Cloning and expression of the gene cluster encoding key proteins involved in acetyl-CoA synthesis in Clostridium thermoaceticum: CO dehydrogenase, the corrinoid/Fe-S protein, and methyltransferase.

Abstract

Acetogenic bacteria fix CO or CO2 by a pathway of autotrophic growth called the acetyl-CoA (or Wood) pathway. Key enzymes in the pathway are a methyltransferase, a corrinoid/Fe-S protein, a disulfide reductase, and a carbon monoxide dehydrogenase. This manuscript describes the isolation of the genes that code for the methyltransferase, the two subunits of the corrinoid/Fe-S protein, and the two subunits of carbon monoxide dehydrogenase. These five genes were found to be clustered within an 10-kilobase segment on the Clostridium thermoaceticum genome. The proteins were expressed at up to 5-10% of Escherichia coli cell protein, and isopropyl .8-D-thiogalactopyranoside had no effect on the levels of expression, implying that the C. thermoaceticum inserts contained transcriptional and translational signals that were recognized by E. coli. The methyltransferase is expressed in E. coli in a fully active dimeric form with a specific activity and heat stability similar to the enzyme expressed in C. thermoaceticum. However, both the corrinoid/Fe-S protein and carbon dioxide dehydrogenase, although expressed in high amounts and with identical subunit molecular weights in E. coli, are inactive and less heat stable than are the native enzymes from C. thermoaceticum. sulfides, and 1-3 Zn atoms per dimer (7). The roles of CODH in the pathway are to bind CO (9, 10), a methyl group (11), and CoA (12, 13) and to catalyze the actual synthesis of acetyl-CoA (12).In this manuscript, we report the cloning of the genes for CODH, MeTr, and C/Fe-SP and the expression of the proteins at high levels in Escherichia coli in the absence of any inducer. We have established that these genes are clustered within a 10-kilobase (kb) DNA segment in the C. thermoaceticum genome and that the CODH genes are directly upstream of the 55-kDa subunit of the C/Fe-SP. We suggest that these clustered genes contain promoter-like sequences and translational signals that are recognized by E. coli. MeTr is expressed in E. coli as a heat-stable dimer and is fully active. Both the C/Fe-SP and CODH, although expressed in high amounts by E. coli, are inactive and are much less heat stable than are the active enzymes from C. thermoaceticum.A preliminary report describing the cloning of the C/Fe-SP and MeTr-encoding genes has been published (14). MATERIALS AND METHODSBacterial Strains, Plasmids, and Growth Conditions. C. thermoaceticum, DSM 521, was cultured in 20-liter carboys at 55°C under CO2 as described by Ljungdahl and Andreesen (15). E. coli K-12 strain JM109 (F' traD36 proAB laclqZAM-15/supE44 thi) was cultured on SOB medium (16). Colonies transformed with pUC9 (17) were grown at pH 7.5 at 37°C on LB-ampicillin medium (16) containing 0.8% tryptone, 0.5% yeast extract, 0.5% sodium chloride, and ampicillin at 0.1 mg/ml. 5-Bromo-4-chloro-3-indolyl (3-D-galactopyranoside and isopropyl B-D-thiogalactopyranoside (IPTG) (both from Boehringer Mannheim) were added to the LB-ampicillin medium to final concentrations of 0.032% and 1.6 mM, respectively, for colo...

The reductive acetyl coenzyme A pathway: sequence and heterologous expression of active methyltetrahydrofolate:corrinoid/iron-sulfur protein methyltransferase from Clostridium thermoaceticum

“…These results indicate that no frameshift errors occurred during sequence analysis. The deduced subunit molecular weight, 28,640, was identical to that determined by mass-spectroscopic analysis (below), and the sequence of the first 27 amino acids of MeTr originating with N-formylmethionine and ending with PAPV had been determined earlier and was identical to that area in the deduced sequence (24). The deduced and experimentally determined amino acid compositions were in agreement.…”

“…The acsE gene encoding MeTr has previously been cloned from C. thermoaceticum DSM521 into plasmid pCtJ9A and expressed at low levels in Escherichia coli (24). The acsE gene is part of a cluster containing the genes encoding CO dehydrogenase (acsA and acsB) (21) and the C/Fe-SP (acsC and acsD) (18).…”

“…For DNA sequencing and increasing the level of heterologous expression, the acsE gene was subcloned by digesting pCtJ9A (24) with PstI and EcoRI and ligating the fragment into Bluescript vectors SK-and KS-digested with the same enzymes. Plasmid DNA was transformed into E. coli (13), and colonies were selected and screened by growth on plates containing ampicillin, 5-bromo-4-chloro-3-indolyl-p-Dgalactopyranoside (X-Gal) and isopropyl-3-D-thiogalactopyranoside (IPTG) and by colony hybridization with _P-labeled oligonucleotide probes specific to MeTr (24). Plasmid DNA was prepared on a large scale by alkali lysis (6) followed by precipitation with polyethylene glycol (20).…”

“…The same procedure was used to purify the enzyme from E. coli except that, after the disrupted cells were centrifuged, the supernatant was incubated at 70'C for 10 min and centrifuged at 10,000 X g before being applied to the DEAE column. MeTr was routinely measured by Western hybridization (24), and the concentration of protein was determined by the Rose Bengal method (9). The specific activity of purified MeTr from both E. coli and C. thermoaceticum was between 150 and 200 U mg-1 at 550C.…”

New methods for tightly regulated gene expression and highly efficient chromosomal integration of cloned genes for Methanosarcina species

“…However, it remains to be shown if this regulation is dose-dependent for the whole Methanosarcina population, as is the case for tetO/TetR systems in bacteria and eukaryotes, or an autocatalytic induction of expression due to active uptake of the inducer, as is the case for Plac-and Para-dependent gene expression (Novick andWeiner 1957, Morgan-Kiss et al 2002). With the Pmcr(tetO)/TetR system established for Methanosarcina it seems feasible now to overproduce enzymes in a catalytically active form where other host/overexpresion systems have resulted in partially inactive protein (Roberts et al 1989, Sauer et al 1997, Sauer and Thauer Rudolf 1998, Loke et al 2000. Furthermore, even toxic genes can probably be overproduced because of the tight repression of the hybrid promoter in the absence of tetracycline.…”