Cloning, sequencing, and expression of genes encoding phosphotransacetylase and acetate kinase from Clostridium acetobutylicum ATCC 824

Boynton, Zhuang L.; Bennett, George N.; Rudolph, Frederick B.

doi:10.1128/aem.62.8.2758-2766.1996

Cited by 62 publications

(24 citation statements)

References 50 publications

(63 reference statements)

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“…As expected, expression of the genes involved in acetate formation [phosphotransacetylase (pta) and acetate kinase (ack) which form an operon; Boynton et al, 1996] and butyrate formation [phosphotransbutyrylase (ptb) and butyrate kinase (buk) which also form an operon; Cary et al, 1988] is very similar in each comparison. Similarly, all of the genes involved in the conversion of acetyl-CoA to butyryl-CoA show a similar expression pattern for each comparison.…”

Section: Gene Expression Versus Flux Patterns Of Primary Metabolismsupporting

confidence: 71%

Transcriptional analysis of product‐concentration driven changes in cellular programs of recombinant Clostridium acetobutylicumstrains

Tummala

Junne

Paredes

et al. 2003

Biotech & Bioengineering

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Antisense RNA (asRNA) downregulation alters protein expression without changing the regulation of gene expression. Downregulation of primary metabolic enzymes possibly combined with overexpression of other metabolic enzymes may result in profound changes in product formation, and this may alter the large-scale transcriptional program of the cells. DNA-array based large-scale transcriptional analysis has the potential to elucidate factors that control cellular fluxes even in the absence of proteome data. These themes are explored in this study of large-scale transcriptional analysis programs and the in vivo primarymetabolism fluxes of several related recombinant C. acetobutylicum strains: C. acetobutylicum ATCC 824 (pSOS95del) (plasmid control; produces high levels of butanol and acetone), 824(pCTFB1AS) (expresses antisense RNA against CoA transferase (ctfb1-asRNA); produces very low levels of butanol and acetone), and 824(pAADB1) (expresses ctfb1-asRNA and the alcoholaldehyde dahydrogenase gene (aad); produce high alcohol and low acetone levels). DNA-array based transcriptional analysis revealed that the large changes in product concentrations (and notably butanol concentration) due to ctfb1-asRNA expression alone and in combination with aad overexpression resulted in dramatic changes of the cellular transcriptome. Cluster analysis and gene expression patterns of established and putative operons involved in stress response, motility, sporulation, and fatty-acid biosynthesis indicate that these simple genetic changes dramatically alter the cellular programs of C. acetobutylicum. Comparison of gene expression and flux analysis data may point to possible flux-controling steps and suggest unknown regulatory mechanisms. B

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Section: Gene Expression Versus Flux Patterns Of Primary Metabolismsupporting

confidence: 71%

Transcriptional analysis of product‐concentration driven changes in cellular programs of recombinant Clostridium acetobutylicumstrains

Tummala

Junne

Paredes

et al. 2003

Biotech & Bioengineering

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“…, 1981), but the gene sequence has not been reported. Moorella thermoacetica lacks any gene that is homologous to the phosphotransacetylase genes whose products are used for phosphorylation of acetate from acetyl‐CoA in related organisms, like Clostridium acetobutyricum (Boynton et al. , 1996).…”

Section: Resultsmentioning

confidence: 99%

The complete genome sequence ofMoorella thermoacetica(f.Clostridium thermoaceticum)

Pierce

Xie

Barabote

et al. 2008

Environmental Microbiology

258

283

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SummaryThis paper describes the genome sequence of M. thermoacetica (f. Clostridium thermoaceticum), which is the model acetogenic bacterium that has been widely used for elucidating the WoodLjungdahl pathway of CO and CO 2 fixation. This pathway, which is also known as the reductive acetyl-CoA pathway, allows acetogenic (often called homoacetogenic) bacteria to convert glucose stoichiometrically into three mol of acetate and to grow autotrophically using H 2 and CO as electron donors and CO 2 as an electron acceptor. Methanogenic archaea use this pathway in reverse to grow by converting acetate into methane and CO 2 . Acetogenic bacteria also couple the Wood-Ljungdahl pathway to a variety of other pathways to allow the metabolism of a wide variety of carbon sources and electron donors (sugars, carboxylic acids, alcohols, and aromatic compounds) and electron acceptors (CO 2 , nitrate, nitrite, thiosulfate, dimethylsulfoxide, and aromatic carboxyl groups). The genome consists of a single circular 2628784 bp chromosome encoding 2615 open reading frames, which includes 2523 predicted protein-encoding genes. Of these, 1834 genes (70.13%) have been assigned tentative functions, 665 (25.43%) matched genes of unknown function, and the remaining 24 (0.92%) had no database match. Two thousand three hundred eighty-four (91.17%) of the ORFs in the M. thermoacetica genome can be grouped in ortholog clusters. This first genome sequence of an acetogenic bacterium provides important information related to how acetogens engage their extreme metabolic diversity by switching among different carbon substrates and electron donors/acceptors and how they conserve energy by anaerobic respiration. Our genome analysis indicates that the key genetic trait for homoacetogenesis is the core acs gene cluster of the Wood-Ljungdahl pathway.

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“…PCR Amplification of ack Gene. Synthetic oligonucleotides were designed as degenerate primers using the codon usage preference for C. tyrobutyricum (http://www.kazusa.or.jp/codon) following previous study (14). The sequences of the PCR primers for ack gene amplification were 5′-GAT AC(A/T) GC(A/T) TT(C/T) CA(C/T) CA(A/G) AC-3′ (forward) and 5′-(G/C)(A/T)(A/G) TT(C/T) TC(A/T) CC(A/T) AT(A/T) CC(A/T) CC 3′ (reverse).…”

Section: Methodsmentioning

confidence: 99%

Construction and Characterization of ack Deleted Mutant of Clostridium tyrobutyricum for Enhanced Butyric Acid and Hydrogen Production

Liu¹,

Zhu²,

Yang³

2006

Biotechnology Progress

168

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Clostridium tyrobutyricum produces butyrate, acetate, H(2), and CO(2) as its main fermentation products from glucose and xylose. To improve butyric acid and hydrogen production, integrational mutagenesis was used to create a metabolically engineered mutant with inactivated ack gene, encoding acetate kinase (AK) associated with the acetate formation pathway. A non-replicative plasmid containing the acetate kinase gene (ack) fragment was constructed and introduced into C. tyrobutyricum by electroporation. Integration of the plasmid into the homologous region on the chromosome should inactivate the target ack gene and produce ack-deleted mutant, PAK-Em. Enzyme activity assays showed that the AK activity in PAK-Em decreased by approximately 50%; meanwhile, phosphotransacetylase (PTA) and hydrogenase activities each increased by approximately 40%. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) results showed that the expression of protein with approximately 32 kDa molecular mass was reduced significantly in the mutant. Compared to the wild type, the mutant grew more slowly at pH 6.0 and 37 degrees C, with a lower specific growth rate of 0.14 h(-1) (vs 0.21 h(-1) for the wild type), likely due to the partially impaired PTA-AK pathway. However, the mutant produced 23.5% more butyrate (0.42 vs 0.34 g/g glucose) at a higher final concentration of 41.7 g/L (vs 19.98 g/L) as a result of its higher butyrate tolerance as indicated in the growth kinetics study using various intial concentrations of butyrate in the media. The mutant also produced 50% more hydrogen (0.024 g/g) from glucose than the wild type. Immobilized-cell fermentation of PAK-Em in a fibrous-bed bioreactor (FBB) further increased the final butyric acid concentration (50.1 g/L) and the butyrate yield (0.45 g/g glucose). Furthermore, in the FBB fermentation at pH 5.0 with xylose as the substrate, only butyric acid was produced by the mutant, whereas the wild type produced large amounts of acetate (0.43 g/g xylose) and lactate (0.61 g/g xylose) and little butyrate (0.05 g/g xylose), indicating a dramatic metabolic pathway shift caused by the ack deletion in the mutant.

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Cloning, sequencing, and expression of genes encoding phosphotransacetylase and acetate kinase from Clostridium acetobutylicum ATCC 824

Cited by 62 publications

References 50 publications

Transcriptional analysis of product‐concentration driven changes in cellular programs of recombinant Clostridium acetobutylicumstrains

Transcriptional analysis of product‐concentration driven changes in cellular programs of recombinant Clostridium acetobutylicumstrains

The complete genome sequence ofMoorella thermoacetica(f.Clostridium thermoaceticum)

Construction and Characterization of ack Deleted Mutant of Clostridium tyrobutyricum for Enhanced Butyric Acid and Hydrogen Production

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