Encoding Acetate Kinase Research Articles

Effects of down-regulation of ackA expression by CRISPR-dCpf1 on succinic acid production in Actinobacillus succinogenes

Succinic acid (SA), a key intermediate in the cellular tricarboxylic acid cycle (TCA), is a 4-carbon dicarboxylic acid of great industrial value. Actinobacillus succinogenes can ferment various carbon sources and accumulate relatively high concentrations of SA, but few reliable genetic engineering tools exist for A. succinogenes and this has hindered strain improvement to increase SA production for industrial application. Two different repressors, endonuclease-deactivated Cas9 (dCas9) from Streptococcus pyogenes and Cpf1 (dCpf1) from Francisella tularensis, were applied to construct a CRISPRi system in A. succinogenes. Codon-optimized Cas9 and native Cpf1 were successfully expressed in A. succinogenes, and the corresponding sgRNA and crRNA expression elements, promoted by the fumarate reductase promoter, frd, were introduced into the CRISPRi plasmid. The highest repression of the ackA gene (encoding acetate kinase) and thereby acetic acid production (~ eightfold) was achieved by the dCpf1-based CRISPRi system, in which the mutation site, E1006A acted at the start of the coding region of ackA, the gene which regulates acetic acid biosynthesis. Compared with the ackA gene knockout mutant, cell growth was moderately improved and SA production increased by 6.3%. Further, the SA titer and productivity in a 3 L fermenter reached 57.06 g/L and 1.87 g/L/h, and there was less acetic acid production. A dCpf1-based CRISPRi-mediated gene repression system was successfully established for the first time, providing a simple and effective tool for studying functional genomics in A. succinogenes and optimizing SA production.

Bacterial and Archaeal Assemblages from Two Size Fractions in Submarine Groundwater Near an Industrial Zone

Nutrients and organic pollutants transported by submarine groundwater discharge (SGD) play a significant role in controlling water quality, and can lead to the concerned deleterious effects on marine ecosystems. Subterranean estuaries are complicated habitats of diverse microbial communities that mediate different biogeochemical processes. However, there is less information on how microorganisms mediate biogeochemical cycles in the submarine groundwater system. In this study, we investigated the changes in bacterial and archaeal assemblages from two size fractions (0.2–0.45 μm and >0.45 μm) in the submarine groundwater of Qinzhou Bay, China. Phylogenetic analysis showed that Bathyarchaeota was dominant in archaeal communities in the >0.45 μm size fraction, but was seldom in the 0.2–0.45 μm fraction. The co-occurrence of sequences belonging to Bathyarchaeota and Methanosaeta was found in the >0.45 μm size fraction. Since a gene encoding acetate kinase of Bathyarchaeota is involved in acetate production, and acetate is also a necessary growth factor for Methanosaeta, the acetate produced by Bathyarchaeota can provide food or energy sources for Methanosaeta in this very >0.45 μm size fraction. The most abundant bacterial sequences in the >0.45 μm size fraction was closely related to biomineral iron-oxidizing Gallionella spp., whereas the dominant bacterial sequences in the 0.2–0.45 μm fraction were affiliated with Limnohabitans spp., which can utilize dissolved organic matter as an important source of growth substrates. Notably, approximately 10% of the bacterial sequences in both of the two size fractions belonged to Novosphingobium spp., which plays an important role in the degradation of pollutants, especially aromatic compounds. Furthermore, the predictive functional profiling also revealed that the pathways involved in the degradation of aromatic compounds by both bacteria and archaea were identified. The presence of nutrients or pollutants in our study site provides different substrates for the growth of the specific microbial groups; in turn, these microbes may help to deplete pollutants to the ocean through submarine groundwater. We suggest that these specific microbial groups could be potential candidates for effective in situ bioremediation of groundwater ecosystems.

Enhanced solvent production by metabolic engineering of a twin-clostridial consortium

The efficient fermentative production of solvents (acetone, n-butanol, and ethanol) from a lignocellulosic feedstock using a single process microorganism has yet to be demonstrated. Herein, we developed a consolidated bioprocessing (CBP) based on a twin-clostridial consortium composed of Clostridium cellulovorans and Clostridium beijerinckii capable of producing cellulosic butanol from alkali-extracted, deshelled corn cobs (AECC). To accomplish this a genetic system was developed for C. cellulovorans and used to knock out the genes encoding acetate kinase (Clocel_1892) and lactate dehydrogenase (Clocel_1533), and to overexpress the gene encoding butyrate kinase (Clocel_3674), thereby pulling carbon flux towards butyrate production. In parallel, to enhance ethanol production, the expression of a putative hydrogenase gene (Clocel_2243) was down-regulated using CRISPR interference (CRISPRi). Simultaneously, genes involved in organic acids reassimilation (ctfAB, cbei_3833/3834) and pentose utilization (xylR, cbei_2385 and xylT, cbei_0109) were engineered in C. beijerinckii to enhance solvent production. The engineered twin-clostridia consortium was shown to decompose 83.2g/L of AECC and produce 22.1g/L of solvents (4.25g/L acetone, 11.5g/L butanol and 6.37g/L ethanol). This titer of acetone-butanol-ethanol (ABE) approximates to that achieved from a starchy feedstock. The developed twin-clostridial consortium serves as a promising platform for ABE fermentation from lignocellulose by CBP.

Impact of deletion of the genes encoding acetate kinase on production of L-tryptophan by Escherichia coli

The production of L-tryptophan was increased by reducing acetate accumulation through a decrease in acetate kinase activity by gene deletion. The effects of disruption of the genes for acetate kinase (ackA) and an enzyme with propionate/acetate kinase activity (tdcD) on L-tryptophan production were investigated. The ackA and/or tdcD deletion mutants accumulated less acetate and more L-tryptophan than the parental strain. Furthermore, the production of L-tryptophan obtained with ackA-tdcD mutant were more than the mutants with a single deletion of ackA or tdcD, while higher production of L-tryptophan and lower concentration of acetate were accumulated in the ackA mutant than the mutant with a lesion in tdcD. In L-tryptophan fed-batch fermentation using the ackA-tdcD mutant, the excretion of acetate was reduced to 1.22 g/L, a 21.79 % reduction compared with the parental strain, and the production of L-tryptophan and glucose conversion rate were increased to 52.5 g/L and 47.9 g/L, respectively, which represented 6.49 % and 10.88 % increases compared with the parental strain, and the glucose conversion rate reached a high level of 21.2 %, which was 8.16 % higher than the parental strain. In addition, the metabolic flux analysis of TRTH and TRTHAT indicated that the carbon flux through EMP was decreased by 8.37 % and the carbon flux through PP was increased by 57.03 % in TRTHAT compared with TRTH. The flux of acetate and tryptophan formation of TRTHAT were 5.2 % and 17.3 %, which were 3.67-times lower and 1.75-times higher than these of TRTH, respectively.

Mechanism for stabilizing mRNAs involved in methanol-dependent methanogenesis of cold-adaptive Methanosarcina mazei zm-15.

Methylotrophic methanogenesis predominates at low temperatures in the cold Zoige wetland in Tibet. To elucidate the basis of cold-adapted methanogenesis in these habitats, Methanosarcina mazei zm-15 was isolated, and the molecular basis of its cold activity was studied. For this strain, aceticlastic methanogenesis was reduced 7.7-fold during growth at 15°C versus 30°C. Methanol-derived methanogenesis decreased only 3-fold under the same conditions, suggesting that it is more cold adaptive. Reverse transcription-quantitative PCR (RT-qPCR) detected <2-fold difference in the transcript abundances of mtaA1, mtaB1, and mtaC1, the methanol methyltransferase (Mta) genes, in 30°C versus 15°C culture, while ackA and pta mRNAs, encoding acetate kinase (Ack) and phosphotransacetylase (Pta) in aceticlastic methanogenesis, were 4.5- and 6.8-fold higher in 30°C culture than in 15°C culture. The in vivo half-lives of mtaA1 and mtaC1B1 mRNAs were similar in 30°C and 15°C cultures. However, the pta-ackA mRNA half-life was significantly reduced in 15°C culture compared to 30°C culture. Using circularized RNA RT-PCR, large 5' untranslated regions (UTRs) (270 nucleotides [nt] and 238 nt) were identified for mtaA1 and mtaC1B1 mRNAs, while only a 27-nt 5' UTR was present in the pta-ackA transcript. Removal of the 5' UTRs significantly reduced the in vitro half-lives of mtaA1 and mtaC1B1 mRNAs. Remarkably, fusion of the mtaA1 or mtaC1B1 5' UTRs to pta-ackA mRNA increased its in vitro half-life at both 30°C and 15°C. These results demonstrate that the large 5' UTRs significantly enhance the stability of the mRNAs involved in methanol-derived methanogenesis in the cold-adaptive M. mazei zm-15.

Activation of glyoxylate pathway without the activation of its related gene in succinate-producing engineered Escherichia coli

For the first time, glyoxylate pathway in the biosynthesis of succinate was activated without the genetic manipulations of any gene related with glyoxylate pathway. Furthermore, the inactivation of succinate biosynthesis by-products genes encoding acetate kinase (ackA) and phosphotransacetylase (pta) was proven to be the key factor to activate glyoxylate pathway in the metabolically engineered Escherichia coli under anaerobic conditions. In order to enhance the succinate biosynthesis specifically, the genes (i.e., ldhA, ptsG, ackA-pta, focA-pflB, adhE) that disrupt by-products biosynthesis pathways were combinatorially deleted, while the E. coli malate dehydrogenase (MDH) was overexpression. The highest succinate production of 150.78mM was obtained with YJ003 (ΔldhA, ptsG, ackA-pta), which were 5-folds higher than that obtained with wild type control strain DY329 (25.13mM). For further understand the metabolic response as a result of several genetic manipulations, an anaerobic stoichiometric model that takes into account the glyoxylate pathway have successfully been implemented to estimate the intracellular fluxes in various recombinant E. coli. The fraction to the glyoxylate pathway from OAA in DY329 was 0 and 31% in YJ003, which indicated that even without the absence of the iclR mutation; the glyoxylate pathway was also activated by deleting the by-products biosynthetic genes, and to be responsible for the higher succinate yields. For further strengthen glyoxylate pathway, a two-stage fed-batch fermentation process was developed by using a 600gl−1 glucose feed to achieve a cell growth rate of 0.07h−1 in aerobic fermentation, and using a 750gl−1 glucose feed to maintain the residual glucose concentration around 40gl−1 when its residual level decreased to 10gl−1 in anaerobic fermentation. The best mutant strain YJ003/pTrc99A-mdh produces final succinate concentration of 274mM by fed-batch culture, which was 10-folds higher than that obtained with wild type control strain DY329. This study discovered that glyoxylate pathway could be activated by deleting glyoxylate pathway irrelevant genes (i.e., genes encoding acetate and lactate) and consequently the succinate biosynthesis was effectively improved. This work provides useful information for the modification of metabolic pathway to improve succinate production.

Metabolic engineering of Escherichia coli for the production of 1-propanol

An engineered Escherichia coli strain that produces 1-propanol under aerobic condition was developed based on an l-threonine-overproducing E. coli strain. First, a feedback resistant ilvA gene encoding threonine dehydratase was introduced and the competing metabolic pathway genes were deleted. Further engineering was performed by overexpressing the cimA gene encoding citramalate synthase and the ackA gene encoding acetate kinase A/propionate kinase II, introducing a modified adhE gene encoding an aerobically functional AdhE, and by deleting the rpoS gene encoding the stationary phase sigma factor. Fed-batch culture of the final engineered strain harboring pBRthrABC-tac-cimA-tac-ackA and pTacDA-tac-adhEmut allowed production of 10.8gL−1 of 1-propanol with the yield and productivity of 0.107gg−1 and 0.144gL−1h−1, respectively, from 100gL−1 of glucose, and 10.3gL−1 of 1-propanol with the yield and productivity of 0.259gg−1 and 0.083gL−1h−1, respectively, from 40gL−1 glycerol.

Production of polyhydroxyalkanoates by Escherichia coli mutants with defected mixed acid fermentation pathways

A series of Escherichia coli BW25113 mutants with reduced mixed acid fermentation were constructed. Genes ackA-pta, poxB, ldhA, adhE, and pflB encoding acetate kinase, phosphate acetyltransferase, pyruvate oxidase, D: -lactate dehydrogenase, acetaldehyde dehydrogenase, and pyruvate formate-lyase, respectively, were deleted successively. When grown under microaerobic condition, the mutants reduced approximately 90% acetate excretion after the deletion of genes ackA-pta and poxB. Production of lactate, ethanol, and formate was also significantly reduced after the deletion of genes ldhA, adhE, and pflB, respectively. The accumulation of biomass and poly(3-hydroxybutyrate) (PHB) were significantly enhanced after deleting the mixed acid fermentation. E. coli mutant BWapld with deletions of ackA-pta, poxB, ldhA, and adhE produced twice the cell dry weight (CDW) and 3.5 times of PHB compared with its wild-type under microaerobic conditions. E. coli mutant BWapl with deletions of ackA-pta, poxB, and ldhA also achieved nearly twice CDW and three times of PHB content in comparison to the wild-type during 48 h static cultivation. Production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] was observed in the mutants under static cultivation. E. coli mutant BWapld could produce approximately 50 wt.% P(3HB-co-3HV) consisting of 5 mol% of 3-hydroxyvalerate (3HV) under aerobic conditions, when the seed culture was inoculated at an appropriate time. When ackA-pta, poxB, ldhA, adhE, and pflB were deleted, E. coli mutant BWapldf accumulated over 70 wt.% P(3HB-co-3HV) consisting of 8 mol% 3HV under aerobic conditions.

Engineering Static and Dynamic Control of Synthetic Pathways

Maximizing the production of a desired small molecule is one of the primary goals in metabolic engineering. Recent advances in the nascent field of synthetic biology have increased the predictability of small-molecule production in engineered cells growing under constant conditions. The next frontier is to create synthetic pathways that adapt to changing environments.

Acetate Consumption Activity Directly Determines the Level of Acetate Accumulation During Escherichia coli W3110 Growth

Escherichia coli excretes acetate during aerobic growth on glycolytic carbon sources, which has been explained as an overflow metabolism when the carbon flux into the cell exceeds the capacity of central metabolic pathways. Nonacetogenic growth of E. coli on gluconeogenic carbon sources like succinate or in carbon-limited slow growth conditions is believed an evidence for the explanation. However, we found that a strain defected in the acs (acetyl Co-A synthetase) gene, the product of which is involved in scavenging acetate, accumulated acetate even in succinate medium and in carbon-limited low growth rate condition, where as its isogenic parental strain did not. The acs promoter was inducible in noncatabolite repression condition, whereas the expression of the ackA-pta operon encoding acetate kinase and phosphotransacetylase for acetate synthesis was constitutive. Results in this study suggest that E. coli excretes and scavenges acetate simultaneously in the carbon-limited low growth condition and in nonacetogenic carbon source, and the activity of the acetate consumption pathway directly affects the accumulation level of acetate in the culture broth.

Analyses of the acetate-producing pathways in Corynebacterium glutamicum under oxygen-deprived conditions

Corynebacterium glutamicum R efficiently produces valuable chemicals from glucose under oxygen-deprived conditions. In an effort to reduce acetate as a byproduct, acetate productivity of several mutant-disrupted genes encoding possible key enzymes for acetate formation was determined. Disruption of the aceE gene that encodes the E1 enzyme of the pyruvate dehydrogenase complex resulted in almost complete elimination of acetate formation under oxygen-deprived conditions, implying that acetate synthesis under these conditions was essentially via acetyl-coenzyme A (CoA). Simultaneous disruption of pta, encoding phosphotransacetylase, and ack, encoding acetate kinase, resulted in no measurable change in acetate productivity. A mutant strain with disruptions in pta, ack and as-yet uncharacterized gene (cgR2472) exhibited 65% reduced acetate productivity compared to the parental strain, although a single disruption of cgR2472 exhibited no effect on acetate productivity. The gene cgR2472 was shown to encode a CoA-transferase (CTF) that catalyzes the formation of acetate from acetyl-CoA. These results indicate that PTA-ACK as well as CTF is involved in acetate production in C. glutamicum. This study provided basic information to reduce acetate production under oxygen-deprived conditions.

Ethanol Catabolism in Corynebacterium glutamicum

Corynebacterium glutamicum grows on a variety of carbohydrates and organic acids as single or combined sources of carbon and energy. Here we show the ability of C. glutamicum to grow on ethanol with growth rates up to 0.24 h^–1 and biomass yields up to 0.47 g dry weight (g ethanol)^–1. Mutants of C. glutamicum deficient in phosphotransacetylase (PTA), isocitrate lyase (ICL) and malate synthase (MS) were unable to grow on ethanol, indicating that acetate activation and the glyoxylate cycle are essential for utilization of this substrate. In accordance, the expression profile of ethanol-grown C. glutamicum cells compared to that of glucose-grown cells revealed an increased expression of genes encoding acetate kinase (AK), PTA, ICL and MS. Furthermore, the specific activities of these four enzymes as well as those of alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) were found to be high in ethanol-grown and low in glucose-grown cells. Growth of C. glutamicum on a mixture of glucose and ethanol led to a biphasic growth behavior, which was due to the sequential utilization of glucose before ethanol. Accordingly, the specific activities of ADH, ALDH, AK, PTA, ICL and MS in cells grown in medium containing both substrates were as low as in glucose-grown cells in the first growth phase, but increased 5- to 100-fold during the second growth phase. The results indicate that ethanol catabolism in C. glutamicum is subject to carbon source-dependent regulation, i.e., to a carbon catabolite control.

Transcription in Mycoplasma pneumoniae: Analysis of the Promoters of the ackA and ldh Genes

The nucleotide sequences that control transcription initiation and regulation in Mycoplasma pneumoniae are poorly understood. Moreover, only few regulatory events have been reported for M. pneumoniae. We have studied changes in the global protein synthesis pattern in M. pneumoniae in response to the presence of glycerol. The ackA and ldh genes, encoding acetate kinase and lactate dehydrogenase, respectively, were controlled in a carbon source-dependent manner. While the ackA gene was strongly expressed in the presence of glucose, transcription of ldh was induced by glycerol. The promoters of both genes were mapped by primer extension analysis. Molecular analysis of transcription regulatory mechanisms in M. pneumoniae has so far not been possible due to the lack of appropriate reporter systems that can be used to study the activity of promoter fragments and their mutant derivatives in vivo. Recently, a reporter system has been developed which allows cloning of promoter fragments in front of a promoterless lacZ gene and inserting this construct into the genome of M. pneumoniae. To study the requirements of M. pneumoniae RNA polymerase for promoter recognition, a series of fusions of deletion and mutant variants of the ldh promoter was constructed and analyzed in vivo. While mutations affecting the −10 region strongly interfered with gene expression, the −35 region seems to be of minor importance in M. pneumoniae.

Large‐loop antisense RNA hairpins provide stabilized and highly efficient antisense regulators in Escherichia coli

Antisense RNAs (ASRs) transcribed from expression plasmids specifically hybridize to target mRNA and inhibit gene expression in bacteria. They are useful tools for functional genomics but in Escherichia coli, the ASR method has shown limited efficiency. We demonstrate that the inhibition efficiency can be improved greatly by placing ASR between an artificial inverted repeat (IR) sequence. In one example, the non-essential ackA gene (encoding acetate kinase) was targeted and inhibited up to 75 %. RNA analysis showed that the ASR was abundant and stable and the target mRNA was degraded. In a second example, the essential fabI gene (encoding enol-ACP reductase) was targeted and the antisense effect prevented growth and sensitize cells to triclosan. We also show some application examples of this new ASR method.

Construction and characterization of ack knock‐out mutants of Propionibacterium acidipropionici for enhanced propionic acid fermentation

Propionibacterium acidipropionici produces propionic acid from glucose with acetic acid, succinic acid, and CO2 as byproducts. In this work, inactivation of ack gene, encoding acetate kinase (AK), by gene disruption and integrational mutagenesis was studied as a method to reduce acetate formation in propionic acid fermentation. The partial ack gene of approximately 750 bp in P. acidipropionici was cloned using a PCR-based method with degenerate primers and sequenced. The deduced amino acid sequence had 88% similarity and 76% identity with the amino acid sequence of AK from Bacillus subtilis. The partial ack gene was used to construct a linear DNA fragment with an inserted tetracycline resistance cassette and a nonreplicative integrational plasmid containing a tetracycline resistance gene cassette. These DNA constructs were then introduced into P. acidipropionici by electroporation, resulting in two mutants, ACK-Tet and TAT-ACK-Tet, respectively. Southern hybridization confirmed that the ack gene in the mutant ACK-Tet was disrupted by the inserted tetracycline resistance gene. As compared to the wild-type, the activities of AK were reduced by 26% and 43% in ACK-Tet and TAT-ACK-Tet mutants, respectively. The specific growth rate of these mutants was reduced by approximately 25% to 0.10/h (0.13/h for the wild-type), probably because of reduced acetate and ATP production. Both mutants produced approximately 14% less acetate from glucose. Although ack disruption alone did not completely eliminate acetate production, the propionate yield was increased by approximately 13%.

Microbial community of a mesophilic propionate-degrading methanogenic consortium in chemostat cultivation analyzed based on 16S rRNA and acetate kinase genes

We constructed a mesophilic anaerobic chemostat that was continuously fed with synthetic wastewater containing propionate as the sole source of carbon and energy. Steady-state conditions were achieved below the critical dilution rate of 0.3 d (-1) with almost complete substrate degradation. The propionate-degrading methanogenic communities in the chemostat at dilution rates of 0.01, 0.08, and 0.3 d (-1) were analyzed using molecular biological techniques. Fluorescence in situ hybridization with archaeal and bacterial domain-specific probes showed that archaeal cells predominated throughout the three dilution rates. Archaeal-16S rRNA gene clone library analysis and quantitative real-time polymerase chain reaction studies showed that hydrogenotrophic methanogen rRNA genes closely related to Methanoculleus was detected at a dilution rate of 0.01 d (-1) , whereas rRNA genes closely related to the Methanoculleus and Methanospirillum genera were detected at dilution rates of 0.08 and 0.3 d (-1) . The aceticlastic methanogen, Methanosaeta , was detected throughout the three dilution rates. Bacterial-rRNA gene clone library analysis and denaturing gradient gel electrophoresis demonstrated that rRNA genes affiliated with the genus Syntrophobacter predominated at the low dilution rate, whereas rRNA genes affiliated with the phylum Firmicutes predominated at the higher dilution rates. A significant number of rRNA genes affiliated with the genus Pelotomaculum were detected at dilution rate of 0.3 d (-1) . The diversity of genes encoding acetate kinase agreed closely with the results of the rRNA gene analysis. The dilution rates significantly altered the archaeal and bacterial communities in the propionate-fed chemostat.

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

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

Butyric acid and hydrogen production by Clostridium tyrobutyricum ATCC 25755 and mutants

Clostridium tyrobutyricum produces butyric acid, acetic acid, hydrogen and carbon dioxide as its main fermentation products. In this work, mutants with inactivated pta gene, encoding phosphotransacetylase (PTA) and ack gene, encoding acetate kinase (AK), were studied for their potential to improve butyric acid production in the fermentation. PTA and AK are two key enzymes in the acetate-producing pathway. PTA and AK activities in the pta-deleted mutant (PPTA-Em) were reduced by 44% and 91%, respectively, whereas AK activity in the ack-deleted mutant (PAK-Em) decreased by 50%. Meanwhile, the activity of butyrate kinase (BK) in PPTA-Em increased by 44% and hydrogenase activity in PAK-Em increased by 40%. As compared with the wild type, the specific growth rate of the mutants decreased by 32% (from 0.28 to 0.19 h −1) because of the impaired PTA-AK pathway. Meanwhile, butyric acid production by these mutants was improved greatly, with higher butyric acid yield (>0.4 g/g versus 0.34 g/g) and final concentration (43 g/L versus 29 g/L), which also indicated that the mutants had better tolerance to butyric acid inhibition. However, acetate production in the mutants was not significantly reduced even though more butyrate was produced from glucose, suggesting the existence of additional acetate forming pathway in C. tyrobutyricum. Also, hydrogen production by PAK-Em mutant increased significantly, with higher hydrogen yield (2.61 mol/mol glucose versus 1.35 mol/mol glucose) and H 2/CO 2 ratio (1.43 versus 1.08). The SDS-PAGE also showed significantly different expression levels of proteins with molecular mass around 32 and 70 kDa. These results suggested that integrational mutagenesis resulted in global metabolic shift and phenotypic changes, which also improved production of butyric acid and hydrogen from glucose in the fermentation.

Cloning of a gene encoding acetate kinase from Methanosarcina mazei 2-P isolated from a Japanese paddy field soil.

The ack gene encoding acetate kinase from the mesophilic Methanosarcina mazei 2-P, isolated from a paddy field soil in Japan, was cloned, sequenced, and functionally expressed in Escherichia coli. The terminal region of the putative pta gene, probably encoding phosphotransacetylase, was found upstream of the ack gene. The deduced amino acid sequence of the acetate kinase is 86.5% identical to that of the Methanosarcina thermophila acetate kinase. The activity of the His(6)-tagged acetate kinase purified from E. coli JM109 was optimal at 35 degrees C.

Adaptation of Lactobacillus sakei to meat: a new regulatory mechanism of ribose utilization?

Lactobacillus sakei is a lactic acid bacterium commonly found on meat and meat products. Meat is a rich substrate but contains small amounts of sugars, mainly glucose and ribose. The phosphotransferase system (PTS) is a multienzymatic complex responsible for sugar uptake. It is also involved in the regulation of metabolism through various mechanisms (catabolite activation and repression, inducer expulsion and exclusion). The ptsHI operon of L. sakei, encoding the general enzymes of the PTS, was studied and mutants were constructed. On ribose, these mutants grow twice as fast as the wild-type strain. This phenotype was never described in other bacteria and suggests that the PTS regulates ribose utilization. When compared to what is known from the regulations involving the PTS in other bacteria, this mechanism might be new. In Bacillus subtilis and Escherichia coli, in which ribose catabolism was investigated, ribose is transported by an ABC transporter, encoded by rbsABCD genes and then phosphorylated by the rbsK encoded ribose kinase. Ribose-5P is then metabolized through the pentose-P pathway involving xylulose-5P phosphoketolase and acetate kinase. Whereas phosphoketolase and acetate kinase activities remained unchanged in L. sakei ptsI mutants, ribose kinase activity and uptake were increased by a factor of 2.5 and 1.5, respectively. The target of the PTS regulation would thus be transport and/or phosphorylation of ribose. The gene cluster encoding a ribose transporter, ribose kinase and a regulator was cloned and sequenced. In L. sakei no gene encoding RbsA, RbsB or RbsC could be found. However, rbsD was present as well as a new gene rbsU, encoding a protein homologous to a glucose transporter responsible for facilitated diffusion of glucose. The rbsUDK operon is induced by ribose via the regulator RbsR encoded by rbsR located downstream of rbsUDK. In ptsI mutants, this operon was not overexpressed on ribose. This shows that the regulation of ribose utilization is not a transcriptional regulation. Upstream from the rbs operon, a gene encoding acetate kinase (ackA) was found. In other bacteria in which these genes were identified, ackA and the rbs operon are not linked. Moreover, in B. subtilis, ackA is regulated by catabolite activation whereas the rbs operon is repressed by catabolite repression, two mechanisms involving the PTS. In L. sakei, ackA and the rbs operon are adjacent on the