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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2018, Vol. 38 Issue (2): 46-53    DOI: 10.13523/j.cb.20180207
研究报告     
重组大肠杆菌转化甘油合成聚3-羟基丙酸-co-乳酸 *
赵志强1,2*,LacmataTamekouStephen1,3*,咸漠1,刘修涛1,2,冯新军1**(),赵广1**()
1 中国科学院青岛生物能源与过程研究所 生物基材料重点实验室 青岛 266101
2 中国科学院大学 北京 100049
3 喀麦隆德尚大学 德尚 999108
Biosynthesis of Poly (3-hydroxypropionate-co-lactate) from Glycerol by Engineered Escherichia coli
Zhi-qiang ZHAO1,2*,Tamekou Stephen LACMATA1,3*,Mo XIAN1,Xiu-tao LIU1,2,Xin-jun FENG1**(),Guang ZHAO1**()
1 CAS Key Laboratory of Biobased Materials, Qingdao Institute of Bioenergy and Bioprocess Technology,Chinese Academy of Sciences, Qingdao 266101, China
2 University of Chinese Academy of Sciences, Beijing 100049, China
3 Department of Biochemistry, University of Dschang, Dschang 999108, Cameroon
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摘要：

聚羟基脂肪酸酯作为性质优良的生物塑料,引起了广泛的关注。由于聚羟基脂肪酸合成酶PhaC特异性较强,难以通过生物合成方法获得含乳酸单体聚合物。为了实现乳酸的聚合,PhaC的筛选至关重要。以甘油为底物,通过引入Klebsiella pneumoniae的甘油脱水酶DhaB123及其激活因子GdrAB以及Salmonella typhimurium LT2的丙醛脱氢酶基因PduP,获得3-羟基丙酰辅酶A;通过引入Megasphaera elsdenii DSM 20460的丙酰辅酶A转移酶PCT,获得乳酰辅酶A;并对3种不同聚羟基脂肪酸合成酶的作用进行考察。在Pseudomonas putida的原始酶PhaC1或者PhaC2的作用下,不能实现乳酸的聚合;而在双位点突变(Ser325Thr和Gln481Lys)的PhaC1(STQK)存在条件下,重组菌可以利用甘油合成聚3-羟基丙酸-co-乳酸。经过对溶氧、有机氮源等发酵条件的优化,聚3-羟基丙酸-co-乳酸的产量可以达到0.22g/L,占细胞干重的3.2%,是含乳酸单体聚合物生物合成研究的一次有益尝试。
关键词： 聚3-羟基丙酸-co-乳酸;  聚羟基脂肪酸合成酶;  丙酰辅酶A转移酶;  位点突变;  甘油    
Abstract:

Polyhydroxyalkanoates (PHAs) have received considerable attention because of their material properties and wide applications for packaging, biofuels, and biomedicine.Polylactate (PLA) isarepresentative bio-based polyester, whichis chemically synthesized rather than synthesized bybacteria, because of the substrate specificity of PHA synthase (PhaC). To produce lactate-based polyesters, itiscriticalto obtain a PhaC capable of LA-polymerization. Glycerol dehydratase DhaB123 and its reactivating factorGdrAB from Klebsiella pneumoniae, and propionaldehyde dehydrogenase PduP from Salmonella typhimurium LT2 were overexpressed to convert glycerol into 3-hydroxypropionyl-CoA;propionyl-CoA transferase (PCT) from Megasphaera elsdenii DSM 20460were used to obtain lactyl-CoA; and three different PhaC were introduced and examined for poly (3-hydroxypropionate-co-lactate) (P(3HP-co-LA)). The wild type enzyme PhaC1, PhaC2 from Pseudomonas putida can’t polymerize lactyl-CoA into polymer. Only with double mutant (Ser325Thr and Gln481Lys) PhaC1(STQK), 3HP-CoA and lactyl-CoA were copolymerized into P(3HP-co-LA)in engineered Escherichia coli. Under optimal conditions, theP(3HP-co-LA) production and contentwere 0.22 g/Land 3.2%(wt/wt[celldryweight]), respectively.A good example was provided for lactate-basedbiopolymerby biosynthesis.
Key words: Poly(3-hydroxypropionate-co-lactate)    Polyhydroxyalkanoate    Synthase Propionyl-CoA transferase    Site mutation    Glycerol
收稿日期: 2017-08-30 出版日期: 2018-03-21
ZTFLH:  Q819  
基金资助: * 国家自然科学基金(31670089);中国科学院重点部署项目资助项目(ZDRW-ZS-2016-3M)
作者简介: 通讯作者 赵广。E-mail: zhaoguang@qibebt.ac.cn;冯新军。E-mail:fengxj@qibebt.ac.cn;
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引用本文:

赵志强,LacmataTamekouStephen,咸漠,刘修涛,冯新军,赵广. 重组大肠杆菌转化甘油合成聚3-羟基丙酸-co-乳酸 *[J]. 中国生物工程杂志, 2018, 38(2): 46-53.

Zhi-qiang ZHAO,Tamekou Stephen LACMATA,Mo XIAN,Xiu-tao LIU,Xin-jun FENG,Guang ZHAO. Biosynthesis of Poly (3-hydroxypropionate-co-lactate) from Glycerol by Engineered Escherichia coli. China Biotechnology, 2018, 38(2): 46-53.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20180207    或    https://manu60.magtech.com.cn/biotech/CN/Y2018/V38/I2/46

图1  以甘油为碳源合成P(3HP-co-LA)的代谢途径示意图
菌株、质粒及引物 特征描述/引物序列 来源/酶切位点
E. coli DH5α 用于重组质粒的构建和保存的菌株 全氏金公司
E. coli JM109(DE3) 蛋白表达和发酵的宿主菌株 TaKaRa
K. pneumoniae ATCC25955 肺炎克雷伯氏菌,携带甘油脱水酶基因dhaB123和甘油脱水酶激活因子gdrAB ATCC
S.typhimurium LT2 鼠伤寒沙门氏菌,携带丙醛脱氢酶基因pduP CGMCC
Pseudomonas putida 恶臭假单胞菌,携带PHA合成酶基因phaC1和phaC2 本实验室保藏
puC57-pct 携带Megasphaera elsdenii DSM 20460来源的丙酰辅酶A转移酶基因pct 金唯智合成
pACYCDuet-f0 重组表达载体,T7启动子,pACYCDuet-1携带dhaB123、gdrAB和pduP基因,氯霉素抗性 本研究构建
pET21a-f1 重组表达载体,T7启动子,pET21a携带phaC1和pct基因,氨苄抗性 本研究构建
pET21a-f2 重组表达载体,T7启动子,pET21a携带phaC2和pct基因,氨苄抗性 本研究构建
pTV118N-pp 重组表达载体,lac启动子,pTV118N携带phac(STQK)和pct基因,氨苄抗性 北海道大学
dhaB123-F CGCCATATGAAAAGATCAAAACGATTTG NdeI
dhaB123-R CACGGTACCGCTTAGCTTCCTTTACGCAG KpnI
gdrA-F GAGAATTCGTGAGCGGAGGTCAGCATGC EcoRI
gdrA-R TTAGATCTCCCACTGACCAAAGCTG
gdrB-F CAGCTTTGGTCAGTGGGAGATCTAAAACGAGGGGACCGTC
gdrB-R CAGAAGCTTCAGTTTCTCTCACTTAACG HindIII
菌株、质粒及引物 特征描述/引物序列 来源/酶切位点
pduP-F CTGAAGCTTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCGCCAT
CGCGGCTATTAAC		 HindIII
pduP-R CAGAAGCTTAGCGAATAGAAAAGCCGTTG HindIII
phaC-1-F GGAATTCCATATGATGAGTAACAAGAACAACGATGAGC NdeI
phaC-1-R CGGGATCCTCAACGCTCGTGAACGTAGGTG BamHI
phaC-2-F GGAATTCCATATGATGACAGACAAACCGGCCAAAGGATC NdeI
phaC-2-R CGGGATCCTCATCGGGTCAGCACGTAGGT BamHI
表1  本研究使用的菌株、载体和引物及其特征描述
图2  本研究使用重组载体示意
图3  基因克隆及重组载体的酶切验证
图4  核磁共振检测结果
图5  溶氧对细胞生长和产量的影响
图6  有机氮源对细胞生长和产量的影响
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