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The mechanisms of energy generation and carbon-source utilization in the syphilis spirochete Treponema pallidum have remained enigmatic despite complete genomic sequence information. Whereas the bacterium harbors enzymes for glycolysis, the apparatus for more efficient use of glucose catabolites, namely the citric-acid cycle, is apparently not present. Yet, the organism's energy needs likely exceed the modest output from glycolysis alone. Recently, building on our structure-function studies of T. pallidum lipoproteins, we proposed a "flavin-centric" metabolic lifestyle for the organism that partially resolves this conundrum. As a part of the hypothesis, we have proposed that T. pallidum contains an acetogenic energy-conservation pathway that catabolizes D-lactate, yielding acetate, reducing equivalents for the generation and maintenance of chemiosmotic potential, and ATP. We already have confirmed the D-lactate dehydrogenase activity in T. pallidum necessary for this pathway to operate. In the current study, we focused on another enzyme ostensibly involved in treponemal acetogenesis, phosphotransacetylase (Pta). This enzyme is putatively identified as TP0094 and, in this study, we determined a high-resolution (1.95 Å) X-ray crystal structure of the protein, finding that its fold comports with other known Pta enzymes. Further studies on its solution behavior and enzyme activity confirmed that it has the properties of a Pta. These results are consistent with the proposed acetogenesis pathway in T. pallidum, and we propose that the protein be referred to henceforth as TpPta., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Brautigam et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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

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

Background: Targetrons are gene targeting vectors derived from mobile group II introns. They consist of an autocatalytic intron RNA (a "ribozyme") and an intron-encoded reverse transcriptase, which use their combined activities to achieve highly efficient site-specific DNA integration with readily programmable DNA target specificity., Methodology/principal Findings: Here, we used a mobile group II intron from the thermophilic cyanobacterium Thermosynechococcus elongatus to construct a thermotargetron for gene targeting in thermophiles. After determining its DNA targeting rules by intron mobility assays in Escherichia coli at elevated temperatures, we used this thermotargetron in Clostridium thermocellum, a thermophile employed in biofuels production, to disrupt six different chromosomal genes (cipA, hfat, hyd, ldh, pta, and pyrF). High integration efficiencies (67-100% without selection) were achieved, enabling detection of disruptants by colony PCR screening of a small number of transformants. Because the thermotargetron functions at high temperatures that promote DNA melting, it can recognize DNA target sequences almost entirely by base pairing of the intron RNA with less contribution from the intron-encoded protein than for mesophilic targetrons. This feature increases the number of potential targetron-insertion sites, while only moderately decreasing DNA target specificity. Phenotypic analysis showed that thermotargetron disruption of the genes encoding lactate dehydrogenase (ldh; Clo1313_1160) and phosphotransacetylase (pta; Clo1313_1185) increased ethanol production in C. thermocellum by decreasing carbon flux toward lactate and acetate., Conclusions/significance: Thermotargetron provides a new, rapid method for gene targeting and genetic engineering of C. thermocellum, an industrially important microbe, and should be readily adaptable for gene targeting in other thermophiles.