CN112226398B - 一种高效生产戊二酸的重组大肠杆菌及其构建方法 - Google Patents
一种高效生产戊二酸的重组大肠杆菌及其构建方法 Download PDFInfo
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Abstract
本发明通过分子生物学手段从大肠杆菌Escherichia coli MG1655基因组中克隆获得赖氨酸脱羧酶基因cadA、丁二胺转氨酶基因patA以及γ‑氨基丁醛脱氢酶基因patD,从荧光假单胞菌Pseudomonas fluorescens基因组中克隆获得4‑氨基丁酸转氨酶基因gabT和琥珀酸半醛脱氢酶基因gabD。将构建好的重组表达载体pETM6R1‑cadA‑RBS01‑patA‑RBS02‑patD‑gabT‑gabD导入大肠杆菌E.coli W3110后,通过氨苄青霉素抗性平板筛选获得高效生产戊二酸的重组菌株Glu‑02。该重组菌株在5‑L发酵罐下采用补料分批发酵策略,发酵60h,戊二酸产量达到56.2g/L,转化率为62.2%。
Description
技术领域
本发明涉及一种高效生产戊二酸的重组大肠杆菌及其构建方法,属于代谢工程技术领域。
背景技术
戊二酸(Glutarate),俗名胶酸,是一种脂肪族二元羧酸,分子式是C5H8O4,分子量132.11,常温下为无色针状结晶固体,易溶于水、乙醇、***等,在水中溶解度可达430g/L。在所有二元羧酸中,戊二酸的熔点最低,为95-98℃,这一良好的特性使其更适合用于尼龙-4,5和尼龙-5,5等聚酯和聚酰胺的生产。此外,戊二酸也是1,5-戊二醇的前体,1,5-戊二醇是用作助焊剂、活化剂以及重要药物中间体的常用增塑剂。总之,戊二酸作为一种重要的C5平台化合物,在药物和化工合成等领域均具有重要的应用价值和发展潜力。
目前,戊二酸的主要合成方式是化学合成法,其中工业生产主要是在硝酸催化下从氧化环己酮和环己醇的混合物中回收利用,在实验室水平也可完成较少剂量的制备工作,比如可分别以γ-丁内酯、二氢吡喃、戊二腈、环己酮等作为底物,通过一系列化学反应制备而得。然而通过传统化学法合成戊二酸具有成本高、污染严重、操作条件要求高等缺点,因此寻找相对环保的生物法合成戊二酸对环境保护和高效生产以及未来戊二酸的生产前景都具有深远的意义。
近年来,国内外研究人员从生化工程和代谢工程两个方面对微生物生产戊二酸进行了探索研究。到目前为止,文献报道过的戊二酸的生物合成途径有以下四种,分别是:戊烯二酸还原途径、碳链延长与脱羧途径、反己二酸降解途径以及赖氨酸降解途径(包括以戊二胺为中间体的降解途径和以5-氨基戊酸为中间体的降解途径)。其中,赖氨酸降解途径的理论产率最高,达到0.75mol/mol葡萄糖。尤其是以戊二胺为中间体的赖氨酸降解途径由于不需要额外的氧气供应,具有更大的改造和生产潜力。进一步提高戊二酸产量成为亟待解决的问题,同时也是当前世界各国科研人员关注的焦点之一。
发明内容
为解决上述问题,本发明的目的是提供一株高产戊二酸的大肠杆菌工程菌,以及应用该工程菌生产戊二酸的方法。本发明通过在大肠杆菌细胞中过量表达合成戊二酸的路径酶赖氨酸脱羧酶、丁二胺转氨酶、γ-氨基丁醛脱氢酶、4-氨基丁酸转氨酶和琥珀酸半醛脱氢酶,随后通过改变RBS的强度精细化调控关键路径酶丁二胺转氨酶和γ-氨基丁醛脱氢酶的表达水平,进而实现了戊二酸的高效生产,为戊二酸的工业化生产奠定了基础。
本发明的第一个目的是提供一种高效生产戊二酸的重组大肠杆菌,所述重组大肠杆菌是在大肠杆菌宿主菌中过表达了赖氨酸脱羧酶cadA、丁二胺转氨酶patA、γ-氨基丁醛脱氢酶patD、4-氨基丁酸转氨酶gabT和琥珀酸半醛脱氢酶gabD;所述丁二胺转氨酶通过核苷酸序列如SEQ ID NO.6所示的核糖体结合位点与表达载体相连。
进一步地,所述赖氨酸脱羧酶的氨基酸序列如SEQ ID NO.1所示;丁二胺转氨酶的氨基酸序列如SEQ ID NO.2所示;γ-氨基丁醛脱氢酶的氨基酸序列如SEQ ID NO.3所示;4-氨基丁酸转氨酶的氨基酸序列如SEQ ID NO.4所示;琥珀酸半醛脱氢酶的氨基酸序列如SEQID NO.5所示。
进一步地,所述γ-氨基丁醛脱氢酶是通过核苷酸序列如SEQ ID NO.7所示的核糖体结合位点与表达载体相连。
进一步地,所述大肠杆菌宿主菌为大肠杆菌E.coli W3110、E.coli MG1655、E.coli B0013或E.coli ATCC 8739。
进一步地,所述表达载体为pETM6R1、pCDR或pCOR。
本发明的第二个目的是提供一种所述重组大肠杆菌的构建方法,包括如下步骤:
(1)获得赖氨酸脱羧酶cadA、丁二胺转氨酶patA、γ-氨基丁醛脱氢酶patD、4-氨基丁酸转氨酶gabT和琥珀酸半醛脱氢酶gabD的单基因表达载体;
(2)将步骤(1)得到的单基因表达载体采用ePathBrick技术对路径酶进行组装,得到重组质粒;
(3)将步骤(2)所得的重组质粒转化到大肠杆菌宿主中,得到大肠杆菌重组菌。
本发明的第三个目的是提供所述重组大肠杆菌在生产戊二酸中的应用。
进一步地,所述应用具体是将所述重组大肠杆菌按照5-10%的接种量接种至发酵培养基中进行培养,培养4-8h,添加0.1-0.4mmol/L的IPTG进行诱导,诱导温度为25-30℃;在发酵过程中,当溶氧急剧上升,开始进行葡萄糖补料,并每隔10-20h补加L-赖氨酸持续至发酵结束。
进一步地,所述发酵培养基包括如下组分:葡萄糖40-60g/L,蛋白胨15-25g/L,酵母提取物5-15g/L,十二水磷酸氢二铵2-3g/L,磷酸二氢铵0.5-1.5g/L,氯化钾0.1-0.2g/L,七水硫酸镁0.2-0.4g/L,和0.5-1.5mL微量金属溶液。
进一步地,所述微量金属溶液包括如下组分:六水氯化铁2-3g/L,六水氯化钴0.2-0.4g/L,氯化铜0.2-0.4g/L,四水氯化锌0.2-0.4g/L,高锰酸钠0.2-0.4g/L,硼酸0.07-0.08g/L,和四水氯化锰0.4-0.6g/L。
本发明的有益效果:
本发明通过改变RBS的强度精细化调控戊二酸合成途径中的关键路径酶丁二胺转氨酶patA和γ-氨基丁醛脱氢酶patD的表达水平,实现了该合成路径的代谢流平衡;最优重组菌株Glu-02在5-L发酵罐水平下发酵60h可产生56.2g/L戊二酸,转化率为62.2%。本发明方法在工业上用于提高戊二酸的产量具有极大的潜力和广泛的价值。
附图说明
图1所示为戊二酸生物合成路径示意图;
图2所示为不同强度RBS的基因表达载体构建示意图;
图3所示为10种重组菌株在摇瓶发酵中的5-氨基戊酸产量及转化率比较;
图4所示为最优重组菌株Glu-02在摇瓶水平补料分批发酵的戊二酸产量;
图5所示为最优重组菌株Glu-02在5-L发酵罐补料分批发酵的戊二酸产量。
具体实施方式
下面结合具体实施例对本发明作进一步说明,以使本领域的技术人员可以更好地理解本发明并能予以实施,但所举实施例不作为对本发明的限定。
实施例1:路径酶相关的单基因表达载体的构建
本发明所用的赖氨酸脱羧酶cadA(NCBI:NP_418555)、丁二胺转氨酶patA(NCBI:NP_417544)和γ-氨基丁醛脱氢酶patD(NCBI:NP_415961)基因来源于大肠杆菌MG1655,4-氨基丁酸转氨酶gabT(NCBI:AEV60208)和琥珀酸半醛脱氢酶gabD(NCBI:AEV60207)基因来源于荧光假单胞菌,如图1所示。大肠杆菌MG1655和荧光假单胞菌接种于25mL的LB液体培养基,在37℃,200rpm培养10h,收集菌体,并利用细菌基因组提取试剂盒提取大肠杆菌菌株和荧光假单胞菌菌株的基因组DNA。
根据已公布的基因组信息序列,分别设计各个路径酶相对应的引物,以上述提取的基因组DNA为模板,利用标准的PCR扩增体系和程序,扩增获得相应的cadA、patA、patD、gabT、gabD基因片段。质粒pETM6R1经过BamHI和XhoI双酶切后,采用琼脂糖核酸电泳进行胶回收,回收获得线性化的质粒pETM6R1。将上述PCR扩增获得的基因片段分别与双酶切后的质粒采用一步同源重组酶进行连接,体系为20μL,条件为37℃,30min。连接产物转化至JM109感受态细胞,挑取单菌落进行PCR验证,阳性转化子进行测序,测序结果与理论序列一致,则证明单基因表达载体构建成功,由此获得5个表达载体,分别为:pETM6R1-cadA、pETM6R1-patA、pETM6R1-patD、pETM6R1-gabT、pETM6R1-gabD。
实施例2:关键路径酶相关的不同强度基因表达载体的构建
将实施例1获得的质粒pETM6R1-patA和pETM6R1-patD经XbaI和BamHI双酶切,获得切除质粒pETM6R1上的原RBS序列的线性化质粒。分别设计RBS01-RBS03的扩增引物共3对,利用PCR仪扩增获得相应的RBS片段,条件为95℃,5min。扩增获得的RBS片段经磷酸化之后与线性化质粒通过T4DNA连接酶相连接,体系为10μL:7.5μL扩增片段,1μL双酶切载体,1μLbuffer,0.5μL T4DNAligase,16℃连接过夜,连接产物转化至JM109感受态细胞,挑取单菌落进行PCR验证,阳性转化子进行测序,测序结果与理论序列一致,则证明不同强度RBS的基因表达载体构建成功,由此获得6个表达载体,分别为:pETM6R1-RBS01-patA、pETM6R1-RBS02-patA、pETM6R1-RBS03-patA、pETM6R1-RBS01-patD、pETM6R1-RBS02-patD、pETM6R1-RBS03-patD。
实施例3:重组表达载体(pETM6R1-RBS01/RBS02/RBS03-patA-RBS01/RBS02/RBS03-patD)的构建
在实施例2构建成功的不同强度单基因表达载体基础上,采用ePathBrick技术对路径酶进行组装。以重组表达载体pETM6R1-RBS01-patA-RBS02-patD的构建为例进行步骤介绍。选取限制性内切酶SpeI和SalI双酶切载体pETM6R1-RBS01-patA以获得线性化的载体并暴露出粘性末端;再选取AvrII和SalI双酶切载体pETM6R1-RBS02-patD,随后进行胶回收,获得带有粘性末端的目的基因;最后经T4连接酶16℃连接过夜,连接产物转化至JM109感受态细胞,挑取单菌落进行PCR验证,条带大小正确,则表明重组表达载体pETM6R1-RBS01-patA-RBS02-patD成功构建,其余9种组合重组表达载体的构建方法均与上述保持一致。由此获得10个重组表达载体,如图2所示,分别为:pETM6R1-原RBS-patA-原RBS-patD、pETM6R1-RBS01-patA-RBS01-patD、pETM6R1-RBS01-patA-RBS02-patD、pETM6R1-RBS01-patA-RBS03-patD、pETM6R1-RB S02-patA-RBS01-patD、pETM6R2-RBS01-patA-RBS02-patD、pETM6R1-RBS02-patA-RBS03-patD、pETM6R1-RBS03-patA-RBS01-patD、pETM6R1-RBS03-patA-RBS02-patD和pETM6R1-RBS03-patA-RBS03-patD。将获得的10种质粒分别转化入大肠杆菌W3110内,获得重组大肠杆菌PW00-PW09。
实施例4:重组菌株PW00-PW09摇瓶发酵
挑取PW00-PW09共10株重组菌株进行摇瓶发酵。单克隆接种于50mL(250mL摇瓶)含有10mg/mL Amp的LB培养基作为摇瓶发酵的种子液,于37℃,200rpm培养10h。摇瓶发酵的接种量为5%,发酵条件为,温度30℃,转速200rpm,初始pH控制在7.0左右。发酵培养基为:葡萄糖50g/L,蛋白胨20g/L,酵母提取物10g/L,十二水磷酸氢二铵2.63g/L,磷酸二氢铵0.87g/L,氯化钾0.15g/L,七水硫酸镁0.37g/L,和1mL微量金属溶液(六水氯化铁2.4g/L,六水氯化钴0.3g/L,氯化铜0.3g/L,四水氯化锌0.3g/L,高锰酸钠0.3g/L,硼酸0.075g/L,四水氯化锰0.5g/L,溶于0.12M盐酸)。培养4h添加0.4mmol/L的IPTG进行诱导,诱导温度为30℃。同时添加30g/L L-赖氨酸作为底物。每隔7.5h取样一次测定OD600、残糖量、L-赖氨酸含量以及5-氨基戊酸产量。
根据5-氨基戊酸的产量测定,结果如图3所示,发酵72h,最优重组菌株PW02可产生18.6g/L 5-氨基戊酸,转化率为68.5%,较对照菌株产量提高了52.2%。
实施例5:重组表达载体(pETM6R1-cadA-RBS01-patA-RBS02-patD-gabT-gabD)的构建
在实施例1和实施例3构建成功的不同强度单基因表达载体的基础上,采用ePathBrick技术对路径酶进行组装。选取限制性内切酶SpeI和SalI双酶切载体pETM6R1-cadA以获得线性化的载体并暴露出粘性末端;再选取AvrII和SalI双酶切载体pETM6R1-RBS01-patA-RBS02-patD,随后进行胶回收,获得带有粘性末端的目的基因;最后经T4连接酶16℃连接过夜,连接产物转化至JM109感受态细胞,挑取单菌落进行PCR验证,条带大小正确,则表明重组表达载体pETM6R1-cadA-RBS01-patA-RBS02-patD成功构建。随后,再采用限制性内切酶SpeI和SalI双酶切上述重组表达载体载体;选取AvrII和SalI双酶切载体pETM6R1-gabT回收片段,同样经过T4连接构建表达载体pETM6R1-cadA-RBS01-patA-RBS02-patD-gabT。同理,最终可获得重组表达载体pETM6R1-cadA-RBS01-patA-RBS02-patD-gabT-gabD。将获得的载体转化入大肠杆菌W3110内,获得重组大肠杆菌Glu-02。
实施例6:最优重组菌株Glu-02在摇瓶水平进行补料分批发酵
挑取Glu-02重组菌株进行摇瓶发酵。单克隆接种于50mL(250mL摇瓶)含有10mg/mLAmp的LB培养基作为摇瓶发酵的种子液,于37℃,200rpm培养10h。摇瓶发酵的接种量为5%,发酵条件为,温度30℃,转速200rpm,初始pH控制在7.0左右。发酵培养基为:葡萄糖50g/L,蛋白胨20g/L,酵母提取物10g/L,十二水磷酸氢二铵2.63g/L,磷酸二氢铵0.87g/L,氯化钾0.15g/L,七水硫酸镁0.37g/L,和1mL微量金属溶液(六水氯化铁2.4g/L,六水氯化钴0.3g/L,氯化铜0.3g/L,四水氯化锌0.3g/L,高锰酸钠0.3g/L,硼酸0.075g/L,四水氯化锰0.5g/L,溶于0.12M盐酸);培养4h添加0.4mmol/L的IPTG进行诱导,诱导温度为30℃。每隔15h补加20g/L L-赖氨酸持续至发酵结束。每隔7.5h取样一次测定OD600、残糖量、L-赖氨酸含量以及戊二酸产量。
根据戊二酸的产量测定,结果如图4所示,发酵72h,重组菌株Glu-02可产生43.2g/L戊二酸,转化率为47.8%。
实施例7:最优重组菌株Glu-02在5L发酵罐进行补料分批发酵
挑取Glu-02重组菌株进行上罐发酵。单克隆接种于50mL(250mL摇瓶)含有10mg/mLAmp的LB培养基作为上罐的种子液,于37℃,200rpm培养10h。发酵罐装液量为2L,发酵培养基为:葡萄糖50g/L,蛋白胨20g/L,酵母提取物10g/L,十二水磷酸氢二铵2.63g/L,磷酸二氢铵0.87g/L,氯化钾0.15g/L,七水硫酸镁0.37g/L,和1mL微量金属溶液(六水氯化铁2.4g/L,六水氯化钴0.3g/L,氯化铜0.3g/L,四水氯化锌0.3g/L,高锰酸钠0.3g/L,硼酸0.075g/L,四水氯化锰0.5g/L,溶于0.12M盐酸);补料培养基为:L-赖氨酸和葡萄糖母液(800g/L);30℃下补料分批发酵。整个发酵过程中的pH控制在7.0左右。按照10%的接种量接种至发酵罐中,培养6-8h,添加0.4mmol/L的IPTG进行诱导,诱导温度为30℃。发酵过程中,当溶氧急剧上升,开始进行葡萄糖补料。每隔12h流加20g/L L-赖氨酸持续至发酵结束。每隔6h取样一次测定OD600、残糖量、L-赖氨酸含量以及戊二酸产量。
根据戊二酸的产量测定,结果如图5所示,发酵60h,重组菌株Glu-02可产生56.2g/L戊二酸,转化率为62.2%。
以上结果说明,本发明技术采用基因工程技术,改变RBS的强度精细化调控戊二酸合成路径中的关键酶丁二胺转氨酶patA和γ-氨基丁醛脱氢酶patD的表达水平能够有效提高戊二酸的产量。
以上所述实施例仅是为充分说明本发明而所举的较佳的实施例,本发明的保护范围不限于此。本技术领域的技术人员在本发明基础上所作的等同替代或变换,均在本发明的保护范围之内。本发明的保护范围以权利要求书为准。
序列表
<110> 江南大学
<120> 一种高效生产戊二酸的重组大肠杆菌及其构建方法
<160> 8
<170> PatentIn version 3.3
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Arg Leu Arg Ala Leu Cys Asp Gln His Gly Ile Leu Leu Ile Ala Asp
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Glu Val Gln Thr Gly Ala Gly Arg Thr Gly Thr Phe Phe Ala Met Glu
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Gln Met Gly Val Ala Ala Asp Leu Thr Thr Phe Ala Lys Ser Ile Ala
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Gly Gly Phe Pro Leu Ala Gly Val Cys Gly Lys Ala Glu Tyr Met Asp
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Ala Ile Ala Pro Gly Gly Leu Gly Gly Thr Tyr Ala Gly Ser Pro Ile
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Ala Cys Ala Ala Ala Leu Ala Val Met Glu Val Phe Glu Glu Glu His
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Leu Leu Asp Arg Cys Lys Ala Val Gly Glu Arg Leu Val Thr Gly Leu
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Lys Ala Ile Gln Lys Lys Tyr Pro Val Ile Gly Glu Val Arg Ala Leu
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Gly Ala Met Ile Ala Leu Glu Leu Phe Glu Asn Gly Asp Thr His Lys
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Pro Asn Ala Ala Ala Val Ala Gln Val Val Ala Lys Ala Arg Asp Lys
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Gly Leu Ile Leu Leu Ser Cys Gly Thr Tyr Gly Asn Val Leu Arg Val
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Ile Leu Glu Glu Cys Phe Ser Glu Leu
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Met Gln Leu Lys Asp Thr Gln Leu Phe Arg Gln Gln Ala Phe Ile Asp
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Arg Ala Leu Thr Ala Lys Glu Arg Ala Asn Lys Leu Arg Arg Trp Tyr
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Glu Leu Leu Ile Glu Asn Gln Asp Asp Leu Gly Arg Leu Met Thr Leu
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Asp Val Ile Pro Gly His Gln Pro Asp Lys Arg Leu Ile Val Ile Lys
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Val Thr Gly Ser Ala Gly Asp Ile Gly Gly Glu Leu Thr Ser Asn Pro
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Ile Val Arg Lys Leu Ser Phe Thr Gly Ser Thr Glu Ile Gly Arg Gln
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Leu Met Ala Glu Cys Ala Lys Asp Ile Lys Lys Val Ser Leu Glu Leu
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Gly Gly Asn Ala Pro Phe Ile Val Phe Asp Asp Ala Asp Leu Asp Lys
260 265 270
Ala Val Glu Gly Ala Ile Ile Ser Lys Tyr Arg Asn Asn Gly Gln Thr
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Cys Val Cys Ala Asn Arg Leu Tyr Ile Gln Asp Ser Val Tyr Asp Ala
290 295 300
Phe Ala Glu Lys Leu Lys Val Ala Val Ala Lys Leu Lys Ile Gly Asn
305 310 315 320
Gly Leu Asp Asp Gly Thr Thr Thr Gly Pro Leu Ile Asp Gly Lys Ala
325 330 335
Val Ala Lys Val Gln Glu His Ile Ala Asp Ala Val Ser Lys Gly Ala
340 345 350
Thr Val Leu Ser Gly Gly Lys Ala Met Glu Gly Asn Phe Phe Glu Pro
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Thr Ile Leu Thr Asn Val Pro Lys Asn Ala Ala Val Ala Lys Glu Glu
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Thr Phe Gly Pro Leu Ala Pro Leu Phe Arg Phe Lys Asp Glu Ala Glu
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Val Ile Ala Met Ser Asn Asp Thr Glu Phe Gly Leu Ala Ser Tyr Phe
405 410 415
Tyr Ala Arg Asp Leu Gly Arg Val Phe Arg Val Ala Glu Ala Leu Glu
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Pro Phe Gly Gly Ile Lys Ala Ser Gly Leu Gly Arg Glu Gly Ser Lys
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<210> 6
<211> 12
<212> DNA
<213> (人工序列)
<400> 6
aaagaggaga aa 12
<210> 7
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<212> DNA
<213> (人工序列)
<400> 7
tcacacagga aag 13
<210> 8
<211> 10
<212> DNA
<213> (人工序列)
<400> 8
agggacagga 10
Claims (9)
1.一种高效生产戊二酸的重组大肠杆菌,其特征在于,所述重组大肠杆菌是在大肠杆菌宿主菌中过表达了赖氨酸脱羧酶cadA、丁二胺转氨酶patA、γ-氨基丁醛脱氢酶patD、4-氨基丁酸转氨酶gabT和琥珀酸半醛脱氢酶gabD;
所述赖氨酸脱羧酶的氨基酸序列如SEQ ID NO.1所示;丁二胺转氨酶的氨基酸序列如SEQ ID NO.2所示;γ-氨基丁醛脱氢酶的氨基酸序列如SEQ ID NO.3所示;4-氨基丁酸转氨酶的氨基酸序列如SEQ ID NO.4所示;琥珀酸半醛脱氢酶的氨基酸序列如SEQ ID NO.5所示。
2.根据权利要求1所述的重组大肠杆菌,其特征在于,所述丁二胺转氨酶通过核苷酸序列如SEQ ID NO.6所示的核糖体结合位点与表达载体相连,所述γ-氨基丁醛脱氢酶是通过核苷酸序列如SEQ ID NO.7所示的核糖体结合位点与表达载体相连。
3.根据权利要求2所述的重组大肠杆菌,其特征在于,所述大肠杆菌宿主菌为大肠杆菌E. coli W3110、E. coli MG1655、E. coli B0013或E. coli ATCC 8739。
4.根据权利要求2所述的重组大肠杆菌,其特征在于,所述表达载体为pETM6R1、pCDR或pCOR。
5.一种权利要求1-4任一项所述重组大肠杆菌的构建方法,其特征在于,包括如下步骤:
(1)获得赖氨酸脱羧酶cadA、丁二胺转氨酶patA、γ-氨基丁醛脱氢酶patD、4-氨基丁酸转氨酶gabT和琥珀酸半醛脱氢酶gabD的单基因表达载体;
(2)将步骤(1)得到的单基因表达载体采用ePathBrick技术对路径酶进行组装,得到重组质粒;
(3)将步骤(2)所得的重组质粒转化到大肠杆菌宿主中,得到大肠杆菌重组菌。
6.权利要求1-4任一项所述重组大肠杆菌在生产戊二酸中的应用。
7.根据权利要求6所述的应用,其特征在于,所述应用具体是将所述重组大肠杆菌按照5-10%的接种量接种至发酵培养基中进行培养,培养4-8 h,添加0.1-0.4 mmol/L的IPTG进行诱导,诱导温度为25-30 ℃;在发酵过程中,当溶氧急剧上升,开始进行葡萄糖补料,并每隔10-20h补加L-赖氨酸持续至发酵结束。
8.根据权利要求7所述的应用,其特征在于,所述发酵培养基包括如下组分:葡萄糖40-60 g/L,蛋白胨15-25 g/L,酵母提取物5-15 g/L,十二水磷酸氢二铵2-3 g/L,磷酸二氢铵0.5-1.5 g/L,氯化钾0.1-0.2 g/L,七水硫酸镁0.2-0.4 g/L,和0.5-1.5 mL 微量金属溶液。
9.根据权利要求8所述的应用,其特征在于,所述微量金属溶液包括如下组分:六水氯化铁2-3 g/L,六水氯化钴0.2-0.4 g/L,氯化铜0.2-0.4 g/L,四水氯化锌0.2-0.4 g/L,高锰酸钠0.2-0.4 g/L,硼酸0.07-0.08 g/L,和四水氯化锰0.4-0.6 g/L。
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