Molecular function of the expansin superfamily has been highlighted for cellulosic biomass conversion. In this report, we identified a new bacterial expansin subfamily by analysis of related bacterial sequences and biochemically examined a member of this new subfamily from Hahella chejuensis (HcEXLX2). Among the various complex polysaccharides tested, HcEXLX2 bound most efficiently to cellulose. The relative binding constant (K( r )) against Avicel was 2.1 L g(-1) at pH 6.0 and 4 degrees C. HcEXLX2 enhanced the activity of cellulase, producing about 4.6 times more hydrolysis product after a 36 h reaction relative to when only cellulase was used. The extension strength test on filter paper indicated that HcEXLX2 has a texture loosening effect on filter paper, which was 53% of that observed for 8 M urea treatment. These activities, compared with a cellulose binding domain from Clostridium thermocellum, implied that the synergistic effect of HcEXLX2 comes from not only binding to cellulose but also disrupting the hydrogen bonds in cellulose. Based on these results, we suggest that the new bacterial expansin subfamily functions by binding to cell wall polysaccharides and increasing the accessibility of cell wall degrading enzymes.

Lactic acid is a platform chemical for the sustainable production of various materials. To develop a robust yeast platform for low-pH production of d-lactic acid (LA), an acid-tolerant yeast strain was isolated from grape skins and named Pichia kudriavzevii NG7 by ribosomal RNA sequencing. This strain could grow at pH 2.0 and 50°C. For the commercial application of P. kudriavzevii NG7 as a lactic acid producer, the ethanol fermentation pathway was redirected to lactic acid by replacing the pyruvate decarboxylase 1 gene (PDC1) with the d-lactate dehydrogenase gene (d-LDH) derived from Lactobacillus plantarum. To enhance lactic acid tolerance, this engineered strain was adapted to high lactic acid concentrations, and a new transcriptional regulator, PAR1, responsible for acid tolerance, was identified by whole-genome resequencing. The final engineered strain produced 135 g/L and 154 g/L of d-LA with productivity over 3.66 g/L/hr at pH 3.6 and 4.16 g/L/hr at pH 4.7, respectively.

The catabolic fate of the major monomeric sugar of red macroalgae, 3,6-anhydro-L-galactose (AHG), is completely unknown in any organisms. AHG is not catabolized by ordinary fermentative microorganisms, and it hampers the utilization of red macroalgae as renewable biomass for biofuel and chemical production. In this study, metabolite and transcriptomic analyses of Vibrio sp., a marine bacterium capable of catabolizing AHG as a sole carbon source, revealed two key metabolic intermediates of AHG, 3,6-anhydrogalactonate (AHGA) and 2-keto-3-deoxy-galactonate; the corresponding genes were verified in vitro enzymatic reactions using their recombinant proteins. Oxidation by an NADP(+) -dependent AHG dehydrogenase and isomerization by an AHGA cycloisomerase are the two key AHG metabolic processes. This newly discovered metabolic route was verified in vivo by demonstrating the growth of Escherichia coli harbouring the genes of these two enzymes on AHG as a sole carbon source. Also, the introduction of only these two enzymes into an ethanologenic E. coli strain increased the ethanol production in E. coli by fermenting both AHG and galactose in an agarose hydrolysate. These findings provide not only insights for the evolutionary adaptation of a central metabolic pathway to utilize uncommon substrates in microbes, but also a metabolic design principle for bioconversion of red macroalgal biomass into biofuels or industrial chemicals.

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A gene, alg7D, from Saccharophagus degradans, coding for a putative alginate lyase belonging to the family of polysaccharide lyase-7, was overexpressed in Escherichia coli. The properties of the recombinant Alg7D were characterized. The enzyme endolytically depolymerized alginate by β-elimination into oligo-alginates with degrees of polymerization of 2-5. Its activity was maximal at 50°C and pH 7 and was slightly increased in the presence of Na(+). The K(M), V(max), k(cat), and k(cat)/K(M) values were: 3 mg ml(-1), 6.2 U mg(-1), 1.9 × 10(-2) s(-1), and 6.3 × 10(-3) mg(-1 )ml s(-1), respectively.

Eubacterium limosum KIST612 is an anaerobic acetogenic bacterium that uses CO as the sole carbon/energy source and produces acetate, butyrate, and ethanol. To evaluate its potential as a syngas microbial catalyst, we have sequenced the complete 4.3-Mb genome of E. limosum KIST612.Synthesis gas (syngas) (H 2 , CO 2 , and CO) has been highlighted for use as a potential feedstock for the production of biofuels and valuable chemicals (9, 16). Eubacterium limosum KIST612 isolated from an anaerobic digester has been considered a microbial syngas catalyst due to its rapid growth under high CO pressure (Ͼ1 atm) and production of acetate and butyrate and ethanol from CO (5-7). To understand its physiological properties (e.g., a high tolerance to CO and production of ethanol) and provide metabolic engineering principles, we attempted to obtain the complete genome sequence information for this microorganism.The genome of E. limosum KIST612 was sequenced by a combination of Illumina Genome Analyzer IIx (GAIIx) and Roche 454 GS FLX (454 GS FLX) platforms. We obtained two libraries of 643,326 single-end (SE) reads and 291,735 pairedend (PE) reads containing 3-kb inserts from 454 GS FLX. The third genomic library of 35,235,888 PE reads containing 400-bp inserts was obtained from GAIIx. To combine these three libraries (454 GS FLX SE and PE and GAIIx PE) into a single procedure, we first assembled GAIIx PE reads into 296 contigs (4,635,997 bases) by the ABySS 1.20 assembler (15) and split into overlapping ϳ1.5-kb fake reads (45,221 reads). We merged these fake reads with 454 SE and PE reads (total 935,061 reads) and assembled into 9 scaffolds (34 contigs) by the Newbler gsAssembler 2.3 (454 Life Sciences, Branford, CT). We determined the actual order of 9 scaffolds in a single contig with a series of PCRs based on a permutation table of scaffolds. The genome was finished by filling gaps with sequencing and primer walking of PCR products using an ABI 3730 capillary sequencer (Applied Biosystems, CA).The complete genome of E. limosum KIST612 consisted of 4,276,902 bp in a single circular chromosome with an average GϩC content of 47.5%. Approximately 91% of the nucleotides were predicted as 4,516 protein-coding regions by the union of Glimmer (8), GeneMarkS (3), and Prodigal (10). The predicted proteins were annotated by BLAST (1) and the RAST server (2). Seventy-eight percent (3,541) of the open reading frames were annotated with known proteins. Five copies of the 16S-23S-5S rRNA operon and a separate 5S rRNA locus were predicted by RNAmmer 1.2 (12), and the 58 tRNA genes were identified by tRNAscan-SE 1.23 (13).Metabolic pathway analysis revealed that E. limosum KIST612 uses the Wood-Ljungdahl pathway to fix CO (or CO 2 ) and converts it into acetyl coenzyme A (acetyl-CoA), like other syngas-utilizing acetogens such as Moorella thermoacetica (14), Clostridium ljungdahlii (11), and Clostridium carboxidivorans strain P7 T (4). E. limosum KIST612 also contains 10 genes annotated as subunits of hydrogenases that may provide reducing...

BsEXLX1 from Bacillus subtilis is the first discovered bacterial expansin as a structural homolog of a plant expansin, and it exhibited synergism with cellulase on the cellulose hydrolysis in a previous study. In this study, binding characteristics of BsEXLX1 were investigated using pretreated and untreated Miscanthus x giganteus in comparison with those of CtCBD3, a cellulose-binding domain from Clostridium thermocellum. The amounts of BsEXLX1 bound to cellulose-rich substrates were significantly lower than those of CtCBD3. However, the amounts of BsEXLX1 bound to lignin-rich substrates were much higher than those of CtCBD3. A binding competition assay between BsEXLX1 and CtCBD3 revealed that binding of BsEXLX1 to alkali lignin was not affected by the presence of CtCBD3. This preferential binding of BsEXLX1 to lignin could be related to root colonization in plants by bacteria, and the bacterial expansin could be used as a lignin blocker in the enzymatic hydrolysis of lignocellulose.

Plant expansin proteins induce plant cell wall extension and have the ability to extend and disrupt cellulose. In addition, these proteins show synergistic activity with cellulases during cellulose hydrolysis. BsEXLX1 originating from Bacillus subtilis is a structural homolog of a β-expansin produced by Zea mays (ZmEXPB1). The Langmuir isotherm for binding of BsEXLX1 to microcrystalline cellulose (i.e., Avicel) revealed that the equilibrium binding constant of BsEXLX1 to Avicel was similar to those of other Type A surface-binding carbohydrate-binding modules (CBMs) to microcrystalline cellulose, and the maximum number of binding sites on Avicel for BsEXLX1 was also comparable to those on microcrystalline cellulose for other Type A CBMs. BsEXLX1 did not bind to cellooligosaccharides, which is consistent with the typical binding behavior of Type A CBMs. The preferential binding pattern of a plant expansin, ZmEXPB1, to xylan, compared to cellulose was not exhibited by BsEXLX1. In addition, the binding capacities of cellulose and xylan for BsEXLX1 were much lower than those for CtCBD3.