Unravelling the role of anaerobic metabolism (pta-ackA) and virulence (misL and ssa) genes in Salmonella Heidelberg shedding using chicken infection model.

The mechanism of colonisation of the chicken intestine by Salmonella remains poorly understood, while the severity of infections vary enormously depending on the serovar and the age of the bird. Several metabolism and virulence genes have been identified in Salmonella Heidelberg; however, information on their roles in infection, particularly in the chicken infection model, remains scarce. In the present publication, we investigated three Salmonella Heidelberg mutants containing deletions in misL, ssa, and pta-ackA genes by using signature-tagged mutagenesis. We found that mutations in these genes of S. Heidelberg result in an increase in fitness in the chicken model. The exception was perhaps the pta-ackA mutant where colonisation was slightly reduced (2, 7, 14, and 21 days post-infection) although some birds were still excreting at the end of the experiment. Our results suggest that for intestinal colonisation of the chicken caecum, substrate-level phosphorylation is likely to be more important than the MisL outer membrane protein or even the secretion system apparatus. These findings validate previous work that demonstrated the contribution of ackA and pta mutants to virulence in chickens, suggesting that the anaerobic metabolism genes such as pta-ackA could be a promising mitigation strategy to reduce S. Heidelberg virulence.

Coordinated expression of acetyl CoA synthetase and the ace operon enzymes in Escherichia coli in preparation for adaptation to acetate.

Successful adaptation of Escherichia coli to constant environmental challenges demands the operation of a wide range of regulatory control mechanisms, some of which are global, while others are specific. Here, we show that the ability of acetate-negative phenotype strains of E. coli devoid of acetate kinase (AK) and phosphotransacetylase (PTA) to assimilate acetate when challenged at the end of growth on acetogenic substrates is explicable by the co-expression of acetyl CoA-synthetase (AcCoA-S) and acetate permease (AP). Furthermore, mRNA transcript measurements for acs and aceA, together with the enzymatic activities of their corresponding enzymes, acetyl CoA synthetase (AcCoA-S) and isocitrate lyase (ICL), clearly demonstrate that the expression of the two enzymes is inextricably linked and triggered in response to growth rate threshold signal (0.4 h-1± 0.03: n4). Interestingly, further restriction of carbon supply to the level of starvation led to the repression of acs (AcCoA-S), ackA (AK) and pta (PTA). Further, we provide evidence that the reaction sequence catalysed by PTA, AK and AcCoA-S is not in operation at low growth rates and that the reaction catalysed by AcCoA-S is not merely an ATP-dissipating reaction but rather advantageous, as it elevates the available free energy (ΔG°) in central metabolism. Moreover, the transcriptomic data reinforce the view that the expression of PEP carboxykinase is essential in gluconeogenic phenotypes.

Improvement of Streptococcus suis glutamate dehydrogenase expression in Escherichia coli through genetic modification of acetate synthesis pathway.

Escherichia coli generates acetate as an undesirable by-product that has several negative effects on protein expression, and the reduction of acetate accumulation by modifying genes of acetate synthesis pathway can improve the expression of recombinant proteins. In the present study, the effect of phosphotransacetylase (pta) or/and acetate kinase (ackA) deletion on glutamate dehydrogenase (GDH) expression was investigated. The results indicated that the disruptions of pta or/and ackA decreased the acetate accumulation and synthesis of per gram cell, and increased cell density, and GDH expression and synthesis of per gram cell. The pta gene was more important for acetate formation than the ackA gene. Using the strain with deletions of pta-ackA (SSGPA) for GDH expression, acetate accumulation (2·61 g l-1 ) and acetate synthesis of per gram cell (0·229 g g-1 ) were lowest, decreasing by 28·29 and 41·43% compared with those of the parental strain (SSG) respectively. The flux of acetate synthesis (6·6%) was decreased by 72·15% compared with that of SSG, and the highest cell density (11·38 g l-1 ), GDH expression (2·78 mg ml-1 ), and GDH formation of per gram cell (0·2442 mg mg-1 ) were obtained, which were 1·22-, 1·43- and 1·17-times higher than the parental strain respectively. SIGNIFICANCE AND IMPACT OF THE STUDY: Significance and Impact of the Study: Acetate is the key undesirable by-product in Escherichia coli cultivation, and both biomass and production of desired products are increased by the reduction of acetate accumulation. In the present study, the strains with deletions of pta or/and ackA were constructed to reduce the acetate accumulation and improve the GDH expression, and the highest expression level of GDH was obtained using the strain with lesion in pta-ackA that was 1·17-times higher than that of the parental strain. The construction strategy of recombinant E. coli for decreasing the acetate excretion can be used for high expression level of other desired products.

Engineering cytoplasmic acetyl-CoA synthesis decouples lipid production from nitrogen starvation in the oleaginous yeast Rhodosporidium azoricum.

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.

Genetic engineering of Bacillus sp. and fermentation process optimizing for diacetyl production.

Diacetyl, an important flavor extensively used in the food industry, can be produced from the non-enzymatic oxidative decarboxylation of α-acetolactate in bacteria fermentation. In previous work, we obtained a strain of Bacillus sp. DL01-ΔalsD with low diacetyl accumulation. The strain was engineered and optimized for improving the production of diacetyl in this study. First, deletion of the gene encoding phosphotransacetylase (pta), by homologous recombination with high temperature sensitive shuttle plasmid vector pKS1, led to a reduction of acetate and 130% increase of diacetyl production in B. sp. DL01-ΔalsD-Δpta. Then overexpression of α-acetolactate synthase (ALS) from B. subtilis 168 in B. sp. DL01-ΔalsD-Δpta resulted in efficient diacetyl production with a titer of 5.43 g/L. To further increase diacetyl production, single factor and orthogonal experimental data were used to predict the optimal fermentation conditions by Back Propagation neural network. Optimal value of KLa (Dissolved oxygen volume coefficient) was 12.4 h-1 with fermentation parameters of aeration rate 0.66 vvm, agitation speed 179 rpm and temperature 35.7 ℃. A titer of 11.18 g/L diacetyl, the highest reported diacetyl production, was achieved by fed-batch fermentation at the optimal condition using the metabolic engineered strain of B. sp. DL01-ΔalsD-Δpta-als168. These results are of great importance as a new way for the efficient production of diacetyl by food-safety bacteria.

Modification of carbon metabolism in Synechococcus elongatus PCC 7942 by cyanophage-derived sigma factors for bioproduction improvement.

Many cyanophages, which infect cyanobacteria, most of possess putative sigma factors that have high amino acid sequence homology with the σ70-type sigma factor present in cyanobacteria, allowing them to obtain energy and metabolites for their own propagation. In this study, we aimed to modify the carbon metabolism of Synechococcus elongatus PCC 7942 by expressing putative sigma factors from Synechococcus phages to improve bioproduction. Four cyanophage-derived putative sigma factors-putative RpsD4 from Synechococcus phage S-CBS1, putative RpoD and putative RpoS from S-CBS2, and putative RpsD4 from S-CBS3-were selected for this purpose. These were introduced into S. elongatus PCC 7942, and their expression was controlled with a theophylline-dependent riboswitch. The expression of the putative RpoD from S-CBS2 and putative RpsD4 from S-CBS3 resulted in a significant decrease in the growth rate of S. elongatus PCC 7942. In addition, metabolome analysis showed a 3.2-fold increase in acetyl-CoA concentration with the expression of the putative RpoD from S-CBS2 and a 1.9-fold increase with the putative RpsD4 from S-CBS3. The results of RT-qPCR showed that several sugar metabolism genes were repressed by the putative RpoD and activated by the putative RpsD4. In particular, the engineered strain overexpressing the putative RpsD4 and expressing phosphate acetyltransferase succeeded in improving the productivity of the model target product acetate to 217% of its previous value. To the best of our knowledge, this study is the first to modify the metabolism of S. elongatus PCC 7942 by expressing their putative sigma factors from cyanophages.

Cognate sensor kinase-independent activation of Mycobacterium tuberculosis response regulator DevR (DosR) by acetyl phosphate: implications in anti-mycobacterial drug design.

The DevRS/DosT two-component system is essential for mycobacterial survival under hypoxia, a prevailing stress within granulomas. DevR (also known as DosR) is activated by an inducing stimulus, such as hypoxia, through conventional phosphorylation by its cognate sensor kinases, DevS (also known as DosS) and DosT. Here, we show that the DevR regulon is activated by acetyl phosphate under 'non-inducing' aerobic conditions when Mycobacterium tuberculosis devS and dosT double deletion strain is cultured on acetate. Overexpression of phosphotransacetylase caused a perturbation of the acetate kinase-phosphotransacetylase pathway, a decrease in the concentration of acetyl phosphate and dampened the aerobic induction response in acetate-grown bacteria. The operation of two pathways of DevR activation, one through sensor kinases and the other by acetyl phosphate, was established by an analysis of wild-type DevS and phosphorylation-defective DevSH395Q mutant strains under conditions partially mimicking a granulomatous-like environment of acetate and hypoxia. Our findings reveal that DevR can be phosphorylated in vivo by acetyl phosphate. Importantly, we demonstrate that acetyl phosphate-dependent phosphorylation can occur in the absence of DevR's cognate kinases. Based on our findings, we conclude that anti-mycobacterial therapy should be targeted to DevR itself and not to DevS/DosT kinases.

Effect of the pat, fk, stpk Gene Knock-out and mdh Gene Knock-in on Mannitol Production in Leuconostoc mesenteroides.

Leuconostoc mesenteroides can be used to produce mannitol by fermentation, but the mannitol productivity is not high. Therefore, in this study modify the chromosome of Leuconostoc mesenteroides by genetic methods to obtain high-yield strains of mannitol production. In this study, gene knock-out strains and gene knock-in strains were constructed by a two-step homologous recombination method. The mannitol productivity of the pat gene (which encodes phosphate acetyltransferase) deleteon strain (Δpat::amy), fk gene (which encodes fructokinase) deleteon strain (Δfk::amy) and stpk gene (which encodes serine-threonine protein kinase) deleteon strain (Δstpk::amy) were all increased compared to the wild type, and the productivity of mannitol for each strain was 84.8%, 83.5% and 84.1% respectively. The mannitol productivity of the mdh gene (which encodes mannitol dehydrogenase) knock-in strains (Δpat::mdh, Δfk::mdh and Δstpk::mdh) was increased to a higher level than that of the single-gene deletion strains, and the productivity of mannitol for each was 96.5%, 88% and 93.2%, respectively. The multi-mutant strain ΔdtsΔldhΔpat::mdhΔstpk::mdhΔfk::mdh had mannitol productivity of 97.3%. This work shows that multi-gene knock-out and gene knock-in strains have the greatest impact on mannitol production, with mannitol productivity of 97.3% and an increase of 24.7% over wild type. This study used the methods of gene knock-out and gene knock-in to genetically modify the chromosome of Leuconostoc mesenteroides. It is of great significance that we increased the ability of Leuconostoc mesenteroides to produce mannitol and revealed its broad development prospects.

Metabolic engineering of Escherichia coli for the synthesis of polyhydroxyalkanoates using acetate as a main carbon source.

BACKGROUND: High production cost of bioplastics polyhydroxyalkanoates (PHA) is a major obstacle to replace traditional petro-based plastics. To address the challenges, strategies towards upstream metabolic engineering and downstream fermentation optimizations have been continuously pursued. Given that the feedstocks especially carbon sources account up to a large portion of the production cost, it is of great importance to explore low cost substrates to manufacture PHA economically. RESULTS: Escherichia coli was metabolically engineered to synthesize poly-3-hydroxybutyrate (P3HB), poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P3HB4HB), and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) using acetate as a main carbon source. Overexpression of phosphotransacetylase/acetate kinase pathway was shown to be an effective strategy for improving acetate assimilation and biopolymer production. The recombinant strain overexpressing phosphotransacetylase/acetate kinase and P3HB synthesis operon produced 1.27 g/L P3HB when grown on minimal medium supplemented with 10 g/L yeast extract and 5 g/L acetate in shake flask cultures. Further introduction succinate semialdehyde dehydrogenase, 4-hydroxybutyrate dehydrogenase, and CoA transferase lead to the accumulation of P3HB4HB, reaching a titer of 1.71 g/L with a 4-hydroxybutyrate monomer content of 5.79 mol%. When 1 g/L of α-ketoglutarate or citrate was added to the medium, P3HB4HB titer increased to 1.99 and 2.15 g/L, respectively. To achieve PHBV synthesis, acetate and propionate were simultaneously supplied and propionyl-CoA transferase was overexpressed to provide 3-hydroxyvalerate precursor. The resulting strain produced 0.33 g/L PHBV with a 3-hydroxyvalerate monomer content of 6.58 mol%. Further overexpression of propionate permease improved PHBV titer and 3-hydroxyvalerate monomer content to 1.09 g/L and 10.37 mol%, respectively. CONCLUSIONS: The application of acetate as carbon source for microbial fermentation could reduce the consumption of food and agro-based renewable bioresources for biorefineries. Our proposed metabolic engineering strategies illustrate the feasibility for producing polyhydroxyalkanoates using acetate as a main carbon source. Overall, as an abundant and renewable resource, acetate would be developed into a cost-effective feedstock to achieve low cost production of chemicals, materials, and biofuels.

Rediverting carbon flux in Clostridium ljungdahlii using CRISPR interference (CRISPRi).

Clostridium ljungdahlii has emerged as an attractive candidate for the bioconversion of synthesis gas (CO, CO2, H2) to a variety of fuels and chemicals through the Wood-Ljungdahl pathway. However, metabolic engineering and pathway elucidation in this microbe is limited by the lack of genetic tools to downregulate target genes. To overcome this obstacle, here we developed an inducible CRISPR interference (CRISPRi) system for C. ljungdahlii that enables efficient (> 94%) transcriptional repression of several target genes, both individually and in tandem. We then applied CRISPRi in a strain engineered for 3-hydroxybutyrate (3HB) production to examine targets for increasing carbon flux toward the desired product. Downregulating phosphotransacetylase (pta) with a single sgRNA led to a 97% decrease in enzyme activity and a 2.3-fold increase in titer during heterotrophic growth. However, acetate production still accounted for 40% of the carbon flux. Repression of aldehyde:ferredoxin oxidoreductase (aor2), another potential route for acetate production, led to a 5% reduction in acetate flux, whereas using an additional sgRNA targeted to pta reduced the enzyme activity to 0.7% of the wild-type level, and further reduced acetate production to 25% of the carbon flux with an accompanying increase in 3HB titer and yield. These results demonstrate the utility of CRISPRi for elucidating and controlling carbon flow in C. ljungdahlii.

Prediction of reaction knockouts to maximize succinate production by Actinobacillus succinogenes.

Succinate is a precursor of multiple commodity chemicals and bio-based succinate production is an active area of industrial bioengineering research. One of the most important microbial strains for bio-based production of succinate is the capnophilic gram-negative bacterium Actinobacillus succinogenes, which naturally produces succinate by a mixed-acid fermentative pathway. To engineer A. succinogenes to improve succinate yields during mixed acid fermentation, it is important to have a detailed understanding of the metabolic flux distribution in A. succinogenes when grown in suitable media. To this end, we have developed a detailed stoichiometric model of the A. succinogenes central metabolism that includes the biosynthetic pathways for the main components of biomass-namely glycogen, amino acids, DNA, RNA, lipids and UDP-N-Acetyl-α-D-glucosamine. We have validated our model by comparing model predictions generated via flux balance analysis with experimental results on mixed acid fermentation. Moreover, we have used the model to predict single and double reaction knockouts to maximize succinate production while maintaining growth viability. According to our model, succinate production can be maximized by knocking out either of the reactions catalyzed by the PTA (phosphate acetyltransferase) and ACK (acetyl kinase) enzymes, whereas the double knockouts of PEPCK (phosphoenolpyruvate carboxykinase) and PTA or PEPCK and ACK enzymes are the most effective in increasing succinate production.

Production of L-lactic acid from metabolically engineered strain of Enterobacter aerogenes ATCC 29007.

In this study, L-lactic acid production was investigated from metabolically engineered strain of E. aerogenes ATCC 29007. The engineered strain E. aerogenes SUMI01 (Δpta) was generated by the deletion of phosphate acetyltransferase (pta) gene from the chromosome of E. aerogenes ATCC 29007 and deletion was confirmed by colony PCR. Under the optimized fermentation conditions, at 37°C and pH 6 for 84h, the L-lactic acid produced by engineered strain E. aerogenes SUMI01 (Δpta) in flask fermentation using 100g/L mannitol as the carbon source was 40.05g/L as compared to that of the wild type counterpart 20.70g/L. At the end of the batch fermentation in bioreactor the production of L-lactic acid reached to 46.02g/L and yield was 0.41g/g by utilizing 112.32g/L mannitol. This is the first report regarding the production of L-lactic acid from Enterobacter species. We believe that this result may provide valuable guidelines for further engineering Enterobacter strain for the improvement of L-lactic acid production.

Insights from the complete genome sequence of Clostridium tyrobutyricum provide a platform for biotechnological and industrial applications.

Genetic research enables the evolution of novel biochemical reactions for the production of valuable chemicals from environmentally-friendly raw materials. However, the choice of appropriate microorganisms to support these reactions, which must have strong robustness and be capable of a significant product output, is a major difficulty. In the present study, the complete genome of the Clostridium tyrobutyricum strain CCTCC W428, a hydrogen- and butyric acid-producing bacterium with increased oxidative tolerance was analyzed. A total length of 3,011,209 bp of the C. tyrobutyricum genome with a GC content of 31.04% was assembled, and 3038 genes were discovered. Furthermore, a comparative clustering of proteins from C. tyrobutyricum CCTCC W428, C. acetobutylicum ATCC 824, and C. butyricum KNU-L09 was conducted. The results of genomic analysis indicate that butyric acid is produced by CCTCC W428 from butyryl-CoA through acetate reassimilation via CoA transferase, instead of the well-established phosphotransbutyrylase-butyrate kinase pathway. In addition, we identified ten proteins putatively involved in hydrogen production and 21 proteins associated with CRISPR systems, together with 358 ORFs related to ABC transporters and transcriptional regulators. Enzymes, such as oxidoreductases, HNH endonucleases, and catalase, were also found in this species. The genome sequence illustrates that C. tyrobutyricum has several desirable traits, and is expected to be suitable as a platform for the high-level production of bulk chemicals as well as bioenergy.

Metabolic flexibility of a butyrate pathway mutant of Clostridium acetobutylicum.

Clostridium acetobutylicum possesses two homologous buk genes, buk (or buk1) and buk2, which encode butyrate kinases involved in the last step of butyrate formation. To investigate the contribution of buk in detail, an in-frame deletion mutant was constructed. However, in all the Δbuk mutants obtained, partial deletions of the upstream ptb gene were observed, and low phosphotransbutyrylase and butyrate kinase activities were measured. This demonstrates that i) buk (CA_C3075) is the key butyrate kinase-encoding gene and that buk2 (CA_C1660) that is poorly transcribed only plays a minor role; and ii) strongly suggests that a Δbuk mutant is not viable if the ptb gene is not also inactivated, probably due to the accumulation of butyryl-phosphate, which might be toxic for the cell. One of the ΔbukΔptb mutants was subjected to quantitative transcriptomic (mRNA molecules/cell) and fluxomic analyses in acidogenic, solventogenic and alcohologenic chemostat cultures. In addition to the low butyrate production, drastic changes in metabolic fluxes were also observed for the mutant: i) under acidogenic conditions, the primary metabolite was butanol and a new metabolite, 2-hydroxy-valerate, was produced ii) under solventogenesis, 58% increased butanol production was obtained compared to the control strain under the same conditions, and a very high yield of butanol formation (0.3gg-1) was reached; and iii) under alcohologenesis, the major product was lactate. Furthermore, at the transcriptional level, adhE2, which encodes an aldehyde/alcohol dehydrogenase and is known to be a gene specifically expressed in alcohologenesis, was surprisingly highly expressed in all metabolic states in the mutant. The results presented here not only support the key roles of buk and ptb in butyrate formation but also highlight the metabolic flexibility of C. acetobutylicum in response to genetic alteration of its primary metabolism.

Acetate fluxes in Escherichia coli are determined by the thermodynamic control of the Pta-AckA pathway.

Escherichia coli excretes acetate upon growth on fermentable sugars, but the regulation of this production remains elusive. Acetate excretion on excess glucose is thought to be an irreversible process. However, dynamic 13C-metabolic flux analysis revealed a strong bidirectional exchange of acetate between E. coli and its environment. The Pta-AckA pathway was found to be central for both flux directions, while alternative routes (Acs or PoxB) play virtually no role in glucose consumption. Kinetic modelling of the Pta-AckA pathway predicted that its flux is thermodynamically controlled by the extracellular acetate concentration in vivo. Experimental validations confirmed that acetate production can be reduced and even reversed depending solely on its extracellular concentration. Consistently, the Pta-AckA pathway can rapidly switch from acetate production to consumption. Contrary to current knowledge, E. coli is thus able to co-consume glucose and acetate under glucose excess. These metabolic capabilities were confirmed on other glycolytic substrates which support the growth of E. coli in the gut. These findings highlight the dual role of the Pta-AckA pathway in acetate production and consumption during growth on glycolytic substrates, uncover a novel regulatory mechanism that controls its flux in vivo, and significantly expand the metabolic capabilities of E. coli.

CcpA and CodY Coordinate Acetate Metabolism in Streptococcus mutans.

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.

Metabolic engineering for isopropanol production by an engineered cyanobacterium, Synechococcus elongatus PCC 7942, under photosynthetic conditions.

Cyanobacteria engineered for production of biofuels and biochemicals from carbon dioxide represent a promising area of research in relation to a sustainable economy. Previously, we have succeeded in producing isopropanol from cellular acetyl-CoA by means of Synechococcus elongatus PCC 7942 into which a synthetic metabolic pathway was introduced. The isopropanol production by this synthetic metabolic pathway requires acetate; therefore, the cells grown under photosynthetic conditions have to be transferred to a dark and anaerobic conditions to produce acetate. In this study, we achieved acetate production under photosynthetic conditions by S. elongatus PCC 7942 into which we introduced the pta gene encoding phosphate acetyltransferase from Escherichia coli. The metabolic modification (via pta introduction) of the isopropanol-producing strain enabled production of isopropanol under photosynthetic conditions. During 14 days of production, the titer of isopropanol reached 0.55 mM (33.1 mg/l) with an intermediate product, acetone, at 0.21 mM (12.2 mg/l).

Broad substrate specificity of phosphotransbutyrylase from Listeria monocytogenes: A potential participant in an alternative pathway for provision of acyl CoA precursors for fatty acid biosynthesis.

Listeria monocytogenes, the causative organism of the serious food-borne disease listeriosis, has a membrane abundant in branched-chain fatty acids (BCFAs). BCFAs are normally biosynthesized from branched-chain amino acids via the activity of branched chain α-keto acid dehydrogenase (Bkd), and disruption of this pathway results in reduced BCFA content in the membrane. Short branched-chain carboxylic acids (BCCAs) added as media supplements result in incorporation of BCFAs arising from the supplemented BCCAs in the membrane of L. monocytogenes bkd mutant MOR401. High concentrations of the supplements also effect similar changes in the membrane of the wild type organism with intact bkd. Such carboxylic acids clearly act as fatty acid precursors, and there must be an alternative pathway resulting in the formation of their CoA thioester derivatives. Candidates for this are the enzymes phosphotransbutyrylase (Ptb) and butyrate kinase (Buk), the products of the first two genes of the bkd operon. Ptb from L. monocytogenes exhibited broad substrate specificity, a strong preference for branched-chain substrates, a lack of activity with acetyl CoA and hexanoyl CoA, and strict chain length preference (C3-C5). Ptb catalysis involved ternary complex formation. Additionally, Ptb could utilize unnatural branched-chain substrates such as 2-ethylbutyryl CoA, albeit with lower efficiency, consistent with a potential involvement of this enzyme in the conversion of the carboxylic acid additives into CoA primers for BCFA biosynthesis.

Cyclophilin PpiB is involved in motility and biofilm formation via its functional association with certain proteins.

PpiB belongs to the superfamily of peptidyl-prolyl cis/trans isomerases (PPIases, EC: 5.2.1.8), which catalyze the rate-limiting protein folding step at peptidyl-prolyl bonds and control several biological processes. In this study, we show that PpiB acts as a negative effector of motility and biofilm formation ability of Escherichia coli. We identify multicopy suppressors of each ΔppiB phenotype among putative PpiB prey proteins which upon deletion are often characterized by analogous phenotypes. Many putative preys show similar gene expression in wild-type and ΔppiB genetic backgrounds implying possible post-translational modifications by PpiB. We further conducted in vivo and in vitro interaction screens to determine which of them represent true preys. For DnaK, acetyl-CoA carboxylase, biotin carboxylase subunit (AccC) and phosphate acetyltransferase (Pta) we also showed a direct role of PpiB in the functional control of these proteins because it increased the measured enzyme activity of each protein and further interfered with DnaK localization and the correct folding of AccC. Taken together, these results indicate that PpiB is involved in diverse regulatory mechanisms to negatively modulate motility and biofilm formation via its functional association with certain protein substrates.

L-Tryptophan Production in Escherichia coli Improved by Weakening the Pta-AckA Pathway.

Acetate accumulation during the fermentation process of Escherichia coli FB-04, an L-tryptophan production strain, is detrimental to L-tryptophan production. In an initial attempt to reduce acetate formation, the phosphate acetyltransferase gene (pta) from E. coli FB-04 was deleted, forming strain FB-04(Δpta). Unfortunately, FB-04(Δpta) exhibited a growth defect. Therefore, pta was replaced with a pta variant (pta1) from E. coli CCTCC M 2016009, forming strain FB-04(pta1). Pta1 exhibits lower catalytic capacity and substrate affinity than Pta because of a single amino acid substitution (Pro69Leu). FB-04(pta1) lacked the growth defect of FB-04(Δpta) and showed improved fermentation performance. Strain FB-04(pta1) showed a 91% increase in L-tryptophan yield in flask fermentation experiments, while acetate production decreased by 35%, compared with its parent FB-04. Throughout the fed-batch fermentation process, acetate accumulation by FB-04(pta1) was slower than that by FB-04. The final L-tryptophan titer of FB-04(pta1) reached 44.0 g/L, representing a 15% increase over that of FB-04. Metabolomics analysis showed that the pta1 genomic substitution slightly decreased carbon flux through glycolysis and significantly increased carbon fluxes through the pentose phosphate and common aromatic pathways. These results indicate that this strategy enhances L-tryptophan production and decreases acetate accumulation during the L-tryptophan fermentation process.