Metabolism of 3,6-anhydro-L-galactose in the agar-degrading marine bacterium Postechiella marina M091

Abstract

As world energy demand continues to increase and fossil resources are depleted, marine macroalgae are receiving increasing attention as an alternative renewable source for producing biofuels and valuable chemicals. Macroalgae have several advantages over conventional energy crops such as corn and lignocellulosic biomass, due to their high biomass productivity per cultivation area, high carbon dioxide fixation rate, lack of competition with ‘edible’ agriculture for land use, and compatibility with integrated biorefineries commonly used for the production of fuels and co-products. Five genera, Laminaria, Undaria, Porphyra, Euchema, and Gracilaria, represent 76% of the total tonnage of macroalgae production by aquaculture. Recently, breakthroughs in converting various carbohydrates from macroalgae into biofuels through metabolic engineering have demonstrated potential for macroalgae biomass as a promising source for biofuels. In recent years, agarose-containing macroalgae have gained attention as possible renewable sources for bioethanol production because of their high polysaccharide content. The complete hydrolysis of agarose from red algae produces its monomers, D-galactose (D-Gal) and 3,6-anhydro-L-galactose (L-AnG). Although many microorganisms are capable of utilizing agarose as a carbon source and numerous agarases have been identified in microorganisms of various genera, little is known about L-AnG metabolism. Therefore, we conducted this study to characterize the genes encoding the enzymes involved in L-AnG utilization in agar-degrading microorganisms. Specifically, the aim of this thesis was to identify and characterize enzymes catalyzing the first and second steps of L-AnG degradation in agar-degrading bacteria. Comparative genomic analysis of agarolytic bacteria, the marine bacterium Postechiella marina M091 and the soil bacterium Streptomyces coelicolor A3(2), which represent the classes Flavobacteria and Actinobacteria, respectively, revealed that L-AnG metabolizing genes were clustered in these genomes. First, we cloned and characterized a novel L-AnG dehydrogenases (L-AnGDH), an aldehyde dehydrogenase (ALDH), involved in the first step of L-AnG metabolism. L-AnGDH catalyzed the oxidation of L-AnG to 3,6-anhydro-L-galactonate (L-AnGA) using cofactor (NAD(P)+). To confirm the function of this first step enzyme, L-AnGDH, we cloned two bacterial L-AnG dehydrogenases from P. marina (Pm_L-AnGDH) and S. coelicolor (Sc_L-AnGDH). These genes were of similar size and were genetically similar (41 % identity at the amino acid level). Whereas the recombinant Pm_L-AnGDH and Sc_L-AnGDH were similar in their oligomeric state (homotetramer) and optimum reaction conditions (30?C, pH 8.0), the two enzymes were distinguishable by their substrate and cofactor specificities. Sc_L-AnGDH catalyzed the oxidation of L-AnG using both NAD+ and NADP+, with a preference for NAD+. It also catalyzed the dehydrogenation of L-glyceraldehyde, glycolaldehyde, and L-lactaldehyde in the presence of NAD+. On the other hand, Pm_L-AnGDH showed exclusive selectivity towards NADP+ and did not oxidize aldehydes other than L-AnG and L-glyceraldehyde. The phylogenetic analysis of amino sequences indicated that L-AnGDH belongs to a novel subfamily within the ALDH superfamily. We undertook comparative studies of Pm_L-AnGDH and Sc_L-AnGDH to determine which one is more suitable for the conversion of L-AnG to bioethanol. In bioethanol production, cofactor regeneration is very important. Ethanol is produced from acetaldehyde using the reaction catalyzed by alcohol dehydrogenase. Because alcohol dehydrogenases in yeasts and recombinant E. coli use NADH+ as a cofactor, it is desirable to regenerate NADH+ using NAD+-specific L-AnGDH. In view of this, it seems likely that NAD(P)+-dependent Sc_L-AnGDH is more suitable for bioethanol production than NADP+-dependent Pm_L-AnGDH. Second, we investigated the enzyme L-AnGA cycloisomerase (L-AnGACI) that is involved in the second step of L-AnG metabolism. To characterize this novel enzyme, the L-AnGACI gene (M091_0722) from P. marina (Pm_L-AnGACI) was cloned and expressed in E. coli. The conversion of L-AnGA to 2-keto-3-deoxy-L-galactonate (L_KDGal), catalyzed by L-AnGACI, was confirmed by the L-KDGal aldolase (An02g07720) reaction and an LC-MS analysis of the aldolase reaction products. Using the results of the LC-MS analysis, L-glyceraldehyde and pyruvate were confirmed in the in vitro reaction products of L-AnGACI and L-KDGal aldolase. L-AnGACI showed activity only with L-AnGA (100%) and galactarate (1.8%) among the 12 sugar acids and carboxylates tested, and the enzyme activity was maximal at 30°C and pH 8.0. The enzyme activity was enhanced by the addition of divalent ions such as Co2+ and Mg2+, similarly to other enzymes belonging to the enolase superfamily. A sequence alignment enabled us to identify the conserved residues of the enolase superfamily, which include three metal-ion binding ligands (Asp198, Glu224, and Glu250) and the active-site residues (Lys167, Lys169, Asp273, His300, and Glu320). The phylogenetic analysis of the amino acid sequences indicated that Pm_L-AnGACI belongs to a novel family within the mandelate racemase subgroup of the enolase superfamily. Our results not only provide a better understanding of metabolic pathways in bacteria involving the non-fermentable sugars such as L-AnG from red algae, but also highlight the potential for commercial application of metabolic engineering for the bioconversion of various macroalgae resources to produce biofuels and chemicals. These results provide insight into the integrated capabilities that will be necessary to innovate bioprocesses using microorganisms to convert biomass into bio-based products and bioenergy.