Assembly of Synthetic Functional Cellulosomal Structures onto the Cell Surface of Lactobacillus plantarum, a Potent Member of the Gut Microbiome

Abstract

Heterologous display of enzymes on microbial cell surfaces is an extremely desirable approach, since it enables the engineered microbe to interact directly with the plant wall extracellular polysaccharide matrix. In recent years, attempts have been made to endow noncellulolytic microbes with genetically engineered cellulolytic capabilities for improved hydrolysis of lignocellulosic biomass and for advanced probiotics. Thus far, however, owing to the hurdles encountered in secreting and assembling large, intricate complexes on the bacterial cell wall, only free cellulases or relatively simple cellulosome assemblies have been introduced into live bacteria. Here, we employed the "adaptor scaffoldin" strategy to compensate for the low levels of protein displayed on the bacterial cell surface. That strategy mimics natural elaborated cellulosome architectures, thus exploiting the exponential features of their Lego-like combinatorics. Using this approach, we produced several bacterial consortia of , a potent gut microbe which provides a very robust genetic framework for lignocellulosic degradation. We successfully engineered surface display of large, fully active self-assembling cellulosomal complexes containing an unprecedented number of catalytic subunits all produced by the cell consortia. Our results demonstrate that the enzyme stability and performance of the cellulosomal machinery, which are superior to those seen with the equivalent secreted free enzyme system, and the high cellulase-to-xylanase ratios proved beneficial for efficient degradation of wheat straw. The multiple benefits of lactic acid bacteria are well established in health and industry. Here we present an approach designed to extensively increase the cell surface display of proteins via successive assembly of interactive components. Our findings present a stepping stone toward proficient engineering of , a widespread, environmentally important bacterium and potent microbiome member, for improved degradation of lignocellulosic biomass and advanced probiotics.

Metabolic engineering strategies for consolidated production of lactic acid from lignocellulosic biomass

“…(A) Consortium of engineered L. plantarum strains where each strain secretes a different cellulosomal component leading to assembly of designer cellulosomes on the cell surface (modified from ref. ). Cellulosomal components introduced in L. plantarum include wild‐type and chimeric cellulase and hemicellulases from C. papyrosolvens , designer adaptor scaffoldins (Adaptor 1, 2), that is, an intermediate type of scaffoldin able to bind both enzyme subunits and additional scaffoldins, and anchoring scaffoldins (e.g., Anc 4), that is proteins that can tether the protein complex to the cell surface.…”

“…The studies by Mizrahi and Bayer's group in Israel have focused on LAB. Different proteins have been introduced in Lactobacillus plantarum including endoglucanases, xylanases, and different scaffolding proteins . Over years, these studies have gradually improved their achievements leading to an assembly of an L. plantarum consortium that displays mini‐cellulosomes consisting of up to six enzymatic components (Fig.…”

“…Over years, these studies have gradually improved their achievements leading to an assembly of an L. plantarum consortium that displays mini‐cellulosomes consisting of up to six enzymatic components (Fig. 2A) . This outstanding result, leading to engineering of enzyme complexes with significant hydrolysis of wheat straw, was nonetheless insufficient to enable L. plantarum consortium to grow on wheat straw as the sole carbon source.…”

“…This outstanding result, leading to engineering of enzyme complexes with significant hydrolysis of wheat straw, was nonetheless insufficient to enable L. plantarum consortium to grow on wheat straw as the sole carbon source. It has been hypothesized that the enzyme mixture used in engineering of the L. plantarum consortium could release insufficient/unsuitable soluble sugars through biomass hydrolysis for this strain . This focused on the importance of choosing suitable enzymes for RCSs.…”

“…So far, the number of strategies to solve or reduce issues in protein secretion is relatively little . In most cases, they consist of engineering the signal peptide of cellulases by replacing it with host‐specific signal peptides . Inactivation of housekeeping protease(s), such as the unique exported protease HtrA of L. lactis , may be an alternative solution to increase cellulase secretion yield .…”