Salicylic acid (SA) is an important precursor to a wide variety of high-value chemicals and drugs, such as cis,cis-muconic acid (a platform chemical), aspirin and lamivudine (an anti-HIV drug)1. Recently, a novel approach for microbial production of SA has been developed via extending the shikimate pathway in E. coli1. In previous work by our lab, a series of endogenous small molecule sensor-reporter systems were developed by engineering effector specificity of the transcriptional regulator protein AraC234. The AraC variants activate reporter gene expression in response to target small molecules such as mevalonate, triacetic acid lactone, vanillin and SA.
In the present study, we implement our custom-made SA biosensor (AraC-SA) to improve SA production by recombinant E. coli. The ribosomal binding site calculator56was used to design a spectrum of predicted translation rates for six genes involved in the heterologous SA biosynthesis pathway, and the resulting combinatorial expression library of all genes was subjected to AraC-SA-based high-throughput screening and selection. The degradation pathway of the intermediate chorismate was also attenuated to increase flux toward SA. Improved SA production and altered gene expression profile results will be presented. Finally, the influence of increasing expression of a multidrug efflux pump to increase SA tolerance and yield will be discussed.
1. Lin, Y., Sun, X., Yuan, Q. & Yan, Y. Extending shikimate pathway for the production of muconic acid and its precursor salicylic acid in Escherichia coli. Metab. Eng. 23,62–9 (2014).
2. Tang, S. Y. & Cirino, P. C. Design and application of a mevalonate-responsive regulatory protein. Angew. Chemie, Int. Ed. 50,1084–1086 (2011).
3. Tang, S. Y. et al. Screening for enhanced triacetic acid lactone production by recombinant Escherichia coli expressing a designed triacetic acid lactone reporter. J. Am. Chem. Soc. 135,10099–103 (2013).
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6. Espah Borujeni, A., Channarasappa, A. S. & Salis, H. M. Translation rate is controlled by coupled trade-offs between site accessibility, selective RNA unfolding and sliding at upstream standby sites. Nucleic Acids Res. 42, 2646–59 (2014).
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