Characterization of a xylanase-producing Cellvibrio mixtus strain J3-8 and its genome analysis

Abstract

Cellvibrio mixtus strain J3-8 is a gram-negative, xylanase-producing aerobic soil bacterium isolated from giant snails in Singapore. It is able to produce up to 10.1 U ml(-1) of xylanase, which is comparable to xylanase production from known bacterial and fungal strains. Genome sequence analysis of strain J3-8 reveals that the assembled draft genome contains 5,171,890 bp with a G + C content of 46.66%, while open reading frame (ORF) annotations indicate a high density of genes encoding glycoside hydrolase (GH) families involved in (hemi)cellulose hydrolysis. On the basis of 15 identified putative xylanolytic genes, one metabolic pathway in strain J3-8 is constructed for utilization of xylan. In addition, a 1,083 bp xylanase gene from strain J3-8 represents a new member of GH11 family. This gene is verified to be novel via phylogenetic analysis. To utilize this novel gene for hydrolysis of xylan to xylose, it is expressed in recombinant E. coli and characterized for its hydrolytic activity. This study shows that strain J3-8 is a potential candidate for hydrolysis of lignocellulosic materials.

Figure 1

A neighbor-joining phylogenetic tree based on the 16S rRNA gene sequence of Cellvibrio mixtus strain J3-8 by using MEGA (Version 5.05). Distances determined according to Kimura’s two-parameter model and bootstrap values (>50%) based on 1,000 replicates are listed as percentages at nodes. Nucleotide sequence accession numbers are given in parentheses. Scale bar, 0.005 substitutions per 50 nucleotides.

Figure 2

Optimization of xylanase production from C. mixtus J3-8. A: Original culture condition (25 °C, pH=7, 5 g L⁻¹ xylan, without YE addition); B: Same condition as A, except changing temperature to 30 °C; C: Same condition as B, except changing pH to 8; D: Same condition as C, except adding 0.5% yeast extract; E: Same condition as D, except changing the xylan initial concentration to 10 g L⁻¹; F: Same condition as E, except activating the inocula with 10 g L⁻¹ of xylan.

Figure 3

Xylan hydrolysis (3 g L⁻¹) by crude extracellular enzymes (2 U ml⁻¹ of xylanase activity) from strain J3-8. The concentration of generated xylose was detected with or without addition of commercial β-xylosidase (2 U ml⁻¹).

Figure 4

The metabolic pathway overview of C. mixtus J3-8 using xylan as substrate deduced from genome data. Abbreviations: G-1,3-bisP: Glycerate-1,3-bisphosphate; 3-PG: 3-phospho-glycerate; 2-PG: 2-phospho-glycerate; PEP: phosphoenolpyruvate. The numbers indicate the C. mixtus J3-8 annotation and highest identity (in the bracket) from GenBank based on the BLASTP, corresponding to the tag (CMXXXX) in Table S1.

Figure 5

A neighbor-joining phylogenetic tree of xylanases based on the amino acid sequence by using MEGA (Version 5.05). Distances determined according to Kimura’s two-parameter model and bootstrap values (>50%) based on 1,000 replicates are listed as percentages at nodes. Nucleotide sequence accession numbers are given in parentheses.

Figure 6

Sequence alignment of this novel family 11 xylanase. Highlighted blocks indicate the main conserved residues, and the pink color-highlighted amino acids (two glutamic acid residues) are predicted to be the catalytic site. The sequence number is based on C. mixtus J3-8 xylanase amino acid sequence.