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The eight‐carbon fatty acid octanoic acid (OA) is an important platform chemical and precursor of many industrially relevant products. Its microbial biosynthesis is regarded as a promising alternative to current unsustainable production methods. In Saccharomyces cerevisiae, the production of OA had been previously achieved by rational engineering of the fatty acid synthase. For the supply of the precursor molecule acetyl‐CoA and of the redox cofactor NADPH, the native pyruvate dehydrogenase bypass had been harnessed, or the cells had been additionally provided with a pathway involving a heterologous ATP‐citrate lyase. Here, we redirected the flux of glucose towards the oxidative branch of the pentose phosphate pathway and overexpressed a heterologous phosphoketolase/phosphotransacetylase shunt to improve the supply of NADPH and acetyl‐CoA in a strain background with abolished OA degradation. We show that these modifications lead to an increased yield of OA during the consumption of glucose by more than 60% compared to the parental strain. Furthermore, we investigated different genetic engineering targets to identify potential factors that limit the OA production in yeast. Toxicity assays performed with the engineered strains suggest that the inhibitory effects of OA on cell growth likely impose an upper limit to attainable OA yields. [ABSTRACT FROM AUTHOR]

Background: Lipids are important precursors in the biofuel and oleochemical industries. Yarrowia lipolytica is among the most extensively studied oleaginous microorganisms and has been a focus of metabolic engineering to improve lipid production. Yield improvement, through rewiring of the central carbon metabolism of Y. lipolytica from glucose to the lipid precursor acetyl-CoA, is a key strategy for achieving commercial success in this organism. Results: Building on YB-392, a Y. lipolytica isolate known for stable non-hyphal growth and low citrate production with demonstrated potential for high lipid accumulation, we assembled a heterologous pathway that redirects carbon flux from glucose through the pentose phosphate pathway (PPP) to acetyl-CoA. We used phosphofructokinase (Pfk) deletion to block glycolysis and expressed two non-native enzymes, phosphoketolase (Xpk) and phosphotransacetylase (Pta), to convert PPP-produced xylulose-5-P to acetyl-CoA. Introduction of the pathway in a pfk deletion strain that is unable to grow and accumulate lipid from glucose in defined media ensured maximal redirection of carbon flux through Xpk/Pta. Expression of Xpk and Pta restored growth and lipid production from glucose. In 1-L bioreactors, the engineered strains recorded improved lipid yield and cell-specific productivity by up to 19 and 78%, respectively. Conclusions: Yields and cell-specific productivities are important bioprocess parameters for large-scale lipid fermentations. Improving these parameters by engineering the Xpk/Pta pathway is an important step towards developing Y. lipolytica as an industrially preferred microbial biocatalyst for lipid production. [ABSTRACT FROM AUTHOR]

Background: Lipids are important precursors in the biofuel and oleochemical industries. Yarrowia lipolytica is among the most extensively studied oleaginous microorganisms and has been a focus of metabolic engineering to improve lipid production. Yield improvement, through rewiring of the central carbon metabolism of Y. lipolytica from glucose to the lipid precursor acetyl-CoA, is a key strategy for achieving commercial success in this organism. Results: Building on YB-392, a Y. lipolytica isolate known for stable non-hyphal growth and low citrate production with demonstrated potential for high lipid accumulation, we assembled a heterologous pathway that redirects carbon flux from glucose through the pentose phosphate pathway (PPP) to acetyl-CoA. We used phosphofructokinase (Pfk) deletion to block glycolysis and expressed two non-native enzymes, phosphoketolase (Xpk) and phosphotransacetylase (Pta), to convert PPP-produced xylulose-5-P to acetyl-CoA. Introduction of the pathway in a pfk deletion strain that is unable to grow and accumulate lipid from glucose in defined media ensured maximal redirection of carbon flux through Xpk/Pta. Expression of Xpk and Pta restored growth and lipid production from glucose. In 1-L bioreactors, the engineered strains recorded improved lipid yield and cell-specific productivity by up to 19 and 78%, respectively. Conclusions: Yields and cell-specific productivities are important bioprocess parameters for large-scale lipid fermentations. Improving these parameters by engineering the Xpk/Pta pathway is an important step towards developing Y. lipolytica as an industrially preferred microbial biocatalyst for lipid production. [ABSTRACT FROM AUTHOR]

Acetyl phosphate (AcP) is generally produced from acetyl coenzyme A by phosphotransacetylase (Pta), and subsequent reaction with ADP, catalyzed by acetate kinase (Ack), produces ATP. The mechanism of ATP production in Porphyromonas gingivalis is poorly understood. The aim of this study was to explore the molecular basis of the Pta-Ack pathway in this microorganism. Pta and Ack from P. gingivalis ATCC 33277 were enzymatically and structurally characterized. Structural and mutational analyses suggest that Pta is a dimer with two substrate-binding sites in each subunit. Ack is also dimeric, with a catalytic cleft in each subunit, and structural analysis indicates a dramatic domain motion that opens and closes the cleft during catalysis. ATP formation by Ack proceeds via a sequential mechanism. Reverse transcription-PCR analysis demonstrated that the pta (PGN_1179) and ack (PGN_1178) genes, tandemly located in the genome, are cotranscribed as an operon. Inactivation of pta or ack in P. gingivalis by homologous recombination was successful only when the inactivated gene was expressed in trans. Therefore, both pta and ack genes are essential for this microorganism. Insights into the Pta-Ack pathway reported herein would be helpful to understand the energy acquisition in P. gingivalis. [ABSTRACT FROM AUTHOR]

Objectives: To improve the production of 2,3-butanediol (2,3-BD) in Klebsiella pneumoniae, the genes related to the formation of lactic acid, ethanol, and acetic acid were eliminated. Results: Although the cell growth and 2,3-BD production rates of the K. pneumoniae ΔldhA ΔadhE Δpta- ackA strain were lower than those of the wild-type strain, the mutant produced a higher titer of 2,3-BD and a higher yield in batch fermentation: 91 g 2,3-BD/l with a yield of 0.45 g per g glucose and a productivity of 1.62 g/l.h in fed-batch fermentation. The metabolic characteristics of the mutants were consistent with the results of in silico simulation. Conclusions: K. pneumoniae knockout mutants developed with an aid of in silico investigation could produce higher amounts of 2,3-BD with increased titer, yield, and productivity. [ABSTRACT FROM AUTHOR]

The bacterium Chloroflexus aurantiacus excreted significant amounts of acetate during photohetero trophic growth on glucose and in resting cell suspensions. Up to 1.5 mol acetate per mol glucose were formed. In acetate-forming cells, the activities of phosphotransacetylase and acetate kinase, usually involved in acetate formation in Bacteria, could not be detected; instead, the cells contained an acetyl- Co A synthetase ( ADP-forming) ( ACD) (acetyl- Co A + ADP + Pi → acetate + ATP + Co A), an enzyme so far reported in prokaryotes to be specific for acetate-forming Archaea. ACD, which was induced 10-fold during growth on glucose, was purified and the encoding gene was identified as Caur_3920. The recombinant enzyme, a homotetrameric 300-kDa protein composed of 75-kDa subunits, was characterized as functional ACD. Substrate specificities and kinetic constants for acetyl- Co A/acetate and other acyl- Co A esters/acids were determined, showing similarity of the C. aurantiacus ACD to archaeal ACD I isoenzymes, which are involved in acetate formation from sugars. This is the first report of a functional ACD involved in acetate formation in the domain of Bacteria. [ABSTRACT FROM AUTHOR]

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The Escherichia coli genes pta and eutD encode proteins containing the phosphate-acetyltransferase domain. EutD is composed only by this domain and belongs to the ethanolamine operon. This enzyme has not been characterized yet, and its relationship to the multimodular E. coli phosphotransacetylase (Pta) remains unclear. In the present work, a detailed characterization of EutD from E. coli (EcEutD) was performed. The enzyme is a more efficient phosphotransacetylase than E. coli Pta (EcPta) in catalyzing its reaction in either direction and assembles as a dimer, being differentially modulated by EcPta effectors. When comparing EutD and Pta, both from E. coli, certain divergent regions of the primary structure responsible for their unique properties can be found. The growth on acetate of the E. coli pta acs double-mutant strain, was complemented by either introducing EcEutD or by inducing the eut operon with ethanolamine. In this case, the expression of a phosphotransacetylase different from Pta was confirmed by activity assays. Overall, the results indicate that EcEutD and Pta, although able to catalyse the same reaction, display differential efficiency and regulation, and also differ in the induction of their expression. However, under certain growth conditions, they can fulfil equal roles in E. coli metabolism. [ABSTRACT FROM AUTHOR]

Escherichia coli phosphotransacetylase (Pta) catalyzes the reversible interconversion of acetyl-CoA and acetyl phosphate. Both compounds are critical in E. coli metabolism, and acetyl phosphate is also involved in the regulation of certain signal transduction pathways. Along with acetate kinase, Pta plays an important role in acetate production when E. coli grows on rich medium; alternatively, it is involved in acetate utilization at high acetate concentrations. E. coli Pta is composed of three different domains, but only the C-terminal one, called PTA_PTB, is specific for all Ptas. In the present work, the characterization of E. coli Pta and deletions from the N-terminal region were performed. E. coli Pta acetyl phosphate-forming and acetyl phosphate-consuming reactions display different maximum activities, and are differentially regulated by pyruvate and phosphoenolpyruvate. These compounds activate acetyl phosphate production, but inhibit acetyl-CoA production, thus playing a critical role in defining the rates of the two Pta reactions. The characterization of three truncated Ptas, which all display Pta activity, indicates that the substrate-binding site is located at the C-terminal PTA_PTB domain. However, the N-terminal P-loop NTPase domain is involved in expression of the maximal catalytic activity, stabilization of the hexameric native state, and Pta activity regulation by NADH, ATP, phosphoenolpyruvate, and pyruvate. The truncated protein Pta-F3 was able to complement the growth on acetate of an E. coli mutant defective in acetyl-CoA synthetase and Pta, indicating that, although not regulated by metabolites, the Pta C-terminal domain is active in vivo. [ABSTRACT FROM AUTHOR]

Genetic alterations of carbon flux into the acetoin biosynthesis pathway as a possible means to reduce acid accumulation were investigated in the riboflavin-producing Bacillus subtilis during growth on glucose. The lower rates of cell growth and riboflavin production were found in the pta-disrupted mutant while the rate of acetate formation was reduced. In contrast, acid accumulation was significantly reduced, to one-fifth that of the parental strain RH33::[pRB63]n, and a 50% increase in the riboflavin yield was obtained when the expression of the gene encoding acetolactate synthase was increased in the pta-disrupted mutant. Metabolic analysis, together with enzyme activity assays, indicated that the tricarboxylic acid cycle fluxes are significantly increased in response to acetolactate synthase overexpression in pta-disrupted mutant. Moreover, the intracellular ATP-to-ADP ratio also increased 5.8-fold. The high concentration of ATP could explain the increased riboflavin production. [ABSTRACT FROM AUTHOR]

Phosphotransacetylase (Pta) [EC 2.3.1.8] plays a major role in acetate metabolism by catalyzing the reversible transfer of the acetyl group between coenzyme A (CoA) and orthophosphate: CH3COSCoA+HPO $$_{4}^{2-}\rightleftarrows$$ CH3COOPO+CoASH. In this study, we report the crystal structures of Pta from Bacillus subtilis at 2.75 Å resolution and its complex with acetyl phosphate, one of its substrates, at 2.85 Å resolution. In addition, the Pta activity of the enzyme has been assayed. The enzyme folds into an α/β architecture with two domains separated by a prominent cleft, very similar to two other known Pta structures. The enzyme–acetyl phosphate complex structure reveals a few potential substrate binding sites. Two of them are located in the middle of the interdomain cleft: each one is surrounded by a region of strictly and highly conserved residues. High structural similarities are found with 4-hydroxythreonine-4-phosphate dehydrogenase (PdxA), and isocitrate and isopropylmalate dehydrogenases, all of which utilize NADP+ as their cofactor, which binds in the interdomain cleft. Their substrate binding sites are close to the acetyl phosphate binding sites of Pta in the cleft as well. These results suggest that the CoA is likely to bind to the interdomain cleft of Pta in a similar way as NADP+ binds to the other three enzymes. [ABSTRACT FROM AUTHOR]

During growth of Corynebacterium glutamicum on acetate as its carbon and energy source, the expression of the ptaack operon is induced, coding for the acetate-activating enzymes, which are phosphotransacetylase (PTA) and acetate kinase (AK). By transposon rescue, we identified the two genes amrG1 and amrG2 found in the deregulated transposon mutant C. glutamicum G25. The amrG1 gene (NCBI-accession: AF532964) has a size of 732 bp, encoding a polypeptide of 243 amino acids and apparently is partially responsible for the regulation of acetate metabolism in C. glutamicum. We constructed an in-frame deletion mutant and an overexpressing strain of amrG1 in the C. glutamicum ATCC13032 wildtype. The strains were then analyzed with respect to their enzyme activities of PTA and AK during growth on glucose, acetate and glucose or acetate alone as carbon sources. Compared to the parental strain, the amrG1 deletion mutant showed higher specific AK and PTA activities during growth on glucose but showed the same high specific activities of AK and PTA on medium containing acetate plus glucose and on medium containing acetate. In contrast to the gene deletion, overexpression of the amrG1 gene in C. glutamicum 13032 had the adverse regulatory effect. These results indicate that the amrG1 gene encodes a repressor or co-repressor of the ptaack operon. [ABSTRACT FROM AUTHOR]

The efficiency of carbon conversion to biomass and desirable end products in industrial fermentations is diminished by the diversion of carbon to acetate and lactate excretions. In this study, the use of prototrophic and mutant strains of Escherichia coli, as well as enzyme active site directed inhibitors, revealed that flux to acetate excretion is physiologically advantageous to the organism as it facilitates a faster growth rate (μ) and permits growth to high cell densities. Moreover, the abolition of flux to acetate excretion was balanced by the excretion of lactate as well as 2-oxoglutarate, isocitrate and citrate, suggesting a ‘bottle-neck’ effect at the level of 2-oxoglutarate in the Krebs cycle. It is proposed that the acetate excreting enzymes, phosphotransacetylase and acetate kinase, constitute an anaplerotic loop or by-pass, the primary function of which is to replenish the Krebs cycle with reduced CoA, thus relieving the bottle-neck effect at the level of 2-oxoglutarate dehydrogenase. Furthermore, flux to lactate excretion plays a central role in regenerating proton gradient and maintaining the redox balance within the cell. The long-held view that flux to acetate and lactate excretions is merely a function of an ‘over-flow’ in central metabolism should, therefore, be re-evaluated. [ABSTRACT FROM AUTHOR]

Phosphotransa cetylase (Pta) catalyzes the reversible conversion of, acetyl-coenzyme A (CoA) to acetyl phosphate. Polyhydroxybutyrate (PHB) synthase and accumulation were compared between a Pta-deficient mutant and the wild-type Escherichia coli, which were transformed with pAE100, coding for 3-ketothiolase, NADPH-dependenta cetoacetyl-CoA reductase, and PHB synthase from Ralstonia eutropha. During the growth period, PHB synthase activity in the Pta-deficient mutant was lower than that in the wild type. PHB accumulation in the Pta-deficient mutant, however, was higher than that in wild-type cells grown in Luria-Bertani (LB) medium containing 1% glucose (high C:N ratio). The Pta-deficient mutant showed PHB accumulation even in LB medium (low C:N ratio), whereas wild-type cells showed no PHB accumulation. These data suggest the activation of PHB synthase by acetyl phosphate that is synthesized by Pta. A decrease in Pta activity probably causes some increase in acetyl-CoA as substrate for the PHB synthesis pathway, resulting in increased PHB accumulation. [ABSTRACT FROM AUTHOR]

The enzyme, phosphotransacetylase (Pta), catalyzes the conversion of acetyl coenzyme A to acetyl phosphate. The putative pta gene of Bacillus subtilis, which had been sequenced as part of the Genome Project, was cloned and overexpressed in Escherichia coli. We confirmed that the gene encodes Pta by measuring the enzymatic activity of the purified protein. Insertional mutagenesis of the pta gene resulted in complete loss of the Pta activity, indicating that B. subtilis contains only one kind of pta gene. Expression of a pta-lacZ fusion was induced in the presence of excess glucose in the growth medium, and the intact ccpA gene was required for this activation. The transcriptional start site of the pta gene was located at 37 nucleotides upstream of the pta start codon, and a cre (catabolite responsive element) sequence, a cis-acting element that is responsible for the catabolite repression of a number of carbon utilization genes in B. subtilis, was identified upstream of the tentative promoter site. Experiments involving oligonucleotide-directed mutagenesis showed that the cre sequence is involved in glucose-mediated transcriptional activation. [ABSTRACT FROM AUTHOR]

In the amino-acid-producing microorganism Corynebacterium glutamicum, the specific activities of the acetate-activating enzymes acetate kinase and phosphotransacetylase and those of the glyoxylate cycle enzymes isocitrate lyase and malate synthase were found to be high when the cells were grown on acetate (0.8, 2.9, 2.1, and 1.8 U/mg protein, respectively). When the cells were grown on glucose or on other carbon sources such as lactate, succinate, or glutamate, the specific activities were two- to fourfold (acetate kinase and phosphotransacetylase) and 45- to 100-fold (isocitrate lyase and malate synthase) lower, indicating that the synthesis of the four enzymes is regulated by acetate in the growth medium. A comparative Northern (RNA) analysis of the C. glutamicum isocitrate lyase and malate synthase genes ( aceA and aceB) and transcriptional cat fusion experiments revealed that aceA and aceB are transcribed as 1.6- and 2.7-kb monocistronic messages, respectively, and that the regulation of isocitrate lyase and malate synthase synthesis is exerted at the level of transcription from the respective promoters. Surprisingly, C. glutamicum mutants defective in either acetate kinase or phosphotransacetylase showed low specific activities of the other three enzymes (phosphotransacetylase, isocitrate lyase, and malate synthase or acetate kinase, isocitrate lyase, and malate synthase, respectively) irrespective of the presence or absence of acetate in the medium. This result and a correlation of a high intracellular acetyl coenzyme A concentration with high specific activities of isocitrate lyase, malate synthase, acetate kinase, and phosphotransacetylase suggest that acetyl coenzyme A or a derivative thereof may be a physiological trigger for the genetic regulation of enzymes involved in acetate metabolism of C. glutamicum. [ABSTRACT FROM AUTHOR]

Methanobacterium thermoautotrophicum growing on H plus CO as sole carbon and energy source was found to contain acetate thiokinase (Acetyl CoA synthetase; EC 6.2.1.1): Acetate+ATP+CoA → Acetyl CoA+AMP+PPi. The apparent K value for acetate was 40 μM. Acetate kinase (EC 2.7.2.1) and phosphotransacetylase (EC 2.3.1.8) could not be detected. The specific activity of acetate thiokinase was high in cells grown with limited H and CO supply (approximately 100nmol/min · mg protein), it was low in exponentially grown cells (2 nmol/min·mg protein). This corresponded with the finding that cells growing linearly in the presence of acetate assimilated the monocarboxylic acid in high amounts (>10% of the cell carbon was derived from acetate), whereas exponentially growing cells did not (<1% of cell carbon was derived from acetate). These latter observations indicated that acetate thiokinase and free acetate are not involved in autotrophic CO fixation in M. thermoautotrophicum. The presence and some kinetic properties of succinate thiokinase (EC 6.2.1.5), adenylate kinase (EC 2.7.4.3), and inorganic pyrophosphatase (EC 3.6.1.1.) are also described. [ABSTRACT FROM AUTHOR]

The principal energy-yielding reactions of the strict anaerobe Peptococcus prévotii comprised the fermentation of l-serine and l-threonine via the enzymes threonine dehydratase, thioclastic enzyme, phosphotransacetylase and acetate kinase. Threonine dehydratase was purified 700-fold and shown to require pyridoxal 5′-phosphate as co-enzyme, and a reducing agent for optimum activity. The ratio of threonine and serine dehydratase activities was unaltered during purification. The optimum pH was 8.5 to 9.5 and isoleucine did not inhibit. Lineweaver-Burk plots were linear at l-threonine concentrations above 1.35 mM and the Kfor threonine was 2.5 mM and for serine 29 mM. Below this concentration co-operativity occurred which was not nullified by individual adenine nucleotides: Hill plots were biphasic. However, the enzyme was controlled by the adenylate energy charge in a novel manner; only at very low threonine concentrations (<1 mM) was control manifest, when a high energy charge inhibited and a low energy charge stimulated activity. During starvation for 33 hrs in phosphate buffer, pH 6.8, viability fell to zero but, of the enzymes of the energy-generating sequence, only the total units and specific activity of threonine dehydratase decreased (by 35%), which was insufficient to explain the loss of ability to generate ATP. [ABSTRACT FROM AUTHOR]

Background: Oleaginous yeasts are able to accumulate very high levels of neutral lipids especially under condition of excess of carbon and nitrogen limitation (medium with high C/N ratio). This makes necessary the use of two-steps processes in order to achieve high level of biomass and lipid. To simplify the process, the decoupling of lipid synthesis from nitrogen starvation, by establishing a cytosolic acetyl-CoA formation pathway alternative to the one catalysed by ATP-citrate lyase, can be useful. Results: In this work, we introduced a new cytoplasmic route for acetyl-CoA (AcCoA) formation in Rhodosporidium azoricum by overexpressing genes encoding for homologous phosphoketolase (Xfpk) and heterologous phosphotransacetylase (Pta). The engineered strain PTAPK4 exhibits higher lipid content and produces higher lipid concentration than the wild type strain when it was cultivated in media containing different C/N ratios. In a bioreactor process performed on glucose/xylose mixture, to simulate an industrial process for lipid production from lignocellulosic materials, we obtained an increase of 89% in final lipid concentration by the engineered strain in comparison to the wild type. This indicates that the transformed strain can produce higher cellular biomass with a high lipid content than the wild type. The transformed strain furthermore evidenced the advantage over the wild type in performing this process, being the lipid yields 0.13 and 0.05, respectively. Conclusion: Our results show that the overexpression of homologous Xfpk and heterologous Pta activities in R. azoricum creates a new cytosolic AcCoA supply that decouples lipid production from nitrogen starvation. This metabolic modification allows improving lipid production in cultural conditions that can be suitable for the development of industrial bioprocesses using lignocellulosic hydrolysates. [ABSTRACT FROM AUTHOR]

Background: 2-Keto-l-gulonic acid (2-KGA), the precursor of vitamin C, is currently produced by two-step fermentation. In the second step, l-sorbose is transformed into 2-KGA by the symbiosis system composed of Ketogulonicigenium vulgare and Bacillus megaterium. Due to the different nutrient requirements and the uncertain ratio of the two strains, the symbiosis system significantly limits strain improvement and fermentation optimization. Results: In this study, Ketogulonicigenium robustum SPU_B003 was reported for its capability to grow well independently and to produce more 2-KGA than that of K. vulgare in a mono-culture system. The complete genome of K. robustum SPU_B003 was sequenced, and the metabolic characteristics were analyzed. Compared to the four reported K. vulgare genomes, K. robustum SPU_B003 contained more tRNAs, rRNAs, NAD and NADP biosynthetic genes, as well as regulation- and cell signaling-related genes. Moreover, the amino acid biosynthesis pathways were more complete. Two species-specific internal promoters, P1 (orf_01408 promoter) and P2 (orf_02221 promoter), were predicted and validated by detecting their initiation activity. To efficiently produce 2-KGA with decreased CO2 release, an innovative acetyl-CoA biosynthetic pathway (XFP-PTA pathway) was introduced into K. robustum SPU_B003 by expressing heterologous phosphoketolase (xfp) and phosphotransacetylase (pta) initiated by internal promoters. After gene optimization, the recombinant strain K. robustum/pBBR-P1_xfp2502-P2_pta2145 enhanced acetyl-CoA approximately 2.4-fold and increased 2-KGA production by 22.27% compared to the control strain K. robustum/pBBR1MCS-2. Accordingly, the transcriptional level of the 6-phosphogluconate dehydrogenase (pgd) and pyruvate dehydrogenase genes (pdh) decreased by 24.33 ± 6.67 and 8.67 ± 5.51%, respectively. The key genes responsible for 2-KGA biosynthesis, sorbose dehydrogenase gene (sdh) and sorbosone dehydrogenase gene (sndh), were up-regulated to different degrees in the recombinant strain. Conclusions: The genome-based functional analysis of K. robustum SPU_B003 provided a new understanding of the specific metabolic characteristics. The new XFP-PTA pathway was an efficient route to enhance acetyl-CoA levels and to therefore promote 2-KGA production. [ABSTRACT FROM AUTHOR]