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Synthetic operon for (R,R)-2,3-butanediol production in Bacillus subtilis and Escherichia coli

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Abstract

To reduce dependence on petroleum, an alternative route to production of the chemical feedstock 2,3-butanediol (2,3-BD) from renewable lignocellulosic sources is desirable. In this communication, the genes encoding the pathway from pyruvate to 2,3-BD (alsS, alsD, and bdhA encoding acetolactate synthase, acetolactate decarboxylase, and butanediol dehydrogenase, respectively) from Bacillus subtilis were engineered into a single tricistronic operon under control of the isopropyl β-D-1-thiogalactopyranoside (IPTG)-inducible Pspac promoter in a shuttle plasmid capable of replication and expression in either B. subtilis or Escherichia coli. We describe the construction and performance of a shuttle plasmid carrying the IPTG-inducible synthetic operon alsSDbdhA coding for 2,3-BD pathway capable of (i) expression in two important representative model microorganisms, the gram-positive B. subtilis and the gram-negative E. coli; (ii) increasing 2,3-BD production in B. subtilis; and (iii) successfully introducing the B. subtilis 2,3-BD pathway into E. coli. The synthetic alsSDbdhA operon constructed using B. subtilis native genes not only increased the 2,3-BD production in its native host but also efficiently expressed the pathway in the heterologous organism E. coli. Construction of an efficient shuttle plasmid will allow investigation of 2,3-BD production performance in related organisms with industrial potential for production of bio-based chemicals.

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Acknowledgments

We express our thanks to Dr. K.T. Shanmugam for expert assistance and discussion. We also thank Jeff Richards and the late Dr. Lanfang Levine for their excellent technical support with HPLC. This work was supported by grant FLA-MCS-04602 from the US Department of Agriculture, administered through the Florida Agriculture Experiment Station to W.L.N., and support from the UF Department of Microbiology and Cell Science to R.R.O.

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Correspondence to Wayne L. Nicholson.

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de Oliveira, R.R., Nicholson, W.L. Synthetic operon for (R,R)-2,3-butanediol production in Bacillus subtilis and Escherichia coli . Appl Microbiol Biotechnol 100, 719–728 (2016). https://doi.org/10.1007/s00253-015-7030-2

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