A systematic optimization of styrene biosynthesis in Escherichia coli BL21(DE3).
A systematic optimization of styrene biosynthesis in Escherichia coli BL21(DE3).
复制标题
大肠杆菌 BL21(DE3) 中苯乙烯生物合成的系统优化
DOI：
10.1186/s13068-018-1017-z
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发表时间：
2018
影响因子：
6.3
通讯作者：
Xian M
中科院分区：
文献类型：
--
作者：
Liu C;Men X;Chen H;Li M;Ding Z;Chen G;Wang F;Liu H;Wang Q;Zhu Y;Zhang H;Xian M
苯乙烯是一种用途广泛的商品石化产品，用作合成许多有用聚合物的单体组分。虽然在微生物法合成苯乙烯方面已经取得了一些成果，但一些瓶颈问题限制了苯乙烯生产的进一步提高。开发了一种两步法合成苯乙烯的途径，并将其导入大肠杆菌BL21(DE3)中。对苯乙烯生物合成的酶筛选、密码子和质粒优化、代谢流平衡和原位发酵等进行了系统的优化。从拟南芥(AtPAL2)、苦荞麦(FtPAL)、油菜(PcPAL)和黄花蒿(AaPAL)中筛选出限速酶苯丙氨酸解氨酶(PAL)的候选同工酶。经过密码子优化，该工程菌的产量可达55 mg/L，其中AtPAL2最为有效。随后，对工程菌进行了质粒优化，使苯乙烯产量提高到103 mg/L，此外，在工程菌中过表达了两个莽草酸途径基因aroF和phEA，使苯乙烯产量达到210 mg/L。随后，tktA和PPSA的联合过表达使苯乙烯产量增加到275 mg/L。以肉豆蔻酸异丙酯为溶剂，摇瓶发酵48h后，苯乙烯产量达到350m g/L，比出发菌株提高了636%。本研究在摇瓶发酵条件下，实现了在大肠杆菌中从头生产苯乙烯的最高效价。这些结果为以可持续和环境友好的方式发展微生物生产苯乙烯提供了新的见解。本文的在线版本(10.1186/s1306810181017-z)包含补充材料，可供授权用户使用。
Styrene is a versatile commodity petrochemical used as a monomer building-block for the synthesis of many useful polymers. Although achievements have been made on styrene biosynthesis in microorganisms, several bottleneck problems limit factors for further improvement in styrene production. A two-step styrene biosynthesis pathway was developed and introduced into Escherichia coli BL21(DE3). Systematic optimization of styrene biosynthesis, such as enzyme screening, codon and plasmid optimization, metabolic flow balance, and in situ fermentation was performed. Candidate isoenzymes of the rate-limiting enzyme phenylalanine ammonia lyase (PAL) were screened from Arabidopsis thaliana (AtPAL2), Fagopyrum tataricum (FtPAL), Petroselinum crispum (PcPAL), and Artemisia annua (AaPAL). After codon optimization, AtPAL2 was found to be the most effective one, and the engineered strain was able to produce 55 mg/L styrene. Subsequently, plasmid optimization was performed, which improved styrene production to 103 mg/L. In addition, two upstream shikimate pathway genes, aroF and pheA, were overexpressed in the engineered strain, which resulted in styrene production of 210 mg/L. Subsequently, combined overexpression of tktA and ppsA increased styrene production to 275 mg/L. Finally, in situ product removal was used to ease the burden of end-product toxicity. By using isopropyl myristate as a solvent, styrene production reached a final titer of 350 mg/L after 48 h of shake-flask fermentation, representing a 636% improvement, which compared with that achieved in the original strain. This present study achieved the highest titer of de novo production of styrene in E. coli at shake-flask fermentation level. These results obtained provided new insights for the development of microbial production of styrene in a sustainable and environment friendly manner. The online version of this article (10.1186/s13068-018-1017-z) contains supplementary material, which is available to authorized users.
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影响因子：
8.4
作者：
Lee, Hanson;DeLoache, William C.;Dueber, John E.
通讯作者：
Dueber, John E.
影响因子：
4.1
作者：
FORBERG, C;ELIAESON, T;HAGGSTROM, L
通讯作者：
HAGGSTROM, L
影响因子：
5
作者：
Etschmann, M. M. W.;Schrader, J.
通讯作者：
Schrader, J.
影响因子：
2.9
作者：
Chandran, SS;Yi, J;Frost, JW
通讯作者：
Frost, JW
影响因子：
3.8
作者：
Gerigk, M;Bujnicki, R;Takors, R
通讯作者：
Takors, R