Deciphering the lignocellulolytic metabolism and bioethanol production pathway of Klebsiella sp. SWET4 by whole genome and gene expression analyses

The WGS of Klebsiella sp. SWET4 revealed a genome of 5,665,821 bp by assembling 78 scaffolds with N50 and L50 values of 371571 and 5, respectively. BUSCO and ortholog analysis indicated the completeness of the genome and familiarity with Klebsiella variicola. Annotation result confirmed the presence of 5 major cellulose metabolizing genes (bcsZ, bglC, bglA, celA, chbA), besides 4 key xylan degrading genes (xynB, xynT, xylA, xylB) and 4 principal ethanol fermentation (nifJ, adhE, acs, adh1) genes. The lignolytic potential of SWET4 was evident in the kinetic study and the presence of yfeX/efeB, katG, katE, etc. genes were confirmed. Expression study with qPCR indicated overexpression of cellulolytic genes (268.73-fold of bglA, 122.78-fold of bglC, and 45.88-fold of bcsZ) in cellulose broth when compared to glucose. However, less activity was observed for xylanolytic genes (9.19-fold of xylA, 4.29-fold of xylB, and 2.89-fold of xynB) in xylan broth. Additionally, the kinetic study revealed that K_s was significantly lower for cellulose (7.79 and 0.09 g/L) compared to xylan (193.78 and 3.95 g/L) for primary and secondary growth phases, respectively which confirmed the higher preference of cellulose by SWET4. Further, ethanol estimation by HPLC exhibited an increase in ethanol concentration in banana peel fermentation broth from 0.1 g/L (30 h) to 0.35 g/L (56 h). The adh1 gene expression was also found to increase by 84.45-fold, whereas adhE increased by 1.62-fold confirming their influence on ethanol production. This study confirmed the lignocellulolytic and ethanologenic properties of SWET4 enabling the direct biotransformation of cellulosic biomass to ethanol.