Metabolic engineering of Clostridium tyrobutyricum for n-butanol production.

Clostridium tyrobutyricum ATCC 25755, a butyric acid producing bacterium, has been engineered to overexpress aldehyde/alcohol dehydrogenase 2 (adhE2, Genebank no. AF321779) from Clostridium acetobutylicum ATCC 824, which converts butyryl-CoA to butanol, under the control of native thiolase (thl) promoter. Butanol titer of 1.1g/L was obtained in C. tyrobutyricum overexpressing adhE2. The effects of inactivating acetate kinase (ack) and phosphotransbutyrylase (ptb) genes in the host on butanol production were then studied. A high C4/C2 product ratio of 10.6 (mol/mol) was obtained in ack knockout mutant, whereas a low C4/C2 product ratio of 1.4 (mol/mol) was obtained in ptb knockout mutant, confirming that ack and ptb genes play important roles in controlling metabolic flux distribution in C. tyrobutyricum. The highest butanol titer of 10.0g/L and butanol yield of 27.0% (w/w, 66% of theoretical yield) were achieved from glucose in the ack knockout mutant overexpressing adhE2. When a more reduced substrate mannitol was used, the butanol titer reached 16.0 g/L with 30.6% (w/w) yield (75% theoretical yield). Moreover, C. tyrobutyricum showed good butanol tolerance, with >80% and ∼60% relative growth rate at 1.0% and 1.5% (v/v) butanol. These results suggest that C. tyrobutyricum is a promising heterologous host for n-butanol production from renewable biomass.

Multiple control of the acetate pathway in Lactococcus lactis under aeration by catabolite repression and metabolites.

To explore the factors controlling metabolite formation under aeration in Lactococcus lactis, metabolic patterns, enzymatic activities, and transcriptional profiles of genes involved in the aerobic pathway for acetate anabolism were compared between a parental L. lactis strain and its NADH-oxidase-overproducer derivative. Deregulated catabolite repression mutans in the ccpA or pstH genes, encoding CcpA or its co-activator HPr, respectively, were compared with a parental strain, as well. Although the NADH-oxidase activity was derepressed in ccpA, but not in the pstH background, a mixed fermentation was displayed by either mutant, with a higher acetate production in the pstH variant. Moreover, transcription of genes encoding phosphotransacetylase and acetate kinase were derepressed, and the corresponding enzymatic activities increased, in both catabolite repression mutants. These results and the dependence on carbon source for acetate production in the NADH-oxidase-overproducer support the conclusion that catabolite repression, rather than NADH oxidation, plays a critical role to control acetate production. Furthermore, fructose 1,6-bisphosphate influenced the in vitro phosphotransacetylase and acetate kinase activities, while the former was sensitive to diacetyl. Our study strongly supports the model that, under aerobic conditions, the homolactic fermentation in L. lactis MG1363 is maintained by CcpA-mediated repression of mixed acid fermentation.

Genetic manipulation of acid formation pathways by gene inactivation in Clostridium acetobutylicum ATCC 824.

Integrational plasmid technology has been used to disrupt metabolic pathways leading to acetate and butyrate formation in Clostridium acetobutylicum ATCC 824. Non-replicative plasmid constructs, containing either clostridial phosphotransacetylase (pta) or butyrate kinase (buk) gene fragments, were integrated into homologous regions on the chromosome. Integration was assumed to occur by a Campbell-like mechanism, inactivating either pta or buk. Inactivation of the pta gene reduced phosphotransacetylase and acetate kinase activity and significantly decreased acetate production. Inactivation of the buk gene reduced butyrate kinase activity, significantly decreased butyrate production and increased butanol production.

Transcriptional regulation of the phosphotransacetylase-encoding and acetate kinase-encoding genes (pta and ack) from Methanosarcina thermophila.

Phosphotransacetylase and acetate kinase catalyze the activation of acetate to acetyl coenzyme A in the first step of methanogenesis from acetate in Methanosarcina thermophila. The genes encoding these enzymes (pta and ack) have been cloned and sequenced. They are arranged on the chromosome with pta upstream of ack (M.T. Latimer, and J. G. Ferry, J. Bacteriol. 175:6822-6829, 1993). The activities of phosphotransacetylase and acetate kinase are at least 8- to 11-fold higher in acetate-grown cells than in cells grown on methanol, monomethylamine, dimethylamine, or trimethylamine. Northern blot (RNA) analyses demonstrated that pta and ack are transcribed as an approximately 2.4-kb polycistronic message and that the regulation of enzyme synthesis occurs at the mRNA level. Primer extension analyses revealed a transcriptional start site located 27 bp upstream from the translational start of the pta gene and 24 bp downstream from a consensus archaeal boxA promoter sequence. S1 nuclease protection assays detected transcripts with four different 3' ends, each of which mapped to the beginning of four consecutive direct repeats. Northern blot analysis using an ack-specific probe detected both the 2.4-kb polycistronic transcript and a smaller 1.4-kb transcript which is the estimated size of monocistronic ack mRNA. A primer extension product was detected with an ack-specific primer; the 5' end of the product was in the intergenic region between the pta and ack genes but did not follow a consensus archaeal boxA sequence. This result, as well as detection of an additional 1.4-kb mRNA species, suggests processing of the polycistronic 2.4-kb transcript.

Cloning, sequence analysis, and hyperexpression of the genes encoding phosphotransacetylase and acetate kinase from Methanosarcina thermophila.

The genes for the acetate-activating enzymes, acetate kinase and phosphotransacetylase (ack and pta), from Methanosarcina thermophila TM-1 were cloned and sequenced. Both genes are present in only one copy per genome, with the pta gene adjacent to and upstream of the ack gene. Consensus archaeal promoter sequences are found upstream of the pta coding region. The pta and ack genes encode predicted polypeptides with molecular masses of 35,198 and 44,482 Da, respectively. A hydropathy plot of the deduced phosphotransacetylase sequence indicates that it is a hydrophobic polypeptides; however, no membrane-spanning domains are evident. Comparison of the amino acid sequences deduced from the M. thermophila and Escherichia coli ack genes indicate similar subunit molecular weights and 44% identity (60% similarity). The comparison also revealed the presence of several conserved arginine, cysteine, and glutamic acid residues. Arginine, cysteine, and glutamic acid residues have previously been implicated at or near the active site of the E. coli acetate kinase. The pta and ack genes were hyperexpressed in E. coli, and the overproduced enzymes were purified to homogeneity with specific activities higher than those of the enzymes previously purified from M. thermophila. The overproduced phosphotransacetylase and acetate kinase migrated at molecular masses of 37,000 and 42,000 Da, respectively. The activity of the acetate kinase is optimal at 65 degrees C and is protected from thermal inactivation by ATP. Diethylpyrocarbonate and phenylglyoxal inhibited acetate kinase activity in a manner consistent with the presence of histidine and arginine residues at or near the active site; however, the thiol-directed reagents 5,5'-dithiobis (2-nitrobenzoic acid) and N-ethylmaleimide were ineffective.

Cloning of a gene coding for phosphotransacetylase from Escherichia coli.

A phosphotransacetylase gene (pta) has been cloned from a genomic DNA library of Escherichia coli 1100, a derivative of strain K-12. The phosphotransacetylase activities of pta+ plasmid-containing strains were amplified about 150-fold under control of the lac promoter. The molecular weight of the phosphotransacetylase was estimated to be about 81,000 by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The pta gene was found to be downstream of ackA by a combination of restriction analysis and plasmid subcloning. It is located about 13 kb upstream of the purF-folC-hisT region of the chromosome.