Journal of Industrial Microbiology & Biotechnology (1998) 21, 145–149  1998 Society for Industrial Microbiology 1367-5435/98/$12.00 http://www.stockton-press.co.uk/jim Superiority of the PCR-based approach for cloning the acetate kinase gene of Clostridium thermocellum G Ozcengiz 1,2 , J-H Kim 3,4 , WR Lin 1,5 , E Ozcengiz 1,6 , D Westenberg 7 , LR Lynd 3 and AL Demain 1 1 Biology Department, Massachusetts Institute of Technology, Cambridge, MA 02139; 3 Thayer School of Engineering, Dartmouth College, Hanover, NH 03755, USA; 7 Department of Biological Sciences, Dartmouth College, Hanover, NH 03755, USA Cloning of Clostridium thermocellum acetate kinase (ack) and/or phosphotransacetylase (pta) genes in Escherichia coli by functional complementation of ack and/or pta mutants was complicated by an alternative acetate assimilation pathway involving acetyl-CoA synthetase (ACS). In addition to the problems encountered with the complementation approach, cloning of these genes was not readily achieved using heterologous probing with corresponding genes from Escherichia coli and Methanosarcina thermophila due to the lack of sufficient homology. The use of a PCR- based approach, on the other hand, yielded a specific C. thermocellum gene fragment which showed significant sequence identity to the ack gene for which primers were designed. The subcloned ack fragment was then success- fully used as a probe for the isolation of the corresponding gene and restriction analysis of that region. Keywords: Clostridium thermocellum; acetate kinase; phosphotransacetylase; thermophilic bacteria; PCR; gene cloning Introduction The cellulolytic ethanol-producing thermophilic bacterium Clostridium thermocellum has potential for the direct microbial conversion of cellulosic biomass to ethanol by combining cellulase production, hydrolysis and fermen- tation in a single bioreactor [23]. However, coproduction of organic acids (eg acetate and lactate) by the organism imposes an important barrier since high ethanol selectivity is an absolute requirement for a practical process. Strain development has been undertaken to increase selectivity in Clostridium species by mutagenesis combined with screen- ing [1,22] or selection [26,29,34]. However, the failure of this type of approach to yield stable high-yielding strains [17,18,29] suggests that the probem can better be handled using a directed genetic approach. In Escherichia coli and many anaerobic bacteria, acetate formation provides a major source of ATP during anaerobic growth [35]. Acetyl CoA is converted to acetyl phosphate by phosphotransacetylase (PTA) followed by hydrolysis to acetate by acetate kinase (AK). For the aim of developing genetic tools required for pathway engineering in C. therm- ocellum, the cloning and in vitro manipulation of ack and/or pta genes appear to be a logical first priority since the organism typically produces more acetate than lactate [5,37]. Both genes have been cloned and characterized from E. coli [13,24,25,41] and Methanosarcina thermophila [19,33]. The Bacillus subtilis ack gene was also identified Correspondence: AL Demain, Biology Department, Massachusetts Insti- tute of Technology, Cambridge, MA 02139, USA 2 Present address: Biology Dept, Middle East Technical University, Ankara 06531, Turkey 4 Present address: Junior College of Inchon, Korea 5 Present address: Covance Inc, Research Triangle Park, NC 27709, USA 6 Present address: Bacterial Vaccines Production and Research Dept, Refik Saydam Central Institute of Hygiene, Ankara 06100, Turkey Received 22 January 1998; accepted 31 August 1998 on the basis of sequence similarity to the E. coli ack gene [12]. Recently these genes have also been cloned from Clo- stridium acetobutylicum [3,39]. In this organism, acid for- mation pathways were genetically manipulated with the aim of increasing solvent production [11]. PTA has been pur- ified from Clostridium thermoaceticum [7], Clostridium acidiurici [28] and Clostridium kluyveri [15], and the puri- fication of AK from C. thermoaceticum [32] and C. aceto- butylicum [6,39] as well as the properties of AK in cell extracts of C. thermocellum [21] have been reported. In an attempt to clone ack and/or pta genes from C. ther- mocellum in E. coli, we have been investigating three dif- ferent strategies: (i) heterologous complementation; (ii) het- erologous probing; and (iii) PCR-based amplification of a C. thermocellum ack fragment and homologous probing. We now describe amplification of an ack fragment as well as its use as a probe in the isolation of the corresponding gene, and restriction analysis of that region. Materials and methods Materials Restriction endonucleases were purchased from New England Biolabs (Beverly, MA, USA). The pGEM-T vector system was from Promega Corp (Madison, WI, USA). Recombinant plasmids pML702 [19], pML703 [19] and pAK222 [41] were furnished by J Ferry and E Nakano, respectively. Isopropyl-b-d-thiogalactopyranoside (IPTG), 5-bromo-4-chloro-3-indolyl-b-galactoside (X-gal), ampicil- lin, acetyl phosphate and lithium CoA were obtained from Sigma Chemical Co (St Louis, MO, USA). The nonradioac- tive labeling and detection kit was obtained from Boeh- ringer Mannheim Biochemicals (Indianapolis, IN, USA). Oligonucleotide primers were synthesized in a Perkin Elmer Applied Biosystems (Norwalk, CT, USA) Model 392 DNA synthesizer. The target DNA was amplified using the PCR core kit supplied by Boehringer Mannheim, in a