Dynamic regulation is a promising strategy for fine-tuning metabolic fluxes in microbial cell factories. However, few of these synthetic regulatory systems have been developed for central carbon metabolites. Here we created a set of programmable and bifunctional pyruvate-responsive genetic circuits for dynamic dual control (activation and inhibition) of central metabolism in Bacillus subtilis. We used these genetic circuits to design a feedback loop control system that relies on the intracellular concentration of pyruvate to fine-tune the target metabolic modules, leading to the glucaric acid titer increasing from 207 to 527 mg l−1. The designed logic gate-based circuits were enabled by the characterization of a new antisense transcription mechanism in B. subtilis. In addition, a further increase to 802 mg l−1 was achieved by blocking the formation of by-products. Here, the constructed pyruvate-responsive genetic circuits are presented as effective tools for the dynamic control of central metabolism of microbial cell factories.
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All data produced or analyzed for this study are included in the published article, supplementary information files and source data files, or are available from the corresponding authors upon reasonable request. Sequence data in this article can be found in NCBI under the following accession numbers: pdhR (GenBank ID: 944827), yeeZ (GenBank ID: 946538), udh (GenBank ID: SPD80607.1), gene ino1 (GenBank ID: CAA89448.1). Source data are provided with this paper.
The Python code used in this study can be found in Supplementary Note 1.
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This work was financially supported by the National Natural Science Foundation of China (grant nos 31870069, 31930085 and 21676119), the National Key Research and Development Program of China (grant nos 2018YFA0900300 and 2018YFA0900504), the Fundamental Research Funds for the Central Universities (grant no. JUSRP51713B) and by BBSRC (grant no. BB/R01602X/1).
The authors declare no competing interests.
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Xu, X., Li, X., Liu, Y. et al. Pyruvate-responsive genetic circuits for dynamic control of central metabolism. Nat Chem Biol 16, 1261–1268 (2020). https://doi.org/10.1038/s41589-020-0637-3