Production of polyhydroxyalkanoate copolymers containing 4-hydroxybutyrate in engineered Bacillus megaterium.

Despite being used as a common platform for the commercial production of many biochemicals, Bacilli are often overlooked as a source of industrial polyhydroxyalkanoates (PHAs), biodegradable plastic replacements. In addition to having a robust expression system, the lack of lipopolysaccharides and ease of lysis make Bacilli an attractive host for the production of PHAs. In this work, a Bacillus megaterium strain was engineered to generate poly(3-hydroxybutyrate-co-4-hydroxybutryate) (P[3HB-co-4HB]) copolymers, which are among the most useful and industrially-relevant copolymers. These copolymers had lower modulus and increased toughness, thus making the copolymer suitable for a broader range of applications. Due to high metabolic flux through succinate, the engineered B. megaterium strain produced P(3HB-co-4HB) with >10% mol fraction 4HB from glucose, without the use of highly regulated and expensive precursors or potentially damaging truncation of central biochemical pathways.

Biochemical characterization of Caulobacter crescentus xylose dehydrogenase.

d-Xylose sugar is a common component of hemicellulose, the second largest fraction of biomass. Many groups have developed biological conversions of d-xylose to value-added products by recombinant expression of the xylose dehydrogenase enzyme from Caulobacter crescentus. This enzyme uses NAD+ as a cofactor to oxidize d-xylose to d-xylono-1,4-lactone. A detailed understanding of the mechanism of this enzyme could be useful in engineering more efficient versions. Therefore, we have conducted kinetic studies including both the forward and reverse physiological reactions of this enzyme. We demonstrate that the enzyme's substrate binding mode follows a sequential steady state ordered mechanism with NAD+ or NADH binding first. Furthermore, the kcat of the reaction in the direction of NAD+ reduction is 10-fold higher than that of the reverse reaction. From rapid reaction studies, we demonstrate the binding of NAD+ and NADH to the free enzyme and that hydride transfer occurs in a fast step followed by a much slower steady state. We calculate that the dissociations of the sugar products from the enzyme complexes are the major rate limiting steps in both directions.

Absence or presence of metal ion activation in two structurally similar GH43 ß-xylosidases.

Two GH43 ß-xylosidases, RS223-BX from a rice straw metagenomic library, and BoXA from Bacteroides ovatus, that share similar amino acid sequences (81% identical) and 19 of 20 active-site residues, were compared by using site-directed mutagenesis of Asp and His residues implicated in metal binding. Thus, RS223-BX is strongly activated by divalent-metal cations and the previously published X-ray structure of this enzyme shows that a Ca2+ cation is chelated by an active-site Asp carboxyl group and an active-site His. Mutation to Ala causes 90% loss of activity for the Asp mutant and 98% loss of activity for the His mutant, indicating their importance to catalysis. For the other enzyme (BoXA), mutation to Ala causes 20% loss of activity for the His mutant and 40% gain of activity for the Asp mutant, indicating the lack of importance for activity of the native residues and the lack of metal-dependency, given that the Asp residue occupies the active site to secure the metal cation in known metal ion dependent GH43 xylosidases. The high activity of the BoXA mutants compared to that of the analogous RS223-BX mutants further undermines the possibility that BoXA maintains a tightly bound metal cofactor resistant to EDTA extraction. The results strengthen our conclusion that the very similar proteins differ in one being metal ion dependent and one not.

Production of D-Xylonic Acid from Hemicellulose Using Artificial Enzyme Complexes.

Lignocellulosic biomass represents a potentially large resource to supply the world's fuel and chemical feedstocks. Enzymatic bioconversion of this substrate offers a reliable strategy for accessing this material under mild reaction conditions. Owing to the complex nature of lignocellulose, many different enzymatic activities are required to function in concert to perform efficient transformation. In nature, large multienzyme complexes are known to effectively hydrolyze lignocellulose into constituent monomeric sugars. We created artificial complexes of enzymes, called rosettazymes, in order to hydrolyze glucuronoxylan, a common lignocellulose component, into its cognate sugar D-xylose and then further convert the D-xylose into D-xylonic acid, a Department of Energy top-30 platform chemical. Four different types of enzymes (endoxylanase, α-glucuronidase, ß-xylosidase, and xylose dehydrogenase) were incorporated into the artificial complexes. We demonstrated that tethering our enzymes in a complex resulted in significantly more activity (up to 71%) than the same amount of enzymes free in solution. We also determined that varying the enzyme composition affected the level of complex-related activity enhancement as well as overall yield.

Production of Glucaric Acid from Hemicellulose Substrate by Rosettasome Enzyme Assemblies.

Hemicellulose biomass is a complex polymer with many different chemical constituents that can be utilized as industrial feedstocks. These molecules can be released from the polymer and transformed into value-added chemicals through multistep enzymatic pathways. Some bacteria produce cellulosomes which are assemblies composed of lignocellulolytic enzymes tethered to a large protein scaffold. Rosettasomes are artificial engineered ring scaffolds designed to mimic the bacterial cellulosome. Both cellulosomes and rosettasomes have been shown to facilitate much higher rates of biomass hydrolysis compared to the same enzymes free in solution. We investigated whether tethering enzymes involved in both biomass hydrolysis and oxidative transformation to glucaric acid onto a rosettasome scaffold would result in an analogous production enhancement in a combined hydrolysis and bioconversion metabolic pathway. Three different enzymes were used to hydrolyze birchwood hemicellulose and convert the substituents to glucaric acid, a top-12 DOE value added chemical feedstock derived from biomass. It was demonstrated that colocalizing the three different enzymes to the synthetic scaffold resulted in up to 40 % higher levels of product compared to uncomplexed enzymes.

Isolation and characterization of a novel GH67 α-glucuronidase from a mixed culture.

Hemicelluloses represent a large reservoir of carbohydrates that can be utilized for renewable products. Hydrolysis of hemicellulose into simple sugars is inhibited by its various chemical substituents. The glucuronic acid substituent is removed by the enzyme α-glucuronidase. A gene (deg75-AG) encoding a putative α-glucuronidase enzyme was isolated from a culture of mixed compost microorganisms. The gene was subcloned into a prokaryotic vector, and the enzyme was overexpressed and biochemically characterized. The DEG75-AG enzyme had optimum activity at 45 °C. Unlike other α-glucuronidases, the DEG75-AG had a more basic pH optimum of 7-8. When birchwood xylan was used as substrate, the addition of DEG75-AG increased hydrolysis twofold relative to xylanase alone.

Isolation of α-glucuronidase enzyme from a rumen metagenomic library.

α-Glucuronidase enzymes play an essential role in the full enzymatic hydrolysis of hemicellulose. Up to this point, all genes encoding α-glucuronidase enzymes have been cloned from individual, pure culture strains. Using a high-throughput screening strategy, we have isolated the first α-glucuronidase gene (rum630-AG) from a mixed population of microorganisms. The gene was subcloned into a prokaryotic vector, and the enzyme was overexpressed and biochemically characterized. The RUM630-AG enzyme had optimum activity at pH 6.5 and 40 °C. When birchwood xylan was used as substrate, the RUM630-AG functioned synergistically with an endoxylanase enzyme to hydrolyze the substrate.