代谢工程构建重组耶氏解脂酵母生产中长链聚羟基脂肪酸酯

doi:10.13523/j.cb.20160508

中国生物工程杂志

2016, Vol. 36

Issue (5): 53-58 DOI: 10.13523/j.cb.20160508

研究报告

代谢工程构建重组耶氏解脂酵母生产中长链聚羟基脂肪酸酯

高翠娟^1,2,3, 连思琪², 祁庆生³

1. 临沂大学生命科学学院临沂 276000;
2. 香港城市大学环境工程学院香港 999077;
3. 山东大学微生物技术国家重点实验室济南 250100

Production of Medium-chain-length Polyhydroxyalkanoates by Recombinant Yarrowia lipolytica Through Metabolic Engineering

GAO Cui-juan^1,2,3, LIN Carol Sze-ki², QI Qing-sheng³

1. School of Life Science, Linyi University, Linyi 276000, China;
2. School of Energy and Environment, City University of Hong Kong, Kowloon 999077, China;
3. State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, China

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摘要：

中长链聚羟基脂肪酸酯(mcl-PHA)是一大类由微生物合成的天然生物聚酯,因具有可再生性和生物降解性越来越受到人们的关注。Mcl-PHA可由一些假单胞菌类利用自身的脂肪酸合成途径或β-氧化途径来合成。耶氏解脂酵母具有很好的脂/脂肪酸分解代谢能力,但是它体内缺乏PHA合成酶不能合成mcl-PHA。采用代谢工程策略构建重组解脂酵母,外源表达来自铜绿假单胞菌PAO1(Pseudomonas aeruginosa PAO1)的PHA合成酶。在PHA合成酶的C端添加PTS1过氧化物酶体定位信号序列,使其在过氧化物酶体内发挥功能,并对其编码基因PhaC1进行密码子优化得到oPhaC1。利用pINA1312载体构建表达框,借助载体上的zeta序列元件将oPhaC1基因表达框整合至酵母基因组,完成基因的稳定表达。重组菌PSOC在葡萄糖为唯一碳源的培养基中几乎不产PHA,添加0.5%的油酸时可合成占细胞干重0.67%的mcl-PHA。在含三油酸甘油酯的培养基中发酵72h产生1.51% mcl-PHA(wt%)。实验结果充分证明重组解脂酵母作为有潜力的微生物细胞工厂可以用于生产mcl-PHA,也为将来利用富含油脂和其他营养的餐厨垃圾水解液等廉价资源生产mcl-PHA打下基础。

关键词： 代谢工程; mcl-PHA; PHA合成酶; 重组耶氏解脂酵母

Abstract:

Medium-chain-length polyhydroxyalkanoates (mcl-PHAs) are a large class of biopolymers that have attracted extensive attention as renewable and biodegradable bio-plastics. They are naturally synthesized via fatty acid de novo biosynthesis pathway or β-oxidation pathway from Pseudomonads. The unconventional yeast Yarrowia lipolytica has excellent lipid/fatty acid catabolism and anabolism capacity depending of the mode of culture. Nevertheless, it cannot naturally synthesize PHA, as it does not express an intrinsic PHA synthase. Here, a genetically modified strain of Y. lipolytica was constructed by heterologously expressing PhaC1 gene from P. aeruginosa PAO1 encoding PHA synthase with a peroxisomal signal (PTS1) at the C terminal. The gene was coden optimized and linked onto the vector of pINA1312 to construct pINA1312-oPHA. The expression cassette derived from pINA1312-oPHA was integrated onto the genome randomly through zeta element above the vector resulting strain PSOC. The recombinant yeast of PSOC barely produced mcl-PHA when grew in YPD, but can produce 0.67% DCW of PHA after 72 cultivation in YPD medium containing 0.5% oleic acid. Furthermore, when PSOC was grown in the medium containing triolein, PHA accumulated up to 1.51% DCW. It successfully demonstrated the potential use of Y. lipolytica as a promising microbial cell factory for mcl-PHA production, which laid the foundation for using food waste that contains lipids and other essential nutrients to produce mcl-PHA.

Key words: Metabolic engineering Medium-chain-length polyhydroxyalkanoates Recombinant Yarrowia lipolytica PHA synthase

收稿日期: 2016-01-05 出版日期: 2016-03-02

ZTFLH:

Q939.97

基金资助:

山东省优秀中青年科学家科研奖励基金(BS2012SW004),香港研究基金委员会资助项目

通讯作者: 祁庆生 E-mail: qiqingsheng@sdu.edu.cn

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引用本文:

高翠娟, 连思琪, 祁庆生. 代谢工程构建重组耶氏解脂酵母生产中长链聚羟基脂肪酸酯[J]. 中国生物工程杂志, 2016, 36(5): 53-58.

GAO Cui-juan, LIN Carol Sze-ki, QI Qing-sheng. Production of Medium-chain-length Polyhydroxyalkanoates by Recombinant Yarrowia lipolytica Through Metabolic Engineering. China Biotechnology, 2016, 36(5): 53-58.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20160508 或 https://manu60.magtech.com.cn/biotech/CN/Y2016/V36/I5/53

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