Pyruvate Formate-lyase Gene Research Articles

Metal additives for boosting hydrogen production in anaerobic fermentation: Focus on the change of gene expression and cost analysis

Dark fermentation has emerged as a promising strategy for sustainable hydrogen production. However, the low hydrogen yield produced by anaerobic microbes continues to be a challenge. Although metal ion additives have been proposed to enhance hydrogen production, their entire impacts and underlying mechanisms are poorly understood. The present study aimed to examine the effects of Fe2+, Ni2+, Mg2+, and Mo6+ on dark fermentative hydrogen production, as well as their influences on microbial community analysis and gene expression in a batch system. The experiments were conducted under anaerobic conditions at 37 °C, utilizing 100 mM glucose as a substrate and an initial pH of 6.8. The results showed that Mo6+ at 0.015 mg-Mo6+/L significantly enhanced the hydrogen yield (2.21-fold increment) by upregulating nitrogenase gene expression (2.19-fold increment). Fe2+ at 50 mg-Fe2+/L was the second most effective metal ion, with a 1.81-fold increase in hydrogen yield and upregulation of ferredoxin (1.83-fold increment) and pyruvate formate-lyase gene (3.36-fold increment) expression. Mg2+ at 70 mg-Mg2+/L and Ni2+ at 25 mg-Ni2+/L also increased hydrogen yield by 1.51-fold and 1.31-fold, respectively, with upregulation of glyceraldehyde-3-phosphate dehydrogenase (1.02-fold and 1.81-fold increments, respectively) and ATP synthase (1.46-fold and 1.10-fold increments, respectively) expressions for both metal additives, pyruvate formate-lyase (2.77-fold increment) and pyruvate kinase (1.44-fold increment) gene expression for Mg2+. Regarding the mixed metal approach, it was observed that although certain combinations of mixed metals, such as Mg2++Mo6+, resulted in an enhancement of hydrogen yield by a factor of 1.37-fold, the increment was found to be lower than that of an individual metal. Therefore, the results of this study suggest that using a single metal would be more effective. Economic potential calculations further revealed that Mo6+ exhibited the highest economic feasibility, with the lowest expense of 2.02x10−5 $-metal/m3-H2, accounting for 0.002% of the average total production cost. This was followed by Mg2+, Fe2+, and Ni2+. Overall, our findings provide mechanistic insights into the use of metal ion additives for clean hydrogen production and offer a foundation for optimising their economic feasibility in large-scale hydrogen production.

Visible-Light-Driven Enhanced Biohydrogen Production by Photo-Biohybrid System Based on Photoelectron Transfer between Intracellular Photosensitizer Gold Nanoparticles and Clostridium butyricum

Biohydrogen is a clean and renewable energy, but the low yield caused by the lack of reducing power in cells greatly restricts the industrialization of biohydrogen production. Photo-biohybrid systems (PBSs) can integrate the high light energy utilization efficiency of photocatalysts with the excellent catalytic performance of microorganisms. Here, gold nanoparticles were targeted into Clostridium butyricum as intracellular photosensitizers to construct a PBS that could efficiently produce biohydrogen under visible light, with the apparent quantum yield as high as 19.31%. Compared with the dark-fermented C. butyricum, the biohydrogen production of PBS increased by 88.74%. The mechanism of photoelectrons from Au NPs to C. butyricum was elucidated by the transcriptome. Compared with the dark-fermented biohybrids, the expressions of biohydrogen generation-related enzymes, such as hydrogenase and pyruvate formate lyase genes, in the PBS were all upregulated more than 2 times. Furthermore, the genes of riboflavin synthase, electron transfer flavoprotein (ETF), and FAD-dependent oxidoreductase, which are closely related to electron transformation, were all significantly upregulated. The photoelectrons were transferred to the hydrogenase via ETF and FAD2+ to enhance biohydrogen production, independent of pyruvate decomposition. This PBS provides theoretical guidance for constructing an efficient light-driven microbial manufacturing system.

Metabolic Engineering of Central Carbon Metabolism of Bacillus licheniformis for Enhanced Production of Poly-γ-glutamic Acid.

Poly-γ-glutamic acid (γ-PGA) is an anionic polymer with wide-ranging applications in the areas of medicine, light chemical industry, wastewater treatment, and agriculture. However, the production cost of γ-PGA is high for the requirement of adding the expensive precursor L-glutamic acid during fermentation, which hinders its widespread application. In this study, in order to improve γ-PGA yield, central carbon metabolism was engineered to enhance the carbon flux of tricarboxylic acid (TCA) cycle and glutamic acid synthesis in a γ-PGA production strain Bacillus licheniformis WX-02. Firstly, pyruvate dehydrogenase (PdhABCD) and citrate synthase (CitA) were overexpressed to strengthen the flux of pyruvate into TCA cycle, resulting in 34.93% and 11.14% increase of γ-PGA yield in B. licheniformis WX-02, respectively. Secondly, the carbon flux to glyoxylate shunt was rewired via varying the expression of isocitrate lyase (AceA), and a 23.24% increase of γ-PGA yield was obtained in AceA down-regulated strain WXPbacAaceBA. Thirdly, deletion of pyruvate formate-lyase gene pflB led to a 30.70% increase of γ-PGA yield. Finally, combinatorial metabolic engineering was applied, and γ-PGA titer was enhanced to 12.02g/L via overexpressing pdhABCD and citA, repressing aceA, and deleting pflB, with a 69.30% improvement compared to WX-02. Collectively, metabolic engineering of central carbon metabolism is an effective strategy for enhanced γ-PGA production in B. licheniformis, and this research provided a promising strain for industrial production of γ-PGA.

Relative significances of pH and substrate starch level to roles of Streptococcus bovis S1 in rumen acidosis.

To clarify the relative importance of pH and substrate starch level in fermentation characteristics and regulatory mechanism of Streptococcus bovis S1 in rumen acidosis, an in vitro fermentation of three levels of soluble starch (1, 3 and 9 g/L) was established with pH in the media were maintained constant at 5.5 or 6.5. The results showed that the dominant product of S. bovis S1 was lactate at both pH, the production depended on the starch level, and more lactate was produced at pH 6.5 than that at pH 5.5 (P < 0.001). At pH 5.5, the activity of lactate dehydrogenase (LDH) and α-amylase (α-AMY), their abundances, the relative expressions of LDH, PFL (gene encoding pyruvate formate-lyase), CCPA (gene encoding global catabolite control protein A) and α-AMY genes were higher than those at pH 6.5 (P < 0.05), whereas the concentration of fructose-1,6-diphosphate (FDP) was lower. The activity of LDH, α-AMY and FDP, and the relative expressions of LDH, PFL, CCPA and α-AMY genes were, in general, positively related to the starch level. The canonical regression analysis indicated that the pH had more profound effect compared with the starch level, in terms of the acid productions, enzyme activity and gene expressions. It was concluded that the fermentation of S. bovis was regulated at the transcription level in response to both pH and substrate starch concentration, but more sensitive to pH changes.

&lt;i&gt;In silico&lt;/i&gt; Evaluation of the Effect of Pfl Gene Knockout on the Production of D-lactate by &lt;i&gt;Escherichia coli&lt;/i&gt; Genome Scale Model Using the Optflux Software Platform

The increase availability of genome scale metabolic models of Escherichia coli and computational successes is revolutionizing the field of metabolic engineering and synthetic microbiology. E. coli has been experimentally established to produce D-lactate under micro-aerobic conditions when pyruvate formate lyase (PFL) genes are knocked out. However, investigation on the in silico prediction and for evaluation of the effect of PFL genes knockout on the production of D-lactate using E. coli genome scale metabolic model with regulatory on/off minimization (ROOM) under the OptFlux software platform remained under explored. Here, we demonstrate that metabolic engineering strategies using the OptFlux software platform by gene knockout simulation of pflA/b0902, pflB/b0903, pflC/b3952 and pflD/b3951 have been predicted to increase D-lactate production in E. coli and hence maintaining a growth rate that is 96% of the wild-type model. The deletion of the PFL genes have been established to increase D-lactate production in E. coli . The results obtained in this study is in agreement with the previously established experimental studies. These findings suggests that the OptFlux software platform using ROOM as the simulation algorithm, can prospectively and effectively predict future metabolic engineering targets for increased D-lactate production in E. coli and/or other microbial chemical syntheses.

Improvement of 2,3-butanediol yield in Klebsiella pneumoniae by deletion of the pyruvate formate-lyase gene.

Klebsiella pneumoniae is considered a good host strain for the production of 2,3-butanediol, which is a promising platform chemical with various industrial applications. In this study, three genes, including those encoding glucosyltransferase (wabG), lactate dehydrogenase (ldhA), and pyruvate formate-lyase (pflB), were disrupted in K. pneumoniae to reduce both its pathogenic characteristics and the production of several by-products. In flask cultivation with minimal medium, the yield of 2,3-butanediol from rationally engineered K. pneumoniae (ΔwabG ΔldhA ΔpflB) reached 0.461 g/g glucose, which was 92.2% of the theoretical maximum, with a significant reduction in by-product formation. However, the growth rate of the pflB mutant was slightly reduced compared to that of its parental strain. Comparison with similar mutants of Escherichia coli suggested that the growth defect of pflB-deficient K. pneumoniae was caused by redox imbalance rather than reduced level of intracellular acetyl coenzyme A (acetyl-CoA). From an analysis of the transcriptome, it was confirmed that the removal of pflB from K. pneumoniae significantly repressed the expression of genes involved in the formate hydrogen lyase (FHL) system.

Molecular characterization and expression of pyruvate formate-lyase-activating enzyme in a ruminal bacterium, Streptococcus bovis.

To clarify the significance of the activation of pyruvate formate-lyase (PFL) by PFL-activating enzyme (PFL-AE) in Streptococcus bovis, the molecular properties and gene expression of PFL-AE were investigated. S. bovis PFL-AE was deduced to consist of 261 amino acids with a molecular mass of 29.9 kDa and appeared to be a monomer protein. Similar to Escherichia coli PFL-AE, S. bovis PFL-AE required Fe(2+) for activity. The gene encoding PFL-AE (act) was found to be polycistronic, and the PFL gene (pfl) was not included. However, the act mRNA level changed in parallel with the pfl mRNA level, responding to growth conditions, and the change was contrary to the change in the lactate dehydrogenase (LDH) mRNA level. PFL-AE synthesis appeared to change in parallel with PFL synthesis. Introduction of a recombinant plasmid containing S. bovis pfl and the pfl promoter into S. bovis did not affect formate and lactate production, which suggests that the activity of the pfl promoter is low. When the pfl promoter was replaced by the S. bovis ldh promoter, PFL was overexpressed, which caused an increase in the formate-to-lactate ratio. However, when PFL-AE was overexpressed, the formate-to-lactate ratio did not change, suggesting that PFL-AE was present at a level that was high enough to activate PFL. When both PFL-AE and PFL were overexpressed, the formate-to-lactate ratio further increased. It is conceivable that LDH activity is much higher than PFL activity, which may explain why the formate-to-lactate ratio is usually low.

Cloning and sequence analysis of the pfl gene encoding pyruvate formate-lyase from Streptococcus mutans

We have isolated a sorbitol-negative mutant of Streptococcus mutans GS-5 following random mutagenesis with plasmid pVA891 clone banks. This mutant did not metabolize sorbitol anaerobically but did so aerobically. A 10-kb chromosomal DNA fragment flanking the pVA891 insertion was deleted in this mutant. The corresponding region from the parental strain GS-5 was then recovered by a marker rescue method with Escherichia coli. The pyruvate formate-lyase gene, pfl, was identified within a 3-kb PstI-XbaI fragment located in the middle of the deleted region of the chromosome, and its inactivation in S. mutans produced the same sorbitol-negative phenotype. Nucleotide sequence analysis of the pfl gene revealed a 2.3-kb open reading frame (ORF) preceded by potential ribosome-binding and promoter-like sequences. The ORF specified a putative protein of 775 amino acid residues with a calculated molecular weight of 87,533. The amino acid sequence deduced from the ORF exhibited significant similarity to that of the E. coli pfl gene.

Integration host factor is required for anaerobic pyruvate induction of pfl operon expression in Escherichia coli.

The expression of the pyruvate formate-lyase gene (pfl) is induced by anaerobic growth, and this is increased further by growth in pyruvate. Previous work has shown that anaerobic induction is strongly dependent on the activator FNR and partially dependent on a second transcription factor, ArcA, while pyruvate induction only required FNR. Anaerobic and pyruvate regulation both require the presence of a 5' nontranslated regulatory sequence which spans approximately 500 bp of DNA. A mobility shift assay was developed to identify proteins that bind to this regulatory region. Several binding activities were separated by heparin agarose chromatography, and one of these activities was characterized and shown to be integration host factor (IHF). Mobility shift and DNase I footprinting experiments defined a single IHF binding site in the pfl promoter-regulatory region. With pfl-lacZ fusions, it could be shown that introduction of a himD mutation abolished pyruvate-dependent induction of anaerobic expression in vivo. The same result was observed when the pfl IHF binding site was mutated. In addition, the partial anaerobic induction of expression found in an fnr strain was completely blocked in an fnr himD double mutant and in an fnr IHF binding site double mutant. Taken together, these data suggest that IHF is necessary for both pyruvate induction and the anaerobic induction mediated by ArcA.

Anaerobic induction of pyruvate formate-lyase gene expression is mediated by the ArcA and FNR proteins.

The pyruvate formate-lyase (pfl) gene of Escherichia coli is transcribed from seven promoters which are coordinately induced 12- to 15-fold by anaerobiosis. The FNR protein plays a major role in the anaerobic control of this system. A mutation in the fnr gene, however, only reduces anaerobic induction fivefold, indicating that FNR is not the only factor involved in the anaerobic activation process (Sawers and Böck, J. Bacteriol. 171:2485-2498, 1989). The residual anaerobic induction could be shown to be imparted by the transcriptional regulator ArcA; an arcA fnr double mutant was incapable of inducing pfl transcription anaerobically. A mutant strain unable to synthesize the membrane-associated histidine kinase (ArcB) that has been proposed to activate ArcA showed the same phenotype as an arcA mutant strain, indicating that a functional ArcB protein is also required for wild-type, anaerobic pfl transcriptional activation. Nuclease S1 analysis revealed that an arcA mutation abolished anaerobic transcription from promoter 7 and reduced expression from promoter 6 but did not affect transcription from promoters 1 to 5. On the other hand, an fnr mutation prevented anaerobic expression from promoters 6 and 7 and reduced transcription from promoters 1 to 5. These data indicate that both ArcA and FNR are essential for anaerobic activation of promoter 7 transcription, which suggests functional interaction between these proteins.

Genetic improvement of Escherichia coli for ethanol production: chromosomal integration of Zymomonas mobilis genes encoding pyruvate decarboxylase and alcohol dehydrogenase II

Zymomonas mobilis genes for pyruvate decarboxylase (pdc) and alcohol dehydrogenase II (adhB) were integrated into the Escherichia coli chromosome within or near the pyruvate formate-lyase gene (pfl). Integration improved the stability of the Z. mobilis genes in E. coli, but further selection was required to increase expression. Spontaneous mutants were selected for resistance to high level of chloramphenicol that also expressed high levels of the Z. mobilis genes. Analogous mutants were selected for increased expression of alcohol dehydrogenase on aldehyde indicator plates. These mutants were functionally equivalent to the previous plasmid-based strains for the fermentation of xylose and glucose to ethanol. Ethanol concentrations of 54.4 and 41.6 g/liter were obtained from 10% glucose and 8% xylose, respectively. The efficiency of conversion exceeded theoretical limits (0.51 g of ethanol/g of sugar) on the basis of added sugars because of the additional production of ethanol from the catabolism of complex nutrients. Further mutations were introduced to inactivate succinate production (frd) and to block homologous recombination (recA).

Novel transcriptional control of the pyruvate formate-lyase gene: upstream regulatory sequences and multiple promoters regulate anaerobic expression.

The sequence of the 5' regulatory region of the gene encoding pyruvate formate-lyase is presented together with a detailed analysis of the transcriptional signals required for its expression. The sequence data revealed that a gene coding for an open reading frame (orf) of unknown function is situated just upstream of the pfl gene. Analysis of RNA transcripts by Northern blot hybridization demonstrated that the genes for orf and pfl were cotranscribed as an operon but that the pfl gene was also transcribed alone. S1 nuclease protection analysis, primer extension, and construction of lacZ fusions with sequential deletions in the pfl 5' regulatory sequence revealed that transcription initiated from at least six promoters which spanned 1.2 kilobases of DNA. Three of these lay within the orf structural gene and were responsible for the high expression of pfl. All transcripts originating from these promoters terminated in the 3' untranslated region of the pfl gene at a strong rho-independent transcription terminator. All of the promoters were coordinately regulated by anaerobiosis, pyruvate, nitrate, and the fnr gene product, and the sequences thought to be responsible for this regulation lay 0.8 to 1.3 kilobases upstream of the translational initiation codon of the pfl gene. There were two sequences within this region which showed strong homology with that proposed to be required for recognition by the Fnr protein.