ORIGINAL PAPER Sequences affecting the regulation of solvent production in Clostridium acetobutylicum Received: 17 December 2002 / Accepted: 27 March 2003 / Published online: 28 May 2003 Ó Society for Industrial Microbiology 2003 Abstract The high solvent phenotype of Clostridium acetobutylicum mutants B and H was complemented by the introduction of a plasmid that contains either an intact or partially-deleted copy of solR, restoring ace- tone and butanol production to wild-type levels. This demonstrates that the solR open reading frame on pSOLThi is not required to restore solvent levels. The promoter region upstream of alcohol dehydrogense E (adhE) was examined in efforts to identify sites that play major roles in the control of expression. A series of adhE promoter fragments was constructed and the expression of each in acid- and solvent-phases of growth was ana- lyzed using a chloramphenicol acetyl-transferase re- porter system. Our results show that a region beyond the 0A box is needed for full induction of the promoter. Additionally, we show that the presence of sequences around a possible processing site designated S2 may have a negative role in the regulation of adhE expres- sion. Keywords Clostridium Solvent Sol operon CAT reporter Alcohol dehydrogenase E Introduction The acetone- and butanol-producing capabilities of several strains of Clostridium have been known for over 80 years. Until the 1950s, solventogenic clostridia were used as the major industrial producers of acetone and butanol [13]. However the boom in the petrochemical industry during the 1950s and 1960s caused a massive reduction in the usage of clostridial fermentation for solvent production in most countries, as crude oil-de- rived acetone and butanol could be produced more cheaply and efficiently [21]. Current concerns regarding the depletion of the world’s crude oil reserves, as well as an increasing desire for more environmentally friendly industrial practices, have led to the suggestion that solventogenic clostridia could be reinstated as industrial-scale solvent producers. If strains of clostridia could be genetically manipulated to increase solvent production from inexpensive carbon sources then clostridia may become a realistic, economic means of solvent production. In the past 20 years, the molecular biology and bio- chemistry behind solvent production in Clostridium acetobutylicum has been investigated and partially elu- cidated. The principal genes involved in solventogenesis (adc, adhE, ctfA, ctfB) reside on the 192-kb pSOL1 megaplasmid, downstream of the solR open reading frame [5, 23]. Loss of pSOL1, forming the strain desig- nated M5, causes a decrease in solvent production [5, 22]. Butyrate kinase (buk) and phosphotransacetylase (pta) knockout mutants both produce significantly in- creased amounts of butanol during exponential growth [8]. The solR gene has also been a subject of study over the past 3 years. Mutants B and H were generated using the plasmid pO1X, bearing a segment of the solR gene that has recombined with the solR locus, found up- stream of the solvent operon, forming a disrupted, non- functional solR derivative. The resulting increase in acetone and butanol production has been documented [10, 24]. SolR overexpression on plasmid pCO1 in ATCC 824 causes a decrease in solvent levels, and a reduction in alcohol dehydrogense E (adhE) mRNA, as detected by primer extension. On the basis of these re- sults, and reported amino acid homology to helix-turn- helix DNA binding proteins, it was suggested that SolR J Ind Microbiol Biotechnol (2003) 30: 414–420 DOI 10.1007/s10295-003-0057-x Miles C. Scotcher Ke-xue Huang Mary L. Harrison Frederick B. Rudolph George N. Bennett M. C. Scotcher K. Huang M. L. Harrison F. B. Rudolph G. N. Bennett (&) Department of Biochemistry and Cell Biology, MS 140, Rice University, 6100 Main Street, Houston, TX 77005, USA E-mail: gbennett@bioc.rice.edu Tel.: +1-713-3484920 Fax: +1-713-3485154 Present address: K. Huang Dow Agrosciences LLC, R & D Discovery, 9330 Zionsville Road, Indianapolis, IN 46268, USA