Spectral deconvolution of redox species in the crotonyl-CoA-dependent NADH:ferredoxin oxidoreductase from Megasphaera elsdenii. A flavin-dependent bifurcating enzyme.

We have undertaken a spectral deconvolution of the three FADs of EtfAB/bcd to the spectral changes seen in the course of reduction, including the spectrally distinct anionic and neutral semiquinone states of electron-transferring and bcd flavins. We also demonstrate that, unlike similar systems, no charge-transfer complex is observed on titration of the reduced M. elsdenii EtfAB with NAD+. Finally, and significantly, we find that removal of the et FAD from EtfAB results in an uncrossing of the half-potentials of the bifurcating FAD that remains in the protein, as reflected in the accumulation of substantial FAD•- in the course of reductive titrations of the depleted EtfAB with sodium dithionite.

Rational design of thiolase substrate specificity for metabolic engineering applications.

Metabolic engineering efforts require enzymes that are both highly active and specific toward the synthesis of a desired output product to be commercially feasible. The 3-hydroxyacid (3HA) pathway, also known as the reverse ß-oxidation or coenzyme-A-dependent chain-elongation pathway, can allow for the synthesis of dozens of useful compounds of various chain lengths and functionalities. However, this pathway suffers from byproduct formation, which lowers the yields of the desired longer chain products, as well as increases downstream separation costs. The thiolase enzyme catalyzes the first reaction in this pathway, and its substrate specificity at each of its two catalytic steps sets the chain length and composition of the chemical scaffold upon which the other downstream enzymes act. However, there have been few attempts reported in the literature to rationally engineer thiolase substrate specificity. In this study, we present a model-guided, rational design study of ordered substrate binding applied to two biosynthetic thiolases, with the goal of increasing the ratio of C6/C4 products formed by the 3HA pathway, 3-hydroxy-hexanoic acid and 3-hydroxybutyric acid. We identify thiolase mutants that result in nearly 10-fold increases in C6/C4 selectivity. Our findings can extend to other pathways that employ the thiolase for chain elongation, as well as expand our knowledge of sequence-structure-function relationship for this important class of enzymes.

Engineering an [FeFe]-Hydrogenase: Do Accessory Clusters Influence O2 Resistance and Catalytic Bias?

[FeFe]-hydrogenases, HydAs, are unique biocatalysts for proton reduction to H2. However, they suffer from a number of drawbacks for biotechnological applications: size, number and diversity of metal cofactors, oxygen sensitivity. Here we show that HydA from Megasphaera elsdenii (MeHydA) displays significant resistance to O2. Furthermore, we produced a shorter version of this enzyme (MeH-HydA), lacking the N-terminal domain harboring the accessory FeS clusters. As shown by detailed spectroscopic and biochemical characterization, MeH-HydA displays the following interesting properties. First, a functional active site can be assembled in MeH-HydA in vitro, providing the enzyme with excellent hydrogenase activity. Second, the resistance of MeHydA to O2 is conserved in MeH-HydA. Third, MeH-HydA is more biased toward proton reduction than MeHydA, as the result of the truncation changing the rate limiting steps in catalysis. This work shows that it is possible to engineer HydA to generate an active hydrogenase that combines the resistance of the most resistant HydAs and the simplicity of algal HydAs, containing only the H-cluster.

Valerate production by Megasphaera elsdenii isolated from pig feces.

Megasphaera elsdenii is able to produce several short-chain fatty acids (SCFAs), such as acetate, propionate, butyrate, and valerate. These SCFAs serve as an energy source for host animals and play an important role in gut health. In this study, M. elsdenii was isolated from pig feces that had been collected from two farms located in distinct areas of Japan. These M. elsdenii isolates were genotyped, and 7 representative strains were selected. When these 7 strains and M. elsdenii JCM 1772T were cultured with lactate for 24 h, all 7 strains produced valerate as a predominant SCFA. Therefore, the valerate-producing M. elsdenii inhabits a wide area of Japan. In contrast, M. elsdenii JCM 1772T produced acetate, propionate, butyrate, and valerate at similar levels. When the Y2 strain, one of the 7 representative strains, was cultured without lactate, low levels of valerate accumulated. In contrast, in a time course of lactate fermentation by the Y2 strain, lactate was rapidly consumed, and acetate and propionate were produced after 6 h of incubation. Thereafter, acetate and propionate were consumed from 6 to 12 h after the start of the incubation, and valerate and butyrate were produced. In most of the previously described M. elsdenii strains, valerate was not a predominant SCFA. Therefore, the M. elsdenii Y2 strain showed an unique metabolism in which valerate was produced as a primary end product of lactate fermentation.

Preferential isolation of Megasphaera elsdenii from pig feces.

Lactic acid produced by intestinal bacteria is fermented by lactate-utilizing bacteria. In this study, we developed a selective culture medium (KMI medium) for Megasphaera elsdenii, a lactate-utilizing bacterium that is abundant in pig intestines. Supplementation of the medium with lactate and beef extract powder was necessary for the preferential growth of M. elsdenii. In addition, we designed a species-specific primer set to detect M. elsdenii. When pig fecal samples were plated on KMI agar medium, approximately 60-100% of the resulting colonies tested positive using the M. elsdenii-specific PCR primers. In fact, nearly all of the large, yellow-white colonies that grew on the KMI agar medium tested positive by PCR with this primer set. The 16S rRNA gene sequences of three representative PCR-positive strains showed strong similarities to that of M. elsdenii ATCC 25940T (98.9-99.2% identity). These three strains were approximately 1.5 µm sized cocci that were primarily arranged in pairs, as was observed for M. elsdenii JCM 1772T. The selective KMI medium and species-specific primer set developed in this study are useful for the isolation and detection of M. elsdenii and will be useful in research aimed at increasing our understanding of intestinal short-chain fatty acid metabolism in pigs.

Biosynthesis of poly(glycolate-co-lactate-co-3-hydroxybutyrate) from glucose by metabolically engineered Escherichia coli.

Metabolically engineered Escherichia coli strains were constructed to effectively produce novel glycolate-containing biopolymers from glucose. First, the glyoxylate bypass pathway and glyoxylate reductase were engineered such as to generate glycolate. Second, glycolate and lactate were activated by the Megasphaera elsdenii propionyl-CoA transferase to synthesize glycolyl-CoA and lactyl-CoA, respectively. Third, ß-ketothiolase and acetoacetyl-CoA reductase from Ralstonia eutropha were introduced to synthesize 3-hydroxybutyryl-CoA from acetyl-CoA. At last, the Ser325Thr/Gln481Lys mutant of polyhydroxyalkanoate (PHA) synthase from Pseudomonas sp. 61-3 was over-expressed to polymerize glycolyl-CoA, lactyl-CoA and 3-hydroxybutyryl-CoA to produce poly(glycolate-co-lactate-co-3-hydroxybutyrate). The recombinant E. coli was able to accumulate the novel terpolymer with a titer of 3.90g/l in shake flask cultures. The structure of the resulting polymer was chemically characterized by proton NMR analysis. Assessment of thermal and mechanical properties demonstrated that the produced terpolymer possessed decreased crystallinity and improved toughness, in comparison to poly(3-hydroxybutyrate) homopolymer. This is the first study reporting efficient microbial production of poly(glycolate-co-lactate-co-3-hydroxybutyrate) from glucose.