Characterization and whole genome sequencing of Klebsiella variicola HSTU-AAM51 strain isolated from Goat rumen contents: prospects of lignocellulase enzyme production

Abstract

Due to shortage of fossil fuels in Bangladesh, we need to source alternative energy one of choices may be the lignocellulosic bioethanol production. However the commercial enzymes for bioethanol production are not available in Bangladesh. Therefore lignocellulase producing strains is the target of our research. The study is aimed to characterize a lignocellose degrading strain and to revealing their mechanism through genome wide investigation. The lignocellulose degrading bacteria was isolated from Goat rumen contents by screening on the CMC, pectin, xylanand starch containing agar media. The isolate was named as Klebsiella variicola HSTU-AAM51. The HSTU-AAM51 showed MR negative, VP negative, catalase positive, oxidase positive, MIU test negative, citrate negative, TSI, , sucrose, dextrose, lactose, urease and maltose positive in vitro test. Klebsiella variicola. HSTU- AAM51 was isolated from goat rumen contents and confirmed to possess xylanase, cellulose and pectinase activity by whole genome sequence analysis. The complete genome ofKlebsiella vaiicola HSTU- AAM51 strain comprises of 5,564,045 bp in a circular chromosome with a G + C content of 57.2%. Among the predicted 5187 protein-coding genes(CDS), 45 genes are involved in the degradation of lignocellulose and other polysaccharides, including6cellulose degrading genes (GH3, GH5, GH13, GH94) , 4xylanose degrading genes (GH10,CE4,GH43), 1 mannose degrading gene (GH5), 4 hemicellulose degrading genes (GH27, GH2, GH13, GH53) , 1 pectin degrading gene (PE1) and 29 lignin degrading genes. Lignocellulose degrading genes are detected by computational analysis, where as some CAZy (GH5, GH94, GH10, GH5, CE4, GH53 and PE1) genes showed signal peptide prediction positive, which proves that they have extracellular enzymes activity. Finally lignocelluloses degrading enzyme activity was ensured by molecular docking analysis where ascellulase enzymes (GH3, GH94 and GH5) showed ability to break down ligand respectively β -D-glucopyranose, alpha & beta –D-Glucose and cellopentaose which release energy respectively ΔG=-10.51K cal/mol, ΔG=-6.8 K cal/mol and ΔG=-10.3 K cal/mol. In addition toxylanase enzymes (GH10 and CE4) showed ability to break down ligand respectivelyβ-D-xylopyranose(XYP),4-O-β -D-xylopyranosyl-β -D-xylopyranose(BXP) and 2-(N-morpholino)-ethanesulfonic acid(MES), 2-(acetylamino)-2-deoxy-A-Dglucopyranose (NDG) which release energy respectively ΔG=-7.3, -6.3 K cal/mol and ΔG=- 7.3, -6.46 K cal/mol. Moreover hemicellulase enzymes (GH53 and GH13) showed ability to break down ligand respectively imidazole(IMD),N-acetyl-D-glucoseamine(NAG), β-Dglactose(GAL) and 2-[bis-(2-hydroxy-ethyl)-amino]-2-hydroxymethyl-propane-1,3-diol(BTB) iii which release energy respectively ΔG=-9.4, -6.7, -7.0 K cal/mol, ΔG=-7.74 K cal/mol and pectinase enzymes (PE1) has capability to break down ligand β-D-galactopyranuronic acid (Pectin) which release energy respectively ΔG=-6.23 K cal/mol. This genome-based analysis facilitates our understanding of the mechanism underlying the biodegradation of lignocelluloses. This genome information including CAZyme repertoire will be useful to understand lingocellulolytic machinery. This study first time reports the whole genome analysis of Klebsiella variicola HSTU-AAM51 which are enriched with carbohydrate and lignin degrading useful enzymes for bioethanol production.