Systems analysis in Cellvibrio japonicus resolves predicted redundancy of β‐glucosidases and determines essential physiological functions

Abstract

SUMMARY Degradation of polysaccharides forms an essential arc in the carbon cycle, provides a percentage of our daily caloric intake, and is a major driver in the renewable chemical industry. Microorganisms proficient at degrading insoluble polysaccharides possess large numbers of carbohydrate active enzymes, many of which have been categorized as functionally redundant. Here we present data that suggests that carbohydrate active enzymes that have overlapping enzymatic activities can have unique, non-overlapping biological functions in the cell. Our comprehensive study to understand cellodextrin utilization in the soil saprophyte Cellvibrio japonicus found that only one of four predicted β-glucosidases is required in a physiological context. Gene deletion analysis indicated that only the cel3B gene product is essential for efficient cellodextrin utilization in C. japonicus and is constitutively expressed at high levels. Interestingly, expression of individual β-glucosidases in Escherichia coli K-12 enabled this non-cellulolytic bacterium to be fully capable of using cellobiose as a sole carbon source. Furthermore, enzyme kinetic studies indicated that the Cel3A enzyme is significantly more active than the Cel3B enzyme on the oligosaccharides but not disaccharides. Our approach for parsing related carbohydrate active enzymes to determine actual physiological roles in the cell can be applied to other polysaccharide-degradation systems.

The complex physiology of Cellvibrio japonicus xylan degradation relies on a single cytoplasmic β‐xylosidase for xylo‐oligosaccharide utilization

“…S1B; Table S1C). It was previously shown, both by microarray and RNAseq studies, that the number of C. japonicus CAZymes that were under growth rate control using glucose as the sole carbon source was very few (Gardner et al ., ; Nelson et al ., ). Analysis of the absolute level of expression of our transcriptomic data, as indicated by the reads per kilobase per million mapped reads (RPKM), did not identify any xylanase genes that were constitutively expressed at a high level.…”

“…The transcriptional response for exponentially growing C. japonicus cells on xylan was very specific for xylanase genes, which differed significantly from the transcriptional response of cells growing on cellulose in the scope and functions of the genes up‐regulated (Gardner et al ., ; Nelson et al ., ). The up‐regulated cohort of CAZyme genes on cellulose consisted of a wide variety of CAZymes predicted to degrade various polysaccharides, whereas on xylan only a select few xylan‐specific CAZymes were represented.…”

Comprehensive functional characterization of the glycoside hydrolase family 3 enzymes from Cellvibrio japonicus reveals unique metabolic roles in biomass saccharification

“…Our previous studies on the functions of the C. japonicus GH3 enzymes tested activity exclusively on β(1→4)‐linked gluco‐oligosaccharides (cellodextrins). Although all GH3 members were competent β(1→4)‐glucosidases in vitro and were able to confer growth on cellobiose when expressed heterologously in E. coli in vivo , individual activities on cello‐oligosaccharides varied by orders‐of‐magnitude amongst the recombinant enzymes (Nelson et al ., ). These results suggested that the true substrates for some of the GH3 enzymes might not be β(1→4)‐linked gluco‐oligosaccharides.…”

Systems analysis of the glycoside hydrolase family 18 enzymes from Cellvibrio japonicus characterizes essential chitin degradation functions

“…An adjusted p-value > 0.01 and a log 2 fold change > 2 were selected as significance cut-off parameters. RNAseq data for glucose grown C. japonicus cells (GSE90955) was used as done previously (26). The chitin-specific RNAseq data that was generated for this study can be found in the NCBI GEO database (GSE108935).…”

“…There is increasing evidence that the CAZymes of C. japonicus that belong to the same GH family are not functionally redundant, but have unique physiological functions (14,26). In the current study, the physiological roles of the four C. japonicus GH18 chitinases were determined focusing on the initial stages of chitin degradation.…”