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Aims Aromatic amines with diverse physical characteristics are often employed as antioxidants and precursors to pharmaceutical products. As the traditional chemical methods pose serious environmental pollution, there is an arising interest in biomanufacturing aromatic amines from renewable feedstocks. Materials and results We report the establishment of a bacterial platform for synthesizing three types of aromatic amines, namely, tyramine, dopamine and phenylethylamine. First, we expressed aromatic amino acid decarboxylase from Enterococcus faecium (pheDC) in an Escherichia coli strain with increasing shikimate (SHK) pathway flux towards L-tyrosine. We found that glycerol served as a better carbon source than glucose, resulting in 940 ± 46 mg/L tyramine from 4% glycerol. Next, the genes of lactate dehydrogenase (ldhA), pyruvate formate lyase (pflB), phosphate acetyltransferase (pta) and alcohol dehydrogenase (adhE) were deleted to mitigate the fermentation by-product formation. The tyramine level was further increased to 1.965 ± 0.205 g/L in the shake flask, which was improved by 2.1 times compared with that of the parental strain. By using a similar strategy, we also managed to produce 703 ± 21 mg/L dopamine and 555 ± 50 mg/L phenethylamine. Conclusions We demonstrated that the knockout of ldhA-pflB-pta-adhE is an effective strategy for improving aromatic amine productions. Significance and Impact of the Study This study achieved the highest aromatic amine titres in E. coli under shake flask reported to date.

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.

Graphical abstract, Microbial communities that metabolise pentose and hexose sugars are useful in producing high-value chemicals, resulting in the effective conversion of raw materials to the product, a reduction in the production cost, and increased yield. Here, we present a computational analysis approach called CAMP (Co-culture/Community Analyses for Metabolite Production) that simulates and identifies appropriate communities to produce a metabolite of interest. To demonstrate this approach, we focus on the optimal production of lactate from various Lactic Acid Bacteria. We used genome-scale metabolic models (GSMMs) belonging to Lactobacillus, Leuconostoc, and Pediococcus species from the Virtual Metabolic Human (VMH; https://vmh.life/) resource and well-curated GSMMs of L. plantarum WCSF1 and L. reuteri JCM 1112. We analysed 1176 two-species communities using a constraint-based modelling method for steady-state flux-balance analysis of communities. Flux variability analysis was used to detect the maximum lactate flux in the communities. Using glucose or xylose as substrates separately or in combination resulted in either parasitism, amensalism, or mutualism being the dominant interaction behaviour in the communities. Interaction behaviour between members of the community was deduced based on variations in the predicted growth rates of monocultures and co-cultures. Acetaldehyde, ethanol, acetate, among other metabolites, were found to be cross-fed between community members. L. plantarum WCSF1 was found to be a member of communities with high lactate yields. In silico community optimisation strategies to predict reaction knock-outs for improving lactate flux were implemented. Reaction knock-outs of acetate kinase, phosphate acetyltransferase, and fumarate reductase in the communities were found to enhance lactate production.

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.

As an emerging pollutant, benzalkonium chlorides (BACs) potentially enriched in waste activated sludge (WAS). However, the microbial response mechanism under chronic effects of BACs on acidogenesis and methanogenesis in anaerobic digestion (AD) has not been clearly disclosed. This study investigated the AD (by-)products and microbial evolution under low to high BACs concentrations from bioreactor startup to steady running. It was found that BACs can lead to an increase of WAS hydrolysis and fermentation, but a disturbance to acidogenic bacteria also occurred at low BACs concentration. A noticeable inhibition to methanogenesis occurred when BAC concentration was up to 15 mg/g TSS. Metagenomic analysis revealed the key genes involved in acetic acid (HAc) biosynthesis (i.e. phosphate acetyltransferase, PTA), β-oxidation pathway (acetyl-CoA C-acetyltransferase) and propionic acid (HPr) conversion was slightly promoted compared with control. Furthermore, BACs inhibited the acetotrophic methanogenesis (i.e. acetyl-CoA synthetase), especially BAC concentration was up to 15 mg/g TSS, thereby enhanced short chain fatty acids (SCFAs) accumulation. Overall, chronic stimulation of functional microorganisms with increasing concentrations of BACs impact WAS fermentation.

In this study, it was found that reducing consumption of acetyl-CoA in mitochondria, peroxisome and lipid biosynthesis could not obviously enhance liamocin biosynthesis by engineered strains of Aureobasidium melanogenm 9-1, but decreased cell growth of the mutants. On the contrary, expression of heterologous PTA gene for phosphotransacetylase in PK pathway and native ALD gene for acetaldehyde dehydrogenase and ACS gene encoding acetyl-CoA synthetase in the PDH bypass pathway reduced liamocin biosynthesis. However, expression the PK gene for phosphoketolase, the PDC gene encoding pyruvate decarboxylase and VHb gene coding for Vitreoscilla hemoglobin (VHb) in the glucose derepression mutants could greatly enhance liamocin production. The resulting strain V33 could produce 55.38 g/L of liamocin and 25.10 g/L of cell dry weight from 117.27 g/L of glucose within 168 h of 10-liter fermentation, leading to the yield of 0.47 g/g of glucose, the productivity of 0.33 g/L/h and rate of glucose utilization of 0.70 ± 0.01 g/L/h. This was a new and efficient strategy for overproduction of liamocin by A. melanogenm.

Acetone-butanol-ethanol (ABE) fermentation by the anaerobic bacterium

The enhanced production of 2,3-butanediol was investigated using a metabolic engineering approach and optimized fermentation conditions. New engineered strains of Enterobacter aerogenes ATCC 29007 were developed by deleting the d -lactate dehydrogenase (ldhA), phosphate acetyltransferase (pta), malate dehydrogenase (mdh), and acetaldehyde dehydrogenase (acdh) genes to block the production of lactate, acetate, succinate, and ethanol, respectively. The resulting engineered strain E. aerogenes SUMI02 (ΔldhAΔpta) produced 36.5 g/L of 2,3-butanediol in flask cultivation, an amount 8.11 times greater than that of its wild type counterpart (4.5 g/L). In addition, the 2,3-butanediol production and productivity reached 38.24 g/L and 0.8 g/L/h, respectively, in the batch fermentation using a bioreactor.

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.

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.

In this study, single-chamber and dual-chamber Microbial electrosynthesis (MES) with carbon fiber brushes as electrodes were operated at 15°C to compare and analyze the difference in methanogenic performance. Metatranscriptomic analysis showed that the relative abundance of electroactive microorganisms Syntrophomonas, Pseudomonas and Bacteroides in each group exceeded 90%, while the abundance of Geobacter was less than 4%. Acetoclastic methanogens Methahnosarcina was more enriched in dual-chamber MES (61.74%~70.42%), and Methanothrix showed higher abundance in single-chamber MES (33.44%~51.71%). Methahnosarcina and Methanothrix could interact with electroactive microorganisms to improve the electron transfer efficiency through direct interspecies electron transfer (DIET). Analysis of the methane metabolic pathways of low-temperature MES found acetoclastic pathway was domination, and single-chamber MES achieved acetate to acetyl-CoA through acetate-CoA ligase (EC: 6.2.1.1), whereas dual-chamber MES was by acetate kinase (EC: 2.7.2.1) and phosphate acetyltransferase (EC: 2.3.1.8). These results are beneficial to further research on the treatment of low-temperature wastewater.

Repurposing existing compounds for new indications may facilitate the discovery of skin prebiotics which have not been well defined. Four compounds that have been registered by the International Nomenclature of Cosmetic Ingredients (INCI) were included to study their abilities to induce the fermentation of Staphylococcusepidermidis (S. epidermidis), a bacterial species abundant in the human skin. Liquid coco-caprylate/caprate (LCC), originally used as an emollient, effectively initiated the fermentation of S. epidermidis ATCC 12228, produced short-chain fatty acids (SCFAs), and provoked robust electricity. Application of LCC plus electrogenic S. epidermidis ATCC 12228 on mouse skin significantly reduced ultraviolet B (UV-B)-induced injuries which were evaluated by the formation of 4-hydroxynonenal (4-HNE), cyclobutane pyrimidine dimers (CPD), and skin lesions. A S. epidermidis S2 isolate with low expressions of genes encoding pyruvate dehydrogenase (pdh), and phosphate acetyltransferase (pta) was found to be poorly electrogenic. The protective action of electrogenic S. epidermidis against UV-B-induced skin injuries was considerably suppressed when mouse skin was applied with LCC in combination with a poorly electrogenic S. epidermidis S2 isolate. Exploring new indication of LCC for promoting S. epidermidis against UV-B provided an example of repurposing INCI-registered compounds as skin prebiotics.

Malic enzymes participate in key metabolic processes, the MaeB-like malic enzymes carry a catalytic inactive phosphotransacetylase domain whose function remains elusive. Here we show that acetyl-CoA directly binds and inhibits MaeB-like enzymes with a saturable profile under physiological relevant acetyl-CoA concentrations. A MaeB-like enzyme from the nitrogen-fixing bacterium Azospirillum brasilense, namely AbMaeB1, binds both acetyl-CoA and unesterified CoASH in a way that inhibition of AbMaeB1 by acetyl-CoA is relieved by increasing CoASH concentrations. Hence, AbMaeB1 senses the acetyl-CoA/CoASH ratio. We revisited E. coli MaeB regulation to determine the inhibitory constant for acetyl-CoA. Our data support that the phosphotransacetylase domain of MaeB-like enzymes senses acetyl-CoA to dictate the fate of carbon distribution at the phosphoenol-pyruvate / pyruvate / oxaloacetate metabolic node.

Acetate is a characteristic by-product of Escherichia coli K-12 growing in batch cultures with glucose, both under aerobic as well as anaerobic conditions. While the reason underlying aerobic acetate production is still under discussion, during anaerobic growth acetate production is important for ATP generation by substrate level phosphorylation. Under both conditions, acetate is produced by a pathway consisting of the enzyme phosphate acetyltransferase (Pta) producing acetyl-phosphate from acetyl-coenzyme A, and of the enzyme acetate kinase (AckA) producing acetate from acetyl-phosphate, a reaction that is coupled to the production of ATP. Mutants in the AckA-Pta pathway differ from each other in the potential to produce and accumulate acetyl-phosphate. In the publication at hand, we investigated different mutants in the acetate pathway, both under aerobic as well as anaerobic conditions. While under aerobic conditions only small changes in growth rate were observed, all acetate mutants showed severe reduction in growth rate and changes in the by-product pattern during anaerobic growth. The AckA– mutant showed the most severe growth defect. The glucose uptake rate and the ATP concentration were strongly reduced in this strain. This mutant exhibited also changes in gene expression. In this strain, the atoDAEB operon was significantly upregulated under anaerobic conditions hinting to the production of acetoacetate. During anaerobic growth, protein acetylation increased significantly in the ackA mutant. Acetylation of several enzymes of glycolysis and central metabolism, of aspartate carbamoyl transferase, methionine synthase, catalase and of proteins involved in translation was increased. Supplementation of methionine and uracil eliminated the additional growth defect of the ackA mutant. The data show that anaerobic, fermentative growth of mutants in the AckA-Pta pathway is reduced but still possible. Growth reduction can be explained by the lack of an important ATP generating pathway of mixed acid fermentation. An ackA deletion mutant is more severely impaired than pta or ackA-pta deletion mutants. This is most probably due to the production of acetyl-P in the ackA mutant, leading to increased protein acetylation.

l-tryptophan (l-trp) is a precursor of various bioactive components and has great pharmaceutical interest. However, due to the requirement of several precursors and complex regulation of the pathways involved, the development of an efficient l-trp production strain is challenging. In this study, Escherichia coli (E. coli) strain KW001 was designed to overexpress the l-trp operator sequences (trpEDCBA) and 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase (aroGfbr). To further improve the production of l-trp, pyruvate kinase (pykF) and the phosphotransferase system HPr (ptsH) were deleted after inactivation of repression (trpR) and attenuation (attenuator) to produce strain KW006. To overcome the relatively slow growth and to increase the transport rate of glucose, strain KW018 was generated by combinatorial regulation of glucokinase (galP) and galactose permease (glk) expression. To reduce the production of acetic acid, strain KW023 was created by repressive regulation of phosphate acetyltransferase (pta) expression. In conclusion, strain KW023 efficiently produced 39.7 g/L of l-trp with a conversion rate of 16.7% and a productivity of 1.6 g/L/h in a 5 L fed-batch fermentation system.

Acetate is a primary inhibitory metabolite in cultures of Escherichia coli, and the production of both biomass and desired products are increased by reducing the accumulation of acetate. In this study, the accumulation of acetate during ʟ-tryptophan production was decreased by genetic modification of ʟ-tryptophan-producing strain (BCTRP) and optimization of the fermentation process. The mutant (BCTRPG), which has a deletion of the integral membrane permease IICBGlc (ptsG), produces a higher concentration of ʟ-tryptophan than mutants with deletions of either phosphate acetyltransferase (pta) or pta–ptsG, due to the low accumulation of acetate and other byproducts, as well as high biomass production. The appropriate dissolved oxygen (DO) level, glucose feeding mode, and pH control strategy were applied to ʟ-tryptophan production using the BCTRPG mutant. The BCTRPG strain with optimized conditions resulted in a reduction in acetate accumulation (71.08% reduction to 0.72 g/L) and an increase in ʟ-tryptophan production (35.81% increase to 17.14 g/L) compared with the BCTRP strain in the original culture condition. Meanwhile, an analysis of the metabolic flux distribution indicated that the acetate synthesis flux decreased from 19.2% (original conditions) to 8.4% (optimized conditions), and the flux of tryptophan formation with the optimized conditions was 18.5%, which was 1.89 times higher than under the original conditions. This study provided the theoretical foundation and technical support for high-level industrialization production of ʟ-tryptophan.

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.

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 Delta buk 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 Delta buk 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 Delta buk Delta ptb 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 (0.3 g g-(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.

Listeria monocytogenes, the causative agent of listeriosis, can build up to dangerous levels in refrigerated foods potentially leading to expensive product recalls. An important aspect of the bacterium’s growth at low temperatures is its ability to increase the branched-chain fatty acid anteiso C15:0 content of its membrane at lower growth temperatures, which imparts greater membrane fluidity. Mutants in the branched-chain α-keto dehydrogenase (bkd) complex are deficient in branched-chain fatty acids (BCFAs,) but these can be restored by feeding C4 and C5 branched-chain carboxylic acids (BCCAs). This suggests the presence of an alternate pathway for production of acyl CoA precursors for fatty acid biosynthesis. We hypothesize that the alternate pathway is composed of butyrate kinase (buk) and phosphotransbutyrylase (ptb) encoded in the bkd complex which produce acyl CoA products by their sequential action through the metabolism of carboxylic acids. We determined the steady state kinetics of recombinant His-tagged Buk using 11 different straight-chain and BCCA substrates in the acyl phosphate forming direction. Buk demonstrated highest catalytic efficiency with pentanoate as the substrate. Low product formation observed with acetate (C2) and hexanoate (C6) as the substrates indicates that Buk is not involved in either acetate metabolism or long chain carboxylic acid activation. We were also able to show that Buk catalysis occurs through a ternary complex intermediate. Additionally, Buk demonstrates a strong preference for BCCAs at low temperatures. These results indicate that Buk may be involved in the activation and assimilation of exogenous carboxylic acids for membrane fatty acid biosynthesis.

Flux analysis is central to understanding cellular metabolism and successful manipulation of metabolic fluxes in microbial cell-factories. Isocitrate dehydrogenase (ICDH) deletion conferred contrasting effects on fluxes through substrate-level phosphorylation (SLP) reactions. While significantly increasing flux through pyruvate kinase, it diminishes flux through succinyl CoA synthetase and upregulates phosphotransacetylase (PTA) and acetate kinase (AK). In addition to acetate, the ICDH-less strain excretes pyruvate, citrate and isocitrate. While efflux to acetate excretion by the Escherichia coli parental strain and its ICDH-less derivative is a reflection of high throughput of glycolytic intermediates, excretion of pyruvate is a reflection of high throughput via pyruvate kinase. On the other hand, citrate and isocitrate excretion is a reflection of truncating the Krebs cycle at the level of ICDH. Furthermore, another striking finding is the inability of the ICDH-less cultures to utilize acetate as a source of carbon despite the availability of an adequate supply of extracellular glutamate (for biosynthesis) and elevated levels of AK and PTA (for acetate uptake). This striking observation is now explicable in the light of the newly proposed hypothesis that the expression of the ace operon enzymes is controlled in response to a minimum threshold signal (ATP), which could not be achieved in the ICDH-less strain.

Growth of the anaerobic thermophile Thermacetogenium phaeum with methanol, ethanol, ethanolamine, and acetate was investigated in axenic cultures and in syntrophic cultures with Methanothermobacter thermautotrophicus. Microcompartment genes were identified in the T. phaeum genome, and presence of microcompartments was confirmed by transmission electron microscopy and proteome analysis. These genes were expressed only during growth with ethanolamine. Proteome data were compared after growth with all four substrates, and activities of key enzymes of the Wood–Ljungdahl pathway and of enzyme systems leading to production or degradation of acetaldehyde such as alcohol dehydrogenase, aldehyde:ferredoxin oxidoreductase, acetate kinase, and phosphate acetyltransferase were measured in cytoplasmic fractions. Accounting of fermentation stoichiometries and growth yields with all four substrates showed that ethanol and methanol oxidation follow the same stoichiometries as in Acetobacterium woodii. On the other hand, the pathways of ethanol and methanol degradations vary between both organisms. Growth yields of T. phaeum were substantially lower than reported for A. woodii. Since T. phaeum has no Rnf complex encoded in its genome, the mechanisms of ATP synthesis have to be different from those of A. woodii. In addition to the central degradation pathways also found in A. woodii, T. phaeum maintains enzyme systems that compensate for the absence of an Rnf-complex but which on the other hand cause a loss of energy. On the basis of our data, pathways of methanol and ethanol degradation in T. phaeum are discussed. published

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.

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.

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.

We previously reported the metabolic analysis of butanol and acetone production from exogenous acetate by (13)C tracer experiments (Gao et al., RSC Adv., 5, 8486-8495, 2015). To clarify the influence of acetate on acetone-butanol-ethanol (ABE) production, we first performed an enzyme assay in Clostridium saccharoperbutylacetonicum N1-4. Acetate addition was found to drastically increase the activities of key enzymes involved in the acetate uptake (phosphate acetyltransferase and CoA transferase), acetone formation (acetoacetate decarboxylase), and butanol formation (butanol dehydrogenase) pathways. Subsequently, supplementation of acetate during acidogenesis and early solventogenesis resulted in a significant increase in ABE production. To establish an efficient ABE production system using acetate as a co-substrate, several shot strategies were investigated in batch culture. Batch cultures with two substrate shots without pH control produced 14.20 g/L butanol and 23.27 g/L ABE with a maximum specific butanol production rate of 0.26 g/(g h). Furthermore, pH-controlled (at pH 5.5) batch cultures with two substrate shots resulted in not only improved acetate consumption but also a further increase in ABE production. Finally, we obtained 15.13 g/L butanol and 24.37 g/L ABE at the high specific butanol production rate of 0.34 g/(g h) using pH-stat co-feeding method. Thus, in this study, we established a high ABE production system using glucose and acetate as co-substrates in a pH-stat co-feeding system with C. saccharoperbutylacetonicum N1-4.

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.

The phosphotransacetylase-acetate kinase (Pta-AckA) pathway is thought to be a vital ATP generating pathway for Staphylococcus aureus. Disruption of the Pta-AckA pathway during overflow metabolism causes significant reduction in growth rate and viability, albeit not due to intracellular ATP depletion. Here, we demonstrate that toxicity associated with inactivation of the Pta-AckA pathway resulted from an altered intracellular redox environment. Growth of the pta and ackA mutants under anaerobic conditions partially restored cell viability. NMR metabolomics analyses and (13)C6-glucose metabolism tracing experiments revealed the activity of multiple pathways that promote redox (NADH/NAD(+)) turnover to be enhanced in the pta and ackA mutants during anaerobic growth. Restoration of redox homeostasis in the pta mutant by overexpressing l- lactate dehydrogenase partially restored its viability under aerobic conditions. Together, our findings suggest that during overflow metabolism, the Pta-AckA pathway plays a critical role in preventing cell viability defects by promoting intracellular redox homeostasis.

The bacterium Corynebacterium glutamicum is utilized during industrial fermentation to produce amino acids such as l‐glutamate. During l‐glutamate fermentation, C. glutamicum changes the flux of central carbon metabolism to favor l‐glutamate production, but the molecular mechanisms that explain these flux changes remain largely unknown. Here, we found that the profiles of two major lysine acyl modifications were significantly altered upon glutamate overproduction in C. glutamicum; acetylation decreased, whereas succinylation increased. A label‐free semi‐quantitative proteomic analysis identified 604 acetylated proteins with 1328 unique acetylation sites and 288 succinylated proteins with 651 unique succinylation sites. Acetylation and succinylation targeted enzymes in central carbon metabolic pathways that are directly related to glutamate production, including the 2‐oxoglutarate dehydrogenase complex (ODHC), a key enzyme regulating glutamate overproduction. Structural mapping revealed that several critical lysine residues in the ODHC components were susceptible to acetylation and succinylation. Furthermore, induction of glutamate production was associated with changes in the extent of acetylation and succinylation of lysine, suggesting that these modifications may affect the activity of enzymes involved in glutamate production. Deletion of phosphotransacetylase decreased the extent of protein acetylation in nonproducing condition, suggesting that acetyl phosphate‐dependent acetylation is active in C. glutamicum. However, no effect was observed on the profiles of acetylation and succinylation in glutamate‐producing condition upon disruption of acetyl phosphate metabolism or deacetylase homologs. It was considered likely that the reduced acetylation in glutamate‐producing condition may reflect metabolic states where the flux through acid‐producing pathways is very low, and substrates for acetylation do not accumulate in the cell. Succinylation would occur more easily than acetylation in such conditions where the substrates for both acetylation and succinylation are limited. This is the first study investigating the acetylome and succinylome of C. glutamicum, and it provides new insight into the roles of acyl modifications in C. glutamicum biology.