Search

For the efficient production of target metabolites from carbohydrates, syngas, or H 2 -CO 2 by genetically engineered Moorella thermoacetica , the control of acetate production (a main metabolite of M. thermoacetica ) is desired. Although propanediol utilization protein (PduL) was predicted to be a phosphotransacetylase (PTA) involved in acetate production in M. thermoacetica , this has not been confirmed. Our findings described herein directly demonstrate that two putative PduL proteins, encoded by Moth_0864 ( pduL1 ) and Moth_1181 ( pduL2 ), are involved in acetate formation as PTAs. To disrupt these genes, we replaced each gene with a lactate dehydrogenase gene from Thermoanaerobacter pseudethanolicus ATCC 33223 ( T-ldh ). The acetate production from fructose as the sole carbon source by the pduL1 deletion mutant was not deficient, whereas the disruption of pduL2 significantly decreased the acetate yield to approximately one-third that of the wild-type strain. The double-deletion (both pduL genes) mutant did not produce acetate but produced only lactate as the end product from fructose. These results suggest that both pduL genes are associated with acetate formation via acetyl-coenzyme A (acetyl-CoA) and that their disruption enables a shift in the homoacetic pathway to the genetically synthesized homolactic pathway via pyruvate. IMPORTANCE This is the first report, to our knowledge, on the experimental identification of PTA genes in M. thermoacetica and the shift of the native homoacetic pathway to the genetically synthesized homolactic pathway by their disruption on a sugar platform., (Copyright © 2017 American Society for Microbiology.)

In the dental caries pathogen Streptococcus mutans , phosphotransacetylase (Pta) and acetate kinase (Ack) convert pyruvate into acetate with the concomitant generation of ATP. The genes for this pathway are tightly regulated by multiple environmental and intracellular inputs, but the basis for differential expression of the genes for Pta and Ack in S. mutans had not been investigated. Here, we show that inactivation in S. mutans of ccpA or codY reduced the activity of the ackA promoter, whereas a ccpA mutant displayed elevated pta promoter activity. The interactions of CcpA with the promoter regions of both genes were observed using electrophoretic mobility shift and DNase protection assays. CodY bound to the ackA promoter region but only in the presence of branched-chain amino acids (BCAAs). DNase footprinting revealed that the upstream region of both genes contains two catabolite-responsive elements ( cre1 and cre2 ) that can be bound by CcpA. Notably, the cre2 site of ackA overlaps with a CodY-binding site. The CcpA- and CodY-binding sites in the promoter region of both genes were further defined by site-directed mutagenesis. Some differences between the reported consensus CodY binding site and the region protected by S. mutans CodY were noted. Transcription of the pta and ackA genes in the ccpA mutant strain was markedly different at low pH relative to transcription at neutral pH. Thus, CcpA and CodY are direct regulators of transcription of ackA and pta in S. mutans that optimize acetate metabolism in response to carbohydrate, amino acid availability, and environmental pH. IMPORTANCE The human dental caries pathogen Streptococcus mutans is remarkably adept at coping with extended periods of carbohydrate limitation during fasting periods. The phosphotransacetylase-acetate kinase (Pta-Ack) pathway in S. mutans modulates carbohydrate flux and fine-tunes the ability of the organisms to cope with stressors that are commonly encountered in the oral cavity. Here, we show that CcpA controls transcription of the pta and ackA genes via direct interaction with the promoter regions of both genes and that branched-chain amino acids (BCAAs), particularly isoleucine, enhance the ability of CodY to bind to the promoter region of the ackA gene. A working model is proposed to explain how regulation of pta and ackA genes by these allosterically controlled regulatory proteins facilitates proper carbon flow and energy production, which are essential functions during infection and pathogenesis as carbohydrate and amino acid availability continually fluctuate., (Copyright © 2017 American Society for Microbiology.)

In the dental caries pathogen Streptococcus mutans, phosphotransacetylase (Pta) catalyzes the conversion of acetyl coenzyme A (acetyl-CoA) to acetyl phosphate (AcP), which can be converted to acetate by acetate kinase (Ack), with the concomitant generation of ATP. A ΔackA mutant displayed enhanced accumulation of AcP under aerobic conditions, whereas little or no AcP was observed in the Δpta or Δpta ΔackA mutant. The Δpta and Δpta ΔackA mutants also had diminished ATP pools compared to the size of the ATP pool for the parental or ΔackA strain. Surprisingly, when exposed to oxidative stress, the Δpta ΔackA strain appeared to regain the capacity to produce AcP, with a concurrent increase in the size of the ATP pool compared to that for the parental strain. The ΔackA and Δpta ΔackA mutants exhibited enhanced (p)ppGpp accumulation, whereas the strain lacking Pta produced less (p)ppGpp than the wild-type strain. The ΔackA and Δpta ΔackA mutants displayed global changes in gene expression, as assessed by microarrays. All strains lacking Pta, which had defects in AcP production under aerobic conditions, were impaired in their abilities to form biofilms when glucose was the growth carbohydrate. Collectively, these data demonstrate the complex regulation of the Pta-Ack pathway and critical roles for these enzymes in processes that appear to be essential for the persistence and pathogenesis of S. mutans., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Low ethanol yields on xylose hamper economically viable ethanol production from hemicellulose-rich plant material with Saccharomyces cerevisiae. A major obstacle is the limited capacity of yeast for anaerobic reoxidation of NADH. Net reoxidation of NADH could potentially be achieved by channeling carbon fluxes through a recombinant phosphoketolase pathway. By heterologous expression of phosphotransacetylase and acetaldehyde dehydrogenase in combination with the native phosphoketolase, we installed a functional phosphoketolase pathway in the xylose-fermenting Saccharomyces cerevisiae strain TMB3001c. Consequently the ethanol yield was increased by 25% because less of the by-product xylitol was formed. The flux through the recombinant phosphoketolase pathway was about 30% of the optimum flux that would be required to completely eliminate xylitol and glycerol accumulation. Further overexpression of phosphoketolase, however, increased acetate accumulation and reduced the fermentation rate. By combining the phosphoketolase pathway with the ald6 mutation, which reduced acetate formation, a strain with an ethanol yield 20% higher and a xylose fermentation rate 40% higher than those of its parent was engineered.

A new pathway to synthesize poly(hydroxyalkanoic acids) (PHA) was constructed by simultaneously expressing butyrate kinase (Buk) and phosphotransbutyrylase (Ptb) genes of Clostridium acetobutylicum and the two PHA synthase genes (phaE and phaC) of Thiocapsa pfennigii in Escherichia coli. The four genes were cloned into the BamHI and EcoRI sites of pBR322, and the resulting hybrid plasmid, pBPP1, conferred activities of all three enzymes to E. coli JM109. Cells of this recombinant strain accumulated PHAs when hydroxyfatty acids were provided as carbon sources. Homopolyesters of 3-hydroxybutyrate (3HB), 4-hydroxybutyrate (4HB), or 4-hydroxyvalerate (4HV) were obtained from each of the corresponding hydroxyfatty acids. Various copolyesters of those hydroxyfatty acids were also obtained when two of these hydroxyfatty acids were fed at equal amounts: cells fed with 3HB and 4HB accumulated a copolyester consisting of 88 mol% 3HB and 12 mol% 4HB and contributing to 68.7% of the cell dry weight. Cells fed with 3HB and 4HV accumulated a copolyester consisting of 94 mol% 3HB and 6 mol% 4HV and contributing to 64.0% of the cell dry weight. Cells fed with 3HB, 4HB, and 4HV accumulated a terpolyester consisting of 85 mol% 3HB, 13 mol% 4HB, and 2 mol% 4HV and contributing to 68.4% of the cell dry weight.

We investigated metabolic engineering of fermentation pathways in Escherichia coli for production of optically pure D- or L-lactate. Several pta mutant strains were examined, and a pta mutant of E. coli RR1 which was deficient in the phosphotransacetylase of the Pta-AckA pathway was found to metabolize glucose to D-lactate and to produce a small amount of succinate by-product under anaerobic conditions. An additional mutation in ppc made the mutant produce D-lactate like a homofermentative lactic acid bacterium. When the pta ppc double mutant was grown to higher biomass concentrations under aerobic conditions before it shifted to the anaerobic phase of D-lactate production, more than 62.2 g of D-lactate per liter was produced in 60 h, and the volumetric productivity was 1.04 g/liter/h. To examine whether the blocked acetate flux could be reoriented to a nonindigenous L-lactate pathway, an L-lactate dehydrogenase gene from Lactobacillus casei was introduced into a pta ldhA strain which lacked phosphotransacetylase and D-lactate dehydrogenase. This recombinant strain was able to metabolize glucose to L-lactate as the major fermentation product, and up to 45 g of L-lactate per liter was produced in 67 h. These results demonstrate that the central fermentation metabolism of E. coli can be reoriented to the production of D-lactate, an indigenous fermentation product, or to the production of L-lactate, a nonindigenous fermentation product.

We examined the effectiveness of antisense RNA (as RNA) strategies for metabolic engineering of Clostridium acetobutylicum. Strain ATCC 824(pRD4) was developed to produce a 102-nucleotide asRNA with 87% complementarity to the butyrate kinase (BK) gene. Strain ATCC 824(pRD4) exhibited 85 to 90% lower BK and acetate kinase specific activities than the control strain. Strain ATCC 824(pRD4) also exhibited 45 to 50% lower phosphotransbutyrylase (PTB) and phosphotransacetylase specific activities than the control strain. This strain exhibited earlier induction of solventogenesis, which resulted in 50 and 35% higher final concentrations of acetone and butanol, respectively, than the concentrations in the control. Strain ATCC 824(pRD1) was developed to putatively produce a 698-nucleotide asRNA with 96% complementarity to the PTB gene. Strain ATCC 824(pRD1) exhibited 70 and 80% lower PTB and BK activities, respectively, than the control exhibited. It also exhibited 300% higher levels of a lactate dehydrogenase activity than the control exhibited. The growth yields of ATCC 824(pRD1) were 28% less than the growth yields of the control. While the levels of acids were not affected in ATCC 824(pRD1) fermentations, the acetone and butanol concentrations were 96 and 75% lower, respectively, than the concentrations in the control fermentations. The lower level of solvent production by ATCC 824(pRD1) was compensated for by approximately 100-fold higher levels of lactate production. The lack of any significant impact on butyrate formation fluxes by the lower PTB and BK levels suggests that butyrate formation fluxes are not controlled by the levels of the butyrate formation enzymes.

Phosphotransbutyrylase (phosphate butyryltransferase [EC 2.3.1.19]) from Clostridium acetobutylicum ATCC 824 was purified approximately 200-fold to homogeneity with a yield of 13%. Steps used in the purification procedure were fractional precipitation with (NH4)2SO4, Phenyl Sepharose CL-4B chromatography, DEAE-Sephacel chromatography, high-pressure liquid chromatography with an anion-exchange column, and high-pressure liquid chromatography with a hydrophobic-interaction column. Gel filtration and denaturing gel electrophoresis data were consistent with a native enzyme having eight 31,000-molecular-weight subunits. Within the physiological range of pH 5.5 to 7, the enzyme was very sensitive to pH change in the butyryl phosphate-forming direction and showed virtually no activity below pH 6. This finding indicates that a change in internal pH may be one important factor in the regulation of the enzyme. The enzyme was less sensitive to pH change in the reverse direction. The enzyme could use a number of substrates in addition to butyryl coenzyme A (butyryl-CoA) but had the highest relative activity with butyryl-CoA, isovaleryl-CoA, and valeryl-CoA. The Km values at 30 degrees C and pH 8.0 for butyryl-CoA, phosphate, butyryl phosphate, and CoASH (reduced form of CoA) were 0.11, 14, 0.26, and 0.077 mM, respectively. Results of product inhibition studies were consistent with a random Bi Bi binding mechanism in which phosphate binds at more than one site.