810306203A CN108546697A CN 108546697 A CN108546697 A CN 108546697A CN 201810306203 A CN201810306203 A CN 201810306203A CN 108546697 A CN108546697 A CN 108546697A
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- Prior art keywords
- alanine
- pandcg
- ala
- enzyme
- beta
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Abstract
本发明通过定向进化的方法获得了一种高酶活力的天冬氨酸脱羧酶突变体,其能高效催化天冬氨酸脱羧生成β‑丙氨酸,通过反应工艺的优化,天冬氨酸底物浓度可以达到250g/L,24小时转化后,转化率可以达到98.3%以上,具有工业应用前景。
Description
技术领域
本发明属于生物催化领域,具体地说,涉及一种天冬氨酸脱羧酶突变体,并且涉及天冬氨酸脱羧酶突变体或者其表达微生物在生产β-丙氨酸中的用途。
背景技术
Beta丙氨酸即β-丙氨酸(β-alanine)是自然界中唯一天然存在的β型氨基酸,近年来,其在医药、化工、食品、环境等领域的作用日渐突出(Biotechnology andBioengineering,2012,109(10):2437-2459.)。在医药领域,主要用于合成泛酸以及泛酸钙,此外还是合成肌肽、帕米酸钠、巴柳氮等药物的原料。在食品领域,可用作调味品。在环境领域,可用于制备水的净化絮凝剂等(Nature Chemical Biology,2012,8(6):536-546.)。有报道称β-丙氨酸是未来全球12种最具开发潜力的三碳化工产品之一(EuropeanPolymer Journal,2013,49(49):1773-1781.)。
目前,国内外生产β-丙氨酸的工业化方法主要采用化学合成法,包括丙烯酸氨化法、丙烯腈氨化水解法及β-氨基丙腈水解法,国内的工业化生产主要采用丙烯腈氨化水解法(氨基酸和生物资源,2005,27(1):52-55)。这些方法大多需要强酸强碱、高温高压的条件,而且产物纯化步骤繁琐,同时存在着腈类物质导致环境污染的问题。随着β-丙氨酸需求量的不断增加,寻求绿色的生产方法具有十分明显的经济和社会效益。酶转化法制备β-丙氨酸具有工艺简单、纯化方便、绿色无污染的特点,该方法现已逐步成为人们研究的热点。编码L-天冬氨酸α-脱羧酶(L-aspartate-alpha-decarboxylase,pand)即天冬氨酸脱羧酶的基因被发现广泛地存在于大肠杆菌、谷氨酸棒杆菌、结核分枝杆菌、沙门氏菌等微生物中(工业微生物,2007,37(5):54-58;Applied and Environmental Microbiology,1999,65(4):1530-1539.Proteins:Structure,Function,and Bioinformatics,2006,65(4):796-802.Protein Expression and Purification,2002,25(3):533-540.Molecular andGeneral Genetics,1975,140(2):159-164.)。高丽娟等将E.coli DH5α来源的pand基因在E.coli BL21(DE3)中表达构建重组菌,酶活为224.96U/L(浙江工业大学硕士学位论文,2007)。Nicole等克隆了C.glutamicum的pand基因(pandC.g.)和E.coli的pand基因(pandE.c.)并在E.coli中表达,结果显示pandC.g.基因表达能消除β-丙氨酸对泛酸合成的限制并获得泛酸大量积累(Applied and Environmental Microbiology,1999,65(4):1530-1539.)。
上述酶转化法的优点主要体现在反应条件温和,易于控制,安全、环保。但要实现工业化,必须大幅度提高作为催化剂的天冬氨酸脱羧酶的酶活性,并要解决降低或消除底物抑制和/或产物抑制、保障天冬氨酸脱羧酶的稳定性和充足供应等等诸多问题。
发明内容
为了获得具有高催化活性的天冬氨酸脱羧酶,本发明选择以谷氨酸棒杆菌C.glutamicum为基因来源,通过易错PCR的方法建立pand基因突变库,通过筛选基因突变库获得酶活力相比野生型明显提高的L-天门冬氨酸-α-脱羧酶。通过扩增突变的pand基因来转化大肠杆菌E.coli,可构建高表达L-天门冬氨酸-α-脱羧酶的重组菌。
因此,本发明的第一个目的在于提供如下用于生产β-丙氨酸的天冬氨酸脱羧酶:
一种天冬氨酸脱羧酶,其氨基酸序列为SEQ ID NO:1:MLRTILGSKIHRATVTQADLDYVGSVTIDADAVHAAGLIRGELVAIVDITNGARLETYVIVGDARTGNICINGAAAHLINPGDLVIIMSYLQATDAEAKAYEPKIVHVDADNRIVALGNDLAEALPGSGLLTSRSI(SEQ ID NO:1)。
本发明的第二个目的在于提供编码上述天冬氨酸脱羧酶的基因。
优选地,上述基因的碱基序列为SEQ ID NO:2:ATGCTGCGCACCATCCTCGGAAGTAAGATTCACCGAGCCACTGTCACTCAAGCTGATCTAGATTATGTTGGCTCTGTAACCATCGACGCCGACGCTGTTCACGCCGCCGGATTGATCCGAGGCGAACTGGTTGCCATCGTAGACATCACCAACGGCGCTCGTCTGGAAACTTATGTCATTGTGGGCGACGCCAGAACGGGCAATATTTGCATCAATGGTGCCGCTGCACACCTTATTAATCCTGGCGATCTTGTGATCATCATGAGCTACCTTCAGGCGACTGATGCGGAAGCCAAGGCGTATGAGCCAAAGATTGTGCACGTGGACGCCGACAACCGCATCGTTGCGCTCGGCAACGATCTTGCGGAAGCACTACCTGGATCCGGGCTTTTGACGTCGAGAAGCATTTAG(SEQ ID NO:2)。
本发明的第三个目的在于提供包含上述基因的质粒。
本发明的第四个目的在于提供转化了上述质粒的微生物。
优选地,上述微生物选自大肠杆菌、酵母、枯草杆菌。更优选大肠杆菌BL21(DE3)。
本发明的第五个目的在于提供上述天冬氨酸脱羧酶或表达微生物在生产β-丙氨酸中的用途。
在一种实施方式中,上述用途是以天冬氨酸为原料生产β-丙氨酸。
本发明使用重组大肠杆菌进行了β-丙氨酸生产实验,天冬氨酸底物浓度可以达到250g/L,24小时转化后,转化率可以达到98%以上,证明具有工业化应用前景。
具体实施方式
以下结合具体实施例对本发明做进一步详细说明。应理解,以下实施例仅用于说明本发明而非用于限定本发明的范围。
本文中涉及到多种物质的添加量、含量及浓度,其中所述的百分含量,除特别说明外,皆指质量百分含量。
本文的实施例中,如果对于反应温度或操作温度没有做出具体说明,则该温度通常指室温(15-25℃)。
作为构建天冬氨酸脱羧酶突变体的基础模板,谷氨酸棒杆菌Corynebacteriumglutamicum ATCC 13869的基因序列为GenBank:CP016335.1:ATGCTGCGCACCATCCTCGGAAGTAAGATTCACCGAGCCACTGTCACTCAAGCTGATCTAGATTATGTTGGCTCTGTAACCATCGACGCCGACCTGGTTCACGCCGCCGGATTGATCGAAGGCGAAAAAGTTGCCATCGTAGACATCACCAACGGCGCTCGTCTGGAAACTTATGTCATTGTGGGCGACGCCAGAACGGGCAATATTTGCATCAATGGTGCCGCTGCACACCTTATTAATCCTGGCGATCTTGTGATCATCATGAGCTACCTTCAGGCGACTGATGCGGAAGCCAAGGCGTATGAGCCAAAGATTGTGCACGTGGACGCCGACAACCGCATCGTTGCGCTCGGCAACGATCTTGCGGAAGCACTACCTGGATCCGGGCTTTTGACGTCGAGAAGCATTTAG(SEQ ID NO:3)。
其编码的野生型天冬氨酸脱羧酶(Pandcg)的氨基酸序列为(GenBank:ANU32426.1):MLRTILGSKIHRATVTQADLDYVGSVTIDADLVHAAGLIEGEKVAIVDITNGARLETYVIVGDARTGNICINGAAAHLINPGDLVIIMSYLQATDAEAKAYEPKIVHVDADNRIVALGNDLAEALPGSGLLTSRSI(SEQID NO:4)。
为了获得酶活性更高的天冬氨酸脱羧酶突变体,本发明对野生型天冬氨酸脱羧酶的基因序列SEQ ID NO:3进行点突变。通过易错PCR技术获得一个或多个氨基酸位点取代的突变体氨基酸序列,筛选出4个可提高天冬氨酸脱羧酶的酶活力或是增加底物特异性的位点,然后以定点组合突变的方式,获得突变体。
在本文中,术语“L-天冬氨酸α-脱羧酶”、“L-天冬氨酸脱羧酶”、“天冬氨酸脱羧酶”和“Pand”表示相同的意义,它们可以互换使用。类似地,术语“L-天门冬氨酸”、“天门冬氨酸”、“L-天冬氨酸”和“天冬氨酸”表示相同的意义,它们可以互换使用。
在本发明中,术语“野生(型)”、“野生酶”、“野生型酶”表示相同的意义,都是指未经基因工程改造的天冬氨酸脱羧酶(GenBank:ANU32426.1)。
本发明的天冬氨酸脱羧酶突变体的氨基酸数量有136个,由于结构明确,因此本领域技术人员很容易获得其编码基因、包含这些基因的表达盒和质粒、以及包含该质粒的转化体。这些基因、表达盒、质粒、转化体可以通过本领域技术人员所熟知的基因工程构建方式获得。
上述转化体宿主可以是任何适合表达天冬氨酸脱羧酶突变体的微生物,括细菌和真菌。优选微生物是大肠杆菌、毕赤酵母、酿酒酵母、或者枯草杆菌,优选大肠杆菌,更优选大肠杆菌BL21(DE3)。
当作为生物催化剂用于生产β-丙氨酸时,本发明的天冬氨酸脱羧酶可以呈现酶的形式或者菌体的形式。所述酶的形式包括游离酶、固定化酶,包括纯化酶、粗酶、发酵液、载体固定的酶等;所述菌体的形式包括存活菌体和死亡菌体。
本发明的天冬氨酸脱羧酶的分离纯化、包括固定化酶制备技术也是本领域技术人员所熟知的。
为简要起见,在实施例中有时将来源于Corynebacterium glutamicum ATCC13869的天冬氨酸脱羧酶简写为Pandcg。
实施例
材料和方法
本文中的全基因合成由苏州金唯智生物科技有限公司完成;表达载体由浙江华睿生物技术有限公司亚克隆制备。引物合成及测序皆由上海生工完成。
本文中的分子生物学实验包括质粒构建、酶切、连接、感受态细胞制备、转化、培养基配制等等,主要参照《分子克隆实验指南》(第三版),J.萨姆布鲁克,D.W.拉塞尔(美)编著,黄培堂等译,科学出版社,北京,2002)进行。必要时可以通过简单试验确定具体实验条件。
PCR扩增实验根据质粒或DNA模板供应商提供的反应条件或试剂盒说明书进行。必要时可以通过简单试验予以调整。
酶活力测定
1.咖啡因脱氢酶酶活力测定
天冬氨酸脱羧酶的酶活力测定方法参照文献(Appl Microbiol Biotechnol 201706DOI10.1007/s00253-017-8337-y)所提供的方法。酶反应体系:pH 7.0、50mmol/L磷酸盐缓冲液2.0mL、酶液1.0mL、L-天冬氨酸(200g/L)1.0mL溶液(天冬氨酸用NaOH调节pH使其溶解)。充分混匀后,37℃,反应2h,然后加入1.0mL的1M的NaOH终止反应,通过HPLC检测β-丙氨酸生成量。
β-丙氨酸衍生产物用HPLC进行测定:色谱柱为La Chrom C18(5μm,4.6×250mm);流动相A液为80%(v/v)腈水溶液,B液为97:3(v/v,pH6.5)的0.1mol/L乙酸钠-乙腈溶液;采用梯度洗脱:0-15min,B液由95%下降65%;15-20min,B液由65%上升到95%;20-30min,B液梯度不变。检测波长为254nm,柱温为40℃。所测得的β-丙氨酸衍生产物的含量等同于衍生前的β-丙氨酸。
酶活力定义:在pH7.0、温度37℃的条件下,每分钟催化L-天冬氨酸产生1微摩尔(μmol)β-丙氨酸所需要的酶量定义为1个单位(U)。
LB培养基:10g/L胰蛋白胨、5g/L酵母提取物、10g/L氯化钠,pH7.2,121℃高温高压灭菌20min。
TB培养基:24g/L酵母提取物、12g/L胰蛋白胨、16.43g/L K2HPO4.3H2O、2.31g/LKH2PO4、5g/L甘油,pH 7.0-7.5,121℃高温高压灭菌20min。
实施例1天冬氨酸脱羧酶基因工程菌构建
通过全基因序列合成序列SEQ ID NO:3,两端设计酶切位点NdeI和BamHI,克隆到pSH质粒上相应位点,得到重组质粒pSH-Pandcg,然后用氯化钙法转化入大肠杆菌表达宿主BL21(DE3)感受态细胞中,涂含卡那霉素的LB培养基平板,37℃培养过夜,挑选单菌落,接种到含有LB培养基的试管中,培养过夜,离心收集菌体,抽提质粒,基因测序确定正确,得到表达野生型Pandcg酶的重组基因工程菌株。
实施例2易错PCR法构建天冬氨酸脱羧酶(Pandcg)随机突变点库
以SEQ ID NO:3为模板,应用易错PCR技术构建随机突变体库。
正向引物Pandcg-Nde-F:5’-ATGTACCTGCGCACCATCCTCGGAAG-3’,
反向引物Pandcg-Xho-R:5’-CTCGAGCTAAATGCTTCTCGACGTCAAAAGC-3’。
50μL易错PCR反应体系包括:50ng质粒模板pSH-Pandcg,30pmol一对引物Pandcg-Nde-F和Pandcg-Xho-R,1×Taq buffer,0.2mM dGTP,0.2mM dATP,1mM dCTP,1mM dTTP,7mMMgCl2,(0mM、0.05mM、0.1mM、0.15mM、0.2mM)MnCl2,2.5个单位的Taq酶(fermentas)。PCR反应条件为:95℃5min;94℃30s,55℃30s,72℃2min/kbp;30个循环;72℃10min。胶回收2.0kb随机突变片段作为大引物,用KOD-plus DNA聚合酶做MegaPrimer PCR:94℃5min,;98℃10s,60℃30s,68℃2min/kbp,25个循环;68℃10min。DpnI消化质粒模板,电转化大肠杆菌E.coliBL21(DE3),得到超过104个克隆的随机突变库
实施例3Pandcg突变体库的高通量筛选
3.1选取突变体库中的转化子,接种到含700μLLB培养基的96孔深孔培养板中,培养基中含100μg/mL卡那霉素,37℃培养6h后,加入终浓度0.1mM IPTG后,降温至25℃,培养过夜。5000rpm离心10min,弃上清,置于-70℃冷冻1h,室温融化30min。加入200μL含0.1M磷酸钾盐缓冲液(pH8.0),重悬菌体,用于Pandcg酶活力测定。
3.2高酶活突变体的测定和筛选
底物反应液:200g/L的L-天冬氨酸,用NaOH调节pH,使其溶解。
终止反应液:0.5ml的1M的NaOH溶液。
将上述步骤3.1中的80μL菌体悬液匀浆破壁,得到的粗酶液加入80μL底物反应液,在37℃的条件下反应2h,加入40μL终止反应液,然后5000rpm离心10min。取上清,用HPLC检测活力。
在随机突变库,通过对约1000个突变体克隆筛选,发现4个氨基酸位点取代的突变体具有提高的酶活力。有5个突变体的Pandcg酶活显著提高,结果如表1所示。
表1.部分天冬氨酸脱羧酶突变体的酶活力比较
突变体 | 突变氨基酸 | 相对酶活力(%) |
Pandcg | -- | 100 |
Pandcg-17 | L32A | 348 |
Pandcg-59 | E40R | 189 |
Pandcg-167 | D19V,L32A | 315 |
Pandcg-398 | L32A,E40R,K43L, | 487 |
Pandcg-677 | E40R | 253 |
表中突变氨基酸D19V表示野生型Pandcg(SEQ ID NO:4)上的第19个氨基酸D(天冬氨酸Asp)替换为V(缬氨酸Val),L32A表示第32个氨基酸L(亮氨酸Leu)替换为A(丙氨酸Ala),E40R表示第40个氨基酸E(谷氨酸Glu)替换为R(精氨酸Arg),K43L表示第43个氨基酸K(赖氨酸Lys)替换为L(亮氨酸Leu)。
其中编号为Pandcg-398的突变体(L32A,E40R,K43L)的酶活力现对于野生型提高了近4倍。
实施例4高酶活基因工程菌的构建
将突变体Pandcg-398的编码基因SEQ ID NO:2按照实施例1中的方法克隆到pSH质粒中,得到重组质粒pSH-Pandcg-398,然后用氯化钙法转化入大肠杆菌BL21(DE3)感受态细胞中,涂含卡那霉素的LB培养基平板,37℃培养过夜,挑选10个单菌落,接种到含有LB培养基的试管中,培养过夜,离心收集菌体,抽提质粒,基因测序确定突变正确,得到重组菌株。
本领域技术人员应当理解,包括SEQ ID NO:2在内的突变体Pandcg-398编码基因、也可在枯草芽孢杆菌、毕赤酵母、酿酒酵母中表达,表达宿主不限于大肠杆菌。
实施例5突变体Pandcg-398菌株的发酵
从含有突变体Pandcg-398工程菌种的平板上挑选单克隆,接种到5mL LB培养基中,37℃培养过夜;1%v/v接种到含有100ml TB培养基的1000mL摇瓶中培养4-6小时,OD达到1.2-1.5,加入0.2mM的IPTG诱导,降温到25℃培养10-16小时,离心获得菌体,-80℃冻存24小时备用。
实施例6突变体Pandcg-398催化L-天冬氨酸制备β-丙氨酸
6.1不同酶量Pandcg-398催化L-天冬氨酸获得β-丙氨酸
反应体系为200mL,底物L-天冬氨酸浓度为250g/L,加酶量分别为2000、4000、6000、8000、10000、12000U/g底物,37℃,200rpm,控制pH7.0,反应24h,测定β-丙氨酸生成量,计算底物转化率,结果见表2。
表2:不同酶量Pandcg-398催化L-天冬氨酸生产β-丙氨酸
6.2突变体Pandcg-398催化L-天冬氨酸脱羧生产β-丙氨酸的时间考察
反应体系200mL,底物L-天冬氨酸浓度为250g/L,加酶量10000U/g底物,37℃,200rpm,控制pH7.0,反应12-36h,测定β-丙氨酸生成量,计算底物转化率,结果见表3。
表3.不同反应时间突变酶Pandcg-398催化L-天冬氨酸获得β-丙氨酸的转化率
6.3规模化生产β-丙氨酸
反应体系50L,底物L-天冬氨酸浓度为250g/L,加酶量10000U/g底物,37℃,200rpm,控制pH7.0,反应24h,测定β-丙氨酸生成量,确定最后的底物转化率超过98.3%。
上述实施例对本发明的天冬氨酸脱羧酶突变体生产β-丙氨酸的工艺进行了验证,相关的工艺条件可以进一步优化。本领域的技术人员应理解,在不违背本发明的思想下,本领域技术人员可以在此基础上做出各种改动或者修改,所做的各种变形或者修改的等价形式,同样应属于本发明的范围。
另外,需说明的是,本说明书中对先前公开的文献的列举和论述不应视为承认该文献是现有技术或者是公知常识。
序列表
<110> 浙江华睿生物技术有限公司
<120> 酶法制备beta丙氨酸
<130> SHPI1810279
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 136
<212> PRT
<213> 人工序列()
<400> 1
Met Leu Arg Thr Ile Leu Gly Ser Lys Ile His Arg Ala Thr Val Thr
1 5 10 15
Gln Ala Asp Leu Asp Tyr Val Gly Ser Val Thr Ile Asp Ala Asp Ala
20 25 30
Val His Ala Ala Gly Leu Ile Arg Gly Glu Leu Val Ala Ile Val Asp
35 40 45
Ile Thr Asn Gly Ala Arg Leu Glu Thr Tyr Val Ile Val Gly Asp Ala
50 55 60
Arg Thr Gly Asn Ile Cys Ile Asn Gly Ala Ala Ala His Leu Ile Asn
65 70 75 80
Pro Gly Asp Leu Val Ile Ile Met Ser Tyr Leu Gln Ala Thr Asp Ala
85 90 95
Glu Ala Lys Ala Tyr Glu Pro Lys Ile Val His Val Asp Ala Asp Asn
100 105 110
Arg Ile Val Ala Leu Gly Asn Asp Leu Ala Glu Ala Leu Pro Gly Ser
115 120 125
Gly Leu Leu Thr Ser Arg Ser Ile
130 135
<210> 2
<211> 411
<212> DNA
<213> 人工序列()
<400> 2
atgctgcgca ccatcctcgg aagtaagatt caccgagcca ctgtcactca agctgatcta 60
gattatgttg gctctgtaac catcgacgcc gacgctgttc acgccgccgg attgatccga 120
ggcgaactgg ttgccatcgt agacatcacc aacggcgctc gtctggaaac ttatgtcatt 180
gtgggcgacg ccagaacggg caatatttgc atcaatggtg ccgctgcaca ccttattaat 240
cctggcgatc ttgtgatcat catgagctac cttcaggcga ctgatgcgga agccaaggcg 300
tatgagccaa agattgtgca cgtggacgcc gacaaccgca tcgttgcgct cggcaacgat 360
cttgcggaag cactacctgg atccgggctt ttgacgtcga gaagcattta g 411
<210> 3
<211> 411
<212> DNA
<213> Corynebacterium glutamicum ATCC 13869
<400> 3
atgctgcgca ccatcctcgg aagtaagatt caccgagcca ctgtcactca agctgatcta 60
gattatgttg gctctgtaac catcgacgcc gacctggttc acgccgccgg attgatcgaa 120
ggcgaaaaag ttgccatcgt agacatcacc aacggcgctc gtctggaaac ttatgtcatt 180
gtgggcgacg ccagaacggg caatatttgc atcaatggtg ccgctgcaca ccttattaat 240
cctggcgatc ttgtgatcat catgagctac cttcaggcga ctgatgcgga agccaaggcg 300
tatgagccaa agattgtgca cgtggacgcc gacaaccgca tcgttgcgct cggcaacgat 360
cttgcggaag cactacctgg atccgggctt ttgacgtcga gaagcattta g 411
<210> 4
<211> 136
<212> PRT
<213> Corynebacterium glutamicum ATCC 13869
<400> 4
Met Leu Arg Thr Ile Leu Gly Ser Lys Ile His Arg Ala Thr Val Thr
1 5 10 15
Gln Ala Asp Leu Asp Tyr Val Gly Ser Val Thr Ile Asp Ala Asp Leu
20 25 30
Val His Ala Ala Gly Leu Ile Glu Gly Glu Lys Val Ala Ile Val Asp
35 40 45
Ile Thr Asn Gly Ala Arg Leu Glu Thr Tyr Val Ile Val Gly Asp Ala
50 55 60
Arg Thr Gly Asn Ile Cys Ile Asn Gly Ala Ala Ala His Leu Ile Asn
65 70 75 80
Pro Gly Asp Leu Val Ile Ile Met Ser Tyr Leu Gln Ala Thr Asp Ala
85 90 95
Glu Ala Lys Ala Tyr Glu Pro Lys Ile Val His Val Asp Ala Asp Asn
100 105 110
Arg Ile Val Ala Leu Gly Asn Asp Leu Ala Glu Ala Leu Pro Gly Ser
115 120 125
Gly Leu Leu Thr Ser Arg Ser Ile
130 135
Claims (10)
1.一种天冬氨酸脱羧酶,其氨基酸序列为SEQ ID NO:1。
2.编码如权利要求1所述天冬氨酸脱羧酶的基因。
3.如权利要求2所述的基因,其特征在于,碱基序列为SEQ ID NO:2。
4.包含如权利要求2或3所述基因的质粒。
5.转化了如权利要求4所述质粒的微生物。
6.如权利要求5所述的微生物,所述微生物选自大肠杆菌、酵母、枯草杆菌。
7.如权利要求6所述的微生物,其为大肠杆菌BL21(DE3)。
8.如权利要求1所述天冬氨酸脱羧酶或者如权利要求6所述微生物在生产β-丙氨酸中的用途。
9.如权利要求8所述的用途,其特征在于,以天冬氨酸为原料生产β-丙氨酸。
10.如权利要求9所述的用途,其特征在于,所述微生物是大肠杆菌BL21(DE3)。
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