A systematic optimization of styrene biosynthesis in Escherichia coli BL21(DE3)

作者：Changqing Liu, Xiao Men, Hai-Lin Chen, Meijie Li, Zhaorui Ding, Guo‐Qiang Chen, Fan Wang, Haobao Liu, Qian Wang, Youshuang Zhu, Haibo Zhang, Mo Xian · 发表于：Biotechnology for Biofuels · 年份：2018 · DOI：10.1186/s13068-018-1017-z · 被引用次数：72 · 研究领域：biodegradable polymer synthesis and properties、Microbial Metabolic Engineering and Bioproduction、Chromium effects and bioremediation

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, ...