title: Synthetic operon for (R,R)-2,3-butanediol production in Bacillus subtilis and Escherichia coli.
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.
authors.affiliation: Department of Microbiology and Cell Science, University of Florida, 505 Odyssey Way, Exploration Park at Kennedy Space Center, Merritt Island, FL, 32953, USA. rafa_rdo@yahoo.com.br.|IPR-PUCRS, Av. Ipiranga, 6681-Prédio 96 J, 90619-900, Porto Alegre, RS, Brazil. rafa_rdo@yahoo.com.br.
authors.forename: Rafael R
authors.initials: RR
authors.lastname: de Oliveira
authors.affiliation: Department of Microbiology and Cell Science, University of Florida, 505 Odyssey Way, Exploration Park at Kennedy Space Center, Merritt Island, FL, 32953, USA. WLN@ufl.edu.
authors.forename: Wayne L
authors.initials: WL
authors.lastname: Nicholson
mesh_terms: D000095:Acetolactate Synthase / Q000235:genetics / Q000378:metabolism; D000429:Alcohol Oxidoreductases / Q000235:genetics / Q000378:metabolism; D001412:Bacillus subtilis / Q000235:genetics* / Q000378:metabolism; D002072:Butylene Glycols / Q000378:metabolism*; D002262:Carboxy-Lyases / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005285:Fermentation; D007218:Industrial Microbiology / Q000379:methods*; D009876:Operon; D010957:Plasmids; D011401:Promoter Regions, Genetic; D058615:Synthetic Biology
chemical_list: D002072:Butylene Glycols; C026978:2,3-butylene glycol; D000429:Alcohol Oxidoreductases; C019801:butanediol dehydrogenase; D000095:Acetolactate Synthase; D002262:Carboxy-Lyases; C023746:acetolactate decarboxylase
keywords: 2,3-Butanediol; Bacillus subtilis; Butanediol dehydrogenase; Escherichia coli; Shuttle plasmid