CN110669713A - 一种合成d-柠檬烯的基因工程菌及其构建方法与应用 - Google Patents
一种合成d-柠檬烯的基因工程菌及其构建方法与应用 Download PDFInfo
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Abstract
一种合成D‑柠檬烯的基因工程菌及其构建方法与应用,涉及基因工程及发酵工程领域,本发明在所述基因工程菌中过表达HMG‑CoA合成酶基因mvaS、乙酰CoA乙酰基转移酶mvaE、牦牛儿基焦磷酸合成酶GPPS基因、D‑柠檬烯合成酶基因ClLS、MVA激酶ERG12基因、MVAP激酶ERG8基因、甲羟戊酸脱羧酶ERG19基因、异戊焦磷酸异构酶IDI基因。在LB培养基中摇瓶发酵后,能够异源合成D‑柠檬烯,D‑柠檬烯的产量27.3mg/L发酵液。本发明可用于D‑柠檬烯工业化生产。
Description
技术领域
本发明涉及基因工程及发酵工程领域,具体涉及一种合成D-柠檬烯的基因工程菌及其构建方法与应用。
背景技术
柠檬烯又称苧烯、苎烯,属于单环萜类化合物,化学式C10H16,是一种来源植物的天然化合物。它主要存在于柑橘类(如柠檬、橙子、柑橘等)的果皮和果肉中,并且已被美国食用香料与提取物制造商协会(FEMA)认定其毒性属GRAS级(一般公认安全),并已被FDA批准食用(刘树文.合成香料技术手册[M].2009.)
在农业上,D-柠檬烯还被用作农作物的生物杀虫剂。在卫生医疗领域中,D-柠檬烯具有抑菌、抗菌活性,可以作为良好的天然抗菌活性物质;同时,它还具有很好的药用价值,如具有抗癌活性、促进消化和减肥作用等(Johnson T J,Jahandideh A,Johnson M D,etal.Producing next-generation biofuels from filamentous cyanobacteria:Aneconomic feasibility analysis[J].Algal Research,2016,20:218-228.Ciriminna R,Lomeli-Rodriguez M,Demma C P,et al.Limonene:a versatile chemical of thebioeconomy[J].Chemical Communications,2014,50(97):15288-15296.Jongedijk E,Cankar K,Buchhaupt M,et al.Biotechnological production of limonene inmicroorganisms[J].Applied Microbiology and Biotechnology,2016,100(7):2927-2938.)柠檬烯还可以作为多种具有重要应用价值的单萜类衍生物如紫苏醇、薄荷醇、松油醇、香芹醇、香芹酮的前体物(Leonard E,Ajikumar P K,Thayer K,et al.Combiningmetabolic and protein engineering of a terpenoid biosynthetic pathway foroverproduction and selectivity control[J].Proceedings of the National Academyof Sciences of the United States of America,2010,107(31):13654-13659.)
D-柠檬烯具有重要的生理功能和很高的应用价值,但由于大量提取D-柠檬烯受来源与工艺的限制,合成生物学为更高效地获取D-柠檬烯提供了新思路。目前关于利用微生物代谢工程技术生产D-柠檬烯的并不多见,主要是有关L-柠檬烯的生物合成报道。2004年,Shimada等从蜜柑Citrusunshiu中分离得到了一个D-柠檬烯合酶基因(GenBank AccessionNo.AB110636.1)[从柑桔中分离到3个新的单萜合酶基因cDNA克隆,分别为CitMTSE1、CitMTS61和CitMTS62。体外翻译功能测试表明,CitMTSE1、CitMTS61和CitMTS62分别编码的D-柠檬烯合成酶、γ-萜烯合成酶和β-蒎烯合成酶。(Shimada T,Endo T,Fujii H,etal.Molecular cloning and functional characterization of four monoterpenesynthase genes from Citrus unshiu Marc.[J].Plant Science(Oxford),2004,166(1):0-58.)2015年,Beekwilder研究组在酿酒酵母中分别引入一个来自紫苏的L-柠檬烯合酶基因和一个来自柠檬的D-柠檬烯合酶基因,再加上一个突变的香叶基焦磷酸合成酶基因,成功构建了L-柠檬烯和D-柠檬烯的合成途径。采用几种方法对酵母生产的柠檬烯进行了捕获。结果表明,表达柠檬烯合酶的菌株在培养过程中添加正十二烷,通过顶空收集的方式,分别为0.12mg/L D-柠檬烯和0.49mg/L L-柠檬烯(Jongedijk E,Cankar K,Ranzijn J,etal.Capturing of the monoterpene olefin limonene produced in\r,Saccharomycescerevisiae[J].Yeast,2014,32(1):159-171)。上述报道的D-柠檬烯生物合成水平较低,制约了D-柠檬烯的工业化生产。
发明内容
为了提高D-柠檬烯的合成水平,本发明提供了一种合成D-柠檬烯的基因工程菌,所述基因工程菌表达HMG-CoA合成酶基因mvaS、乙酰CoA乙酰基转移酶mvaE、牦牛儿基焦磷酸合成酶GPPS基因、D-柠檬烯合成酶基因ClLS、MVA激酶ERG12基因、MVAP激酶ERG8基因、甲羟戊酸脱羧酶ERG19基因、异戊焦磷酸异构酶IDI基因,宿主菌为大肠杆菌。
进一步地限定,所述HMG-CoA合成酶基因mvaS,GeneBank ID为AAG02439,来源于粪肠球菌E.facecalis;乙酰CoA乙酰基转移酶mvaE,GenBank NO.AAG02438,来源于粪肠球菌E.facecalis;牦牛儿基焦磷酸合成酶GPPS基因,Genbank No.AF513112.1,来源于巨冷衫Abies grandis;D-柠檬烯合成酶ClLS基因,Genbank No.AF514287.1,来源于柠檬Citruslimon;MVA激酶ERG12基因,GeneBank ID:855248,来源Saccharomyces cerevisiae;MVAP激酶ERG8基因,GeneBank ID:855260,来源Saccharomyces cerevisiae、甲羟戊酸脱羧酶ERG19基因,GeneBank ID:855779,来源Saccharomyces cerevisiae、异戊焦磷酸异构酶IDI基因,GeneBank ID:855986,来源Saccharomyces cerevisiae。
进一步地限定,所述大肠杆菌为E.coli BL21(DE3)。
本发明还提供了上述基因工程菌的构建方法,步骤如下:
1)构建包含mvaS、mvaE、GPPS、ClLS基因的重组质粒I和包含ERG12、ERG8、ERG19、IDI基因的重组质粒II;
2)将步骤1)构建的重组质粒I和重组质粒II转化入宿主菌,得到合成D-柠檬烯的基因工程菌。
进一步地限定,所述重组质粒I构建所用的中间载体为pET28a(+);所述重组质粒II构建所用的中间载体为pTrcHis2B。
本发明还提供了上述基因工程菌在合成D-柠檬烯中的应用。
进一步地限定,所述合成D-柠檬烯是指将含有上述基因工程菌的种子液接种至发酵培养基中,培养至OD600为0.6-1.0,然后加入诱导剂IPTG,以及正十二烷或邻苯二甲酸二异壬酯中的一种,继续发酵,将发酵液离心后,上清液过滤得到D-柠檬烯。
进一步地限定,所述种子液接种至发酵培养基中培养条件为:培养温度为30-37℃,搅拌速度为220rpm,pH6.0-8.0。
进一步地限定,所述IPTG在发酵液中的终浓度为0.2mmol/L;所述正十二烷或邻苯二甲酸二异壬酯在发酵液中的终浓度为5%-10%(质量体积比)。
进一步地限定,所述继续发酵的时间为60-72h。
有益效果
1、本发明导入了D-柠檬烯合成酶基因首次构建异源合成柠檬烯的大肠杆菌基因工程菌,其生物合成途径见图1,筛选得到菌株生存环境广泛,对发酵设备无特殊要求,具有广泛应用前景,为柠檬烯产业化生产奠定基础。
2、本发明获得的大肠杆菌基因工程菌:在LB培养基中摇瓶发酵后,能够合成D-柠檬烯,发酵72h,产量达到27.3mg/L发酵液,而现有技术通过在酿酒酵母中表达,通过顶空收集法,合成D-柠檬烯产量仅有0.12mg/L(Jongedijk E,Cankar K,Ranzijn J,etal.Capturing of the monoterpene olefin limonene produced in\r,Saccharomycescerevisiae[J].Yeast,2014,32(1):159-171)。
附图说明
图1 D-柠檬烯生物合成路径图;
图2 pET28a-mvaS-mvaE-GPPS-ClLS重组质粒结构示意图。
具体实施方式
定义和缩写
在本文中使用下列的缩写或简称:
异丙基硫代半乳糖苷:IPTG
HMG-CoA合成酶基因:mvaS
乙酰CoA乙酰基转移酶基因:mvaE
牦牛儿基焦磷酸合成酶基因:GPPS
D-柠檬烯合成酶基因:ClLS
MVA激酶ERG12基因:甲羟戊酸激酶基因ERG12
MVAP激酶ERG8基因:甲羟戊酸-5-磷酸激酶基因ERG8
甲羟戊酸脱羧酶基因:ERG19
异戊焦磷酸异构酶基因:IDI
大肠埃希氏杆菌(Escherichia coli):E.coli
“过量表达”或“过表达”指细胞内特定基因受到各种信号调控后,在生物体中超过原有水平表达,可以通过增强内源表达或引入外源基因来实现。
下面通过实例来详细阐明本发明。但本发明并不限于以下实施例。
下述实施例中所涉及的实验方法若无特殊说明,均为常规技术。
下述实施例中所使用的材料、试剂等若无特殊说明,均可从商业途径获得。所用无缝的同源重组酶均购自诺唯赞生物技术公司,质粒提取及胶回收所用试剂盒购自美国OMEGA公司,操作步骤按照产品说明书进行;所有培养基如无特别说明均用去离子水配制。
1)LB种子液培养基:酵母粉5g/L,NaCl 10g/L,蛋白胨10g/L,接种时补加卡那霉素50μg/mL。
2)发酵培养基:K2HPO4·3H2O 9.8g/L,Citric acid·H2O 2.1g/L,柠檬酸铁铵0.3g/L,(NH4)2SO4 3.0g/L,葡萄糖24g/L,MgSO4·7H2O 0.4g/L,1000×微量元素((NH4)6Mo7O24·4H2O 3.7g/L;ZnSO4·7H2O 2.9g/L;H3BO3 24.7g/L;CuSO4·5H2O 2.5g/L;MnCl2·4H2O 15.8g/L),卡那霉素50μg/mL。
其中:K2HPO4·3H2O 9.8g/L,Citric acid·H2O 2.1g/L,柠檬酸铁铵0.3g/L,(NH4)2SO4 3.0g/L混合后调至pH 7.0,121℃,20min高压蒸气灭菌。葡萄糖储液为500g/L,115℃,20min单独灭菌,MgSO4·7H2O储液为200g/L,121℃,20min单独灭菌,1000×微量元素采用0.22μm滤菌膜过滤除菌,转接种子液时分别补加上述单独除菌的葡萄糖、MgSO4·7H2O、1000×微量元素储液及抗生素。
pGH-mvaS、pGH-mvaE、pGH-GPPS、pUC57-ClLS,通过商业化公司根据相应的目的基因序列合成到克隆载体pGH-T中获得,pET28a(+)载体通过商业化途径购买获得。
实施例1.合成D-柠檬烯的基因工程菌的构建。
本实施例所述的合成D-柠檬烯的基因工程菌,表达HMG-CoA合成酶基因mvaS、乙酰CoA乙酰基转移酶mvaE、牦牛儿基焦磷酸合成酶GPPS基因、D-柠檬烯合成酶基因ClLS、MVA激酶ERG12基因、MVAP激酶ERG8基因、甲羟戊酸脱羧酶ERG19基因、异戊焦磷酸异构酶IDI基因,宿主菌为大肠杆菌。所述HMG-CoA合成酶基因mvaS,GeneBank ID为AAG02439,来源于粪肠球菌E.facecalis;乙酰CoA乙酰基转移酶mvaE,GenBank NO.AAG02438,来源于粪肠球菌E.facecalis;牦牛儿基焦磷酸合成酶GPPS基因,Genbank No AF5131121,来源于巨冷衫Abies grandis;D-柠檬烯合成酶ClLS基因,Genbank No.AF514287.1,来源于柠檬Citruslimon;MVA激酶ERG12基因,GeneBank ID:855248,来源Saccharomyces cerevisiae;MVAP激酶ERG8基因,GeneBank ID:855260,来源Saccharomyces cerevisiae、甲羟戊酸脱羧酶ERG19基因,GeneBank ID:855779,来源Saccharomyces cerevisiae、异戊焦磷酸异构酶IDI基因,GeneBank ID:855986,来源Saccharomyces cerevisiae。
具体构建方法如下:
(一)质粒构建:
1.构建重组质粒I:pET28a-mvaS-mvaE-GPPS-ClLS
表1本实例用到的引物
引物名称 | 序列(5’-3’) |
mvaE-F | <u>tcatcaccacagccaggatcc</u>GGATCCAGGAGGTAAAAAAACATGAAAACAGTA |
mvaE-R | <u>cctcctggtatatctcctt</u>TTATTGTTTTCTTAAATCATTTAAAATAGCC |
mvaS-F | <u>AAAGGAGATATAC</u>CAGGAGGTAAAAAAA |
mvaS-R | <u>acctgcaggcgcgccgagctc</u>GAGCTCTTAGTTTCGATAAGAGCGAACGG |
GPPS-F-Bgl II | <u>agatatacatatggcagatct</u>AGATCTATGGAATTCGACTTCAACAAATACA |
GPPS-R | <u>gccgatatccaattgagatc</u>tTTAGTTCTGACGGAAAGCAACG |
ClLS-5Nae I | <u>aacaattggatatcggccggc</u>GCCGGCAAGGAGATATACCATGAGTAGCTGTATTAACC |
ClLS-3Xho I | <u>ggtttctttaccagactcgag</u>CTCGAGTTAACCTTTGGTGCCCGGA |
划线部分为同源臂。
1)分别以mvaE-F、mvaE-R和mvaS-F、mvaS-R为引物,以质粒pGH-mvaS,pGH-mvaE为模板,分别扩增mvaE及mvaS基因,诺唯赞的无缝克隆试剂盒(ClonExpress Ultra One StepCloning Kit C115-01)中的重组酶exnase的作用下,利用同源臂完成mvaE、mvaS和pET28a(+)连接,构建出pET28a-mvaS-mvaE。
2)以GPPS-F和GPPS-R为引物,以质粒pGH-GPPS为模板,扩增GPPS基因,在诺唯赞的无缝克隆试剂盒(ClonExpress Ultra One Step Cloning Kit C115-01)中的重组酶exnase的作用下,利用同源臂完成GPPS和pET28a-mvaS-mvaE的连接,构建出pET28a-mvaS-mvaE-GPPS。
3)以ClLS-5Nae I和ClLS-3Xho I为引物,以质粒pUC57-ClLS为模板,扩增ClLS基因,在诺唯赞的无缝克隆试剂盒(ClonExpress Ultra One Step Cloning Kit C115-01)中的重组酶exnase的作用下,利用同源臂完成ClLS和pET28a-mvaS-mvaE-GPPS的连接,构建出pET28a-mvaS-mvaE-GPPS-ClLS,其重组质粒结构图见图2。
2.构建重组质粒II:pYJM14
本发明所用的重组质粒II为重组质粒pYJM14,包含MVA激酶ERG12基因、MVAP激酶ERG8基因、甲羟戊酸脱羧酶ERG19基因、异戊焦磷酸异构酶IDI基因,该质粒记载在Yang J,Xian M,Su S,et al.Enhancing production of bio-isoprene using hybrid MVApathway and isoprene synthase in E.coli[J].Plos One,2012.详细构建过程参见该文章,公众可通过中国科学院青岛生物能源与过程研究所获得该质粒。
(二)制备合成D-柠檬烯的基因工程菌
按照TAKARA感受态制备试剂盒的操作步骤制备E.coli BL21(DE3)的感受态细胞,将步骤1得到的重组质粒pET28a-mvaS-mvaE-GPPS-ClLS和和步骤2得到的pYJM14质粒通过热激法共同转化至上述制备的感受态细胞,获得重组菌株。
实施例2.利用基因工程菌株发酵生产D-柠檬烯。
摇瓶发酵实验:
1)一级种子液的培养:在LB种子液培养基中分别接种固体LB平板上的实施例1得到重组菌株单菌落,并加入终浓度为50μg/mL卡那霉素和100μg/ml氨苄青霉素,37℃生长12h,得到一级种子液。
2)将步骤1)得到的一级种子液分别按2%(wt)接种量转接至250mL盐水瓶中,含50mL发酵培养基,转接种子液时各补加200g/L的MgSO4·7H2O 100μL、500g/L葡萄糖2.4mL、1000×微量元素50μL((NH4)6MoO24·4H2O 0.37g;ZnSO4·7H2O 0.29g;H3BO4 2.47g;CuSO4·5H2O 0.25g;MuCl2·4H2O 1.58g;用蒸馏水定容到100mL)、终浓度50μg/mL卡那霉素和100μg/ml氨苄青霉素,37℃、180rpm培养。
3)细胞OD600达到0.6-1.0间加入终浓度200μmol/L的IPTG诱导,并加入10%(质量体积分数)正十二烷。诱导后,30℃、180rpm继续培养72h发酵结束。
4)发酵后收集菌液,7500rpm,4℃离心5min,取有机膜过滤,气相色谱-质谱联用备用检测其含量,利用Cyclosil-B手性色谱柱检测其产物结构为D-柠檬烯,经气质检测,发酵72h,产D-柠檬烯异源合成柠檬烯的产量为27.3mg/L发酵液。
气相-质谱联用检测条件:
色谱条件:色谱柱为DB_5MS(30m×0.320mm×0.25um);检测器为氢火焰离子检测器;分析条件如下:柱温50℃保持0min,然后以10℃/min,从50℃升温到250℃,保持10min,汽化温度250℃,检测器温度260℃。
扫描质量数:40-500;离子源温度230度,四级杆150度。
SIM:扫描荷质比68、93m/z
气相色谱检测手性检测条件:
色谱柱:Cyclosil-B(112-6632)
进样量:1μL
进样口:250℃;分流模式,分流比20:1
柱流量:1mL/min,氮气为载气
柱箱:初始50℃,以2℃/min速率,升到190℃(最终温度可以适当降低以缩短采集时间z)
检测器(FID):250℃,空气300mL/min,氢气30mL/min,尾吹气+柱流量25mL/min。
实施例3.利用基因工程菌株发酵生产D-柠檬烯。
摇瓶发酵实验:
1)一级种子液的培养:在LB种子液培养基中分别接种固体LB平板上的实施例3得到重组菌株单菌落,并加入终浓度为50μg/mL卡那霉素和100μg/ml氨苄青霉素,37℃生长12h,得到一级种子液。
2)将步骤1)得到的一级种子液分别按2%(wt)接种量转接至250mL盐水瓶中,含50mL发酵培养基,转接种子液时各补加200g/L的MgSO4·7H2O 100μL、500g/L葡萄糖2.4mL、1000×微量元素50μL((NH4)6MoO24·4H2O 0.37g;ZnSO4·7H2O 0.29g;H3BO4 2.47g;CuSO4·5H2O 0.25g;MuCl2·4H2O 1.58g;用蒸馏水定容到100mL)、终浓度50μg/mL卡那霉素和100μg/ml氨苄青霉素,37℃、180rpm培养。
3)细胞OD600达到0.6-1.0间加入终浓度200μmol/L的IPTG诱导,并加入10%(质量体积分数)DINP(邻苯二甲酸二异壬酯)。诱导后,30℃、180rpm继续培养72h发酵结束
4)发酵后收集菌液,7500rpm,4℃离心5min,取有机膜过滤,气相色谱-质谱联用备用检测其含量,发酵72h,产D-柠檬烯异源合成柠檬烯的产量为10.2mg/L发酵液。
气相-质谱联用检测条件:
色谱条件:色谱柱为DB_5MS(30m×0.320mm×0.25um);检测器为氢火焰离子检测器;分析条件如下:柱温50℃保持0min,然后以10℃/min,从50℃升温到250℃,保持10min,汽化温度250℃,检测器温度260℃。
扫描质量数:40-500;离子源温度230度,四级杆150度。
SIM:扫描荷质比68、93m/z。
本实施例所列数据为多次重复试验的平均数值。虽然本发明已经公开了示例性示范方案,但是本领域人员应该理解,在不背离由后附的权利要求所定义的本发明的精神和范围的条件下,可以进行各种形式和细节的变化,可以进行各种实施方案的任意组合。
核苷酸序列表
<110> 中国科学院青岛生物能源与过程研究所
<120> 一种合成D-柠檬烯的基因工程菌及其构建方法与应用
<130>
<160> 8
<170> PatentIn version 3.5
<210> 1
<211> 54
<212> DNA
<213> mvaE-F
<400> 1
tcatcaccac agccaggatc cggatccagg aggtaaaaaa acatgaaaac agta 54
<210> 2
<211> 50
<212> DNA
<213> mvaE-R
<400> 2
cctcctggta tatctccttt tattgttttc ttaaatcatt taaaatagcc 50
<210> 3
<211> 28
<212> DNA
<213> mvaS-F
<400> 3
aaaggagata taccaggagg taaaaaaa 28
<210> 4
<211> 50
<212> DNA
<213> mvaS-R
<400> 4
acctgcaggc gcgccgagct cgagctctta gtttcgataa gagcgaacgg 50
<210> 5
<211> 52
<212> DNA
<213> GPPS-F-Bgl II
<400> 5
agatatacat atggcagatc tagatctatg gaattcgact tcaacaaata ca 52
<210> 6
<211> 43
<212> DNA
<213> GPPS-R
<400> 6
gccgatatcc aattgagatc tttagttctg acggaaagca acg 43
<210> 7
<211> 59
<212> DNA
<213> ClLS-5Nae I
<400> 7
aacaattgga tatcggccgg cgccggcaag gagatatacc atgagtagct gtattaacc 59
<210> 8
<211> 46
<212> DNA
<213> ClLS-3Xho I
<400> 8
ggtttcttta ccagactcga gctcgagtta acctttggtg cccgga 46
Claims (10)
1.一种合成D-柠檬烯的基因工程菌,其特征在于,所述基因工程菌表达HMG-CoA合成酶基因mvaS、乙酰CoA乙酰基转移酶mvaE、牦牛儿基焦磷酸合成酶GPPS基因、D-柠檬烯合成酶基因ClLS、MVA激酶ERG12基因、MVAP激酶ERG8基因、甲羟戊酸脱羧酶ERG19基因、异戊焦磷酸异构酶IDI基因,宿主菌为大肠杆菌。
2.根据权利要求1所述的基因工程菌,其特征在于,所述HMG-CoA合成酶基因mvaS,GeneBank ID为AAG02439,来源于粪肠球菌E.facecalis;乙酰CoA乙酰基转移酶mvaE,GenBank NO.AAG02438,来源于粪肠球菌E.facecalis;牦牛儿基焦磷酸合成酶GPPS基因,Genbank No.AF513112.1,来源于巨冷衫Abies grandis;D-柠檬烯合成酶ClLS基因,Genbank No.AF514287.1,来源于柠檬Citrus limon;MVA激酶ERG12基因,GeneBank ID:855248,来源Saccharomyces cerevisiae;MVAP激酶ERG8基因,GeneBank ID:855260,来源Saccharomyces cerevisiae、甲羟戊酸脱羧酶ERG19基因,GeneBank ID:855779,来源Saccharomyces cerevisiae、异戊焦磷酸异构酶IDI基因,GeneBank ID:855986,来源Saccharomyces cerevisiae。
3.根据权利要求1所述的基因工程菌,其特征在于,所述大肠杆菌为E.coli BL21(DE3)。
4.权利要求1-3任意一项所述的基因工程菌的构建方法,其特征在于,步骤如下:
1)构建包含mvaS、mvaE、GPPS、ClLS基因的重组质粒I和包含ERG12、ERG8、ERG19、IDI基因的重组质粒II;
2)将步骤1)构建的重组质粒I和重组质粒II转化入宿主菌,得到合成D-柠檬烯的基因工程菌。
5.根据权利要求4所述的构建方法,其特征在于,所述重组质粒I构建所用的中间载体为pET28a(+);所述重组质粒II构建所用的中间载体为pTrcHis2B。
6.权利要求1-3任意一项所述的基因工程菌在合成D-柠檬烯中的应用。
7.根据权利要求6所述的应用,其特征在于,所述合成D-柠檬烯是指将含有权利要求-3任意一项所述的基因工程菌的种子液接种至发酵培养基中,培养至OD600为0.6-1.0,然后加入诱导剂IPTG,以及正十二烷或邻苯二甲酸二异壬酯中的一种,继续发酵,将发酵液离心后,上清液过滤得到D-柠檬烯。
8.根据权利要求7所述的应用,其特征在于,所述种子液接种至发酵培养基中培养条件为:培养温度为30-37℃,搅拌速度为220rpm,pH6.0-8.0。
9.根据权利要求7所述的应用,其特征在于,所述IPTG在发酵液中的终浓度为0.2mmol/L;所述正十二烷或邻苯二甲酸二异壬酯在发酵液中的终浓度为5%-10%(质量体积比)。
10.根据权利要求7所述的应用,其特征在于,所述继续发酵的时间为60-72h。
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