Along with the development of metabolic engineering and the discovery of new metabolic routes, compounds that were previously unobtainable are becoming available through biosynthesis in microbes. Currently, a number of natural and artificial biochemicals, such as succinic acid, lactic acid, isoprene, α-pinene, n-butanol, 2-phenylethanol, sabinene, as well as numerous fatty acids, and their derivatives can be synthesized from renewable substrates such as glucose by microbes. These results obtained provided new insights for the development of microbial production of styrene in a sustainable and environment friendly manner.īiorenewable fuels and chemicals have been receiving more and more attention owing to the increasing depletion of fossil fuel resources and concerns about sustainable development of conventional petrochemical industry. This present study achieved the highest titer of de novo production of styrene in E. 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. Finally, in situ product removal was used to ease the burden of end-product toxicity. Subsequently, combined overexpression of tktA and ppsA increased styrene production to 275 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, plasmid optimization was performed, which improved styrene production to 103 mg/L. After codon optimization, AtPAL2 was found to be the most effective one, and the engineered strain was able to produce 55 mg/L styrene. 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). ![]() Systematic optimization of styrene biosynthesis, such as enzyme screening, codon and plasmid optimization, metabolic flow balance, and in situ fermentation was performed. ResultsĪ two-step styrene biosynthesis pathway was developed and introduced into Escherichia coli BL21(DE3). Although achievements have been made on styrene biosynthesis in microorganisms, several bottleneck problems limit factors for further improvement in styrene production. Styrene is a versatile commodity petrochemical used as a monomer building-block for the synthesis of many useful polymers.
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