Abstract
Engineering microbial strains combining efficient lignocellulose metabolization and high-value chemical production is a cutting-edge strategy towards cost-sustainable 2nd generation biorefining. Here, protein components of the Clostridium cellulovorans cellulosome were introduced in Lactococcus lactis IL1403, one of the most efficient lactic acid producers but unable to directly ferment cellulose. Cellulosomes are protein complexes with high cellulose depolymerization activity whose synergistic action is supported by scaffolding protein(s) (i.e., scaffoldins). Scaffoldins are involved in bringing enzymes close to each other and often anchor the cellulosome to the cell surface. In this study, three synthetic scaffoldins were engineered by using domains derived from the main scaffoldin CbpA and the Endoglucanase E (EngE) of the C. cellulovorans cellulosome. Special focus was on CbpA X2 and EngE S-layer homology (SLH) domains possibly involved in cell-surface anchoring. The recombinant scaffoldins were successfully introduced in and secreted by L. lactis. Among them, only that carrying the three EngE SLH modules was able to bind to the L. lactis surface although these domains lack the conserved TRAE motif thought to mediate binding with secondary cell wall polysaccharides. The synthetic scaffoldins engineered in this study could serve for assembly of secreted or surface-displayed designer cellulosomes in L. lactis.
Highlights
Lignocellulose is the most abundant raw material on the Earth
Only that carrying the three Endoglucanase E (EngE) S-layer homology (SLH) modules was able to bind to the L. lactis surface these domains lack the conserved TRAE motif thought to mediate binding with secondary cell wall polysaccharides
Cloning and expression of synthetic scaffoldins is an essential pre-requisite for introducing designer cellulosomes in heterologous microorganisms and has been performed in a number of microbial models[37,38] including different lactic acid bacteria (LAB).[12,17]
Summary
Its low price makes it an ideal feedstock for 2nd generation biorefining aimed at replacing fossil-derived production of fuels and chemicals.[1] lignocellulose has been selected to be recalcitrant to microbial and enzyme activity; its conversion through biological process is both technically and economically challenging.[1] Nowadays, industrial fermentation of lignocellulose is complex and expensive since multiple bioreactors in series are generally required.[2,3] Development of single-step fermentation (i.e., consolidated bioprocessing, CBP) of biomass is considered as one of the most promising strategies to reduce the costs of 2nd generation biorefinery and make them competitive with those of oil refinery.[3,4] The most straightforward path to achieve this goal is by using microbial strains that can directly ferment plant biomass and produce high-value compounds with high efficiency Since such microbes have not been found in nature, so far, metabolic engineering can hopefully be used to develop them through gene modification techniques.[5,6]. Two of them were secreted in the extracellular medium, while the third one was displayed at L. lactis surface showing that cell-surface binding domains of C. cellulovorans are able to recognize structural motifs on L. lactis cell wall
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