Dataset Information

A combined cell-consortium approach for lignocellulose degradation by specialized Lactobacillus plantarum cells.

ABSTRACT:

Background

Lactobacillus plantarum is an attractive candidate for metabolic engineering towards bioprocessing of lignocellulosic biomass to ethanol or polylactic acid, as its natural characteristics include high ethanol and acid tolerance and the ability to metabolize the two major polysaccharide constituents of lignocellulolytic biomass (pentoses and hexoses). We recently engineered L. plantarum via separate introduction of a potent cellulase and xylanase, thereby creating two different L. plantarum strains. We used these strains as a combined cell-consortium for synergistic degradation of cellulosic biomass.

Results

To optimize enzymatic degradation, we applied the cell-consortium approach to assess the significance of enzyme localization by comparing three enzymatic paradigms prevalent in nature: (i) a secreted enzymes system, (ii) enzymes anchored to the bacterial cell surface and (iii) enzymes integrated into cellulosome complexes. The construction of the three paradigmatic systems involved the division of the production and organization of the enzymes and scaffold proteins into different strains of L. plantarum. The spatial differentiation of the components of the enzymatic systems alleviated the load on the cell machinery of the different bacterial strains. Active designer cellulosomes containing a xylanase and a cellulase were thus assembled on L. plantarum cells by co-culturing three distinct engineered strains of the bacterium: two helper strains for enzyme secretion and one producing only the anchored scaffoldin. Alternatively, the two enzymes were anchored separately to the cell wall. The secreted enzyme consortium appeared to have a slight advantage over the designer cellulosome system in degrading the hypochlorite pretreated wheat straw substrate, and both exhibited significantly higher levels of activity compared to the anchored enzyme consortium. However, the secreted enzymes appeared to be less stable than the enzymes integrated into designer cellulosomes, suggesting an advantage of the latter over longer time periods.

Conclusions

By developing the potential of L. plantarum to express lignocellulolytic enzymes and to control their functional combination and stoichiometry on the cell wall, this study provides a step forward towards optimal biomass bioprocessing and soluble fermentable sugar production. Future expansion of the preferred secreted-enzyme and designer-cellulosome systems to include additional types of enzymes will promote enhanced deconstruction of cellulosic feedstocks.

SUBMITTER: Morais S

PROVIDER: S-EPMC4364503 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Publications

A combined cell-consortium approach for lignocellulose degradation by specialized Lactobacillus plantarum cells.

Moraïs Sarah S Shterzer Naama N Lamed Raphael R Bayer Edward A EA Mizrahi Itzhak I

Biotechnology for biofuels 20140724

<h4>Background</h4>Lactobacillus plantarum is an attractive candidate for metabolic engineering towards bioprocessing of lignocellulosic biomass to ethanol or polylactic acid, as its natural characteristics include high ethanol and acid tolerance and the ability to metabolize the two major polysaccharide constituents of lignocellulolytic biomass (pentoses and hexoses). We recently engineered L. plantarum via separate introduction of a potent cellulase and xylanase, thereby creating two different ...[more]

PMID: 25788977

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Project description:Background:Lignocellulose is the most abundant renewable carbon resource that can be used for biofuels and commodity chemicals production. The ability of complex microbial communities present in natural environments that are specialized in biomass deconstruction can be exploited to develop lignocellulose bioconversion processes. Termites are among the most abundant insects on earth and play an important role in lignocellulose decomposition. Although their digestive microbiome is recognized as a potential reservoir of microorganisms producing lignocellulolytic enzymes, the potential to enrich and maintain the lignocellulolytic activity of microbial consortia derived from termite gut useful for lignocellulose biorefinery has not been assessed. Here, we assessed the possibility of enriching a microbial consortium from termite gut and maintaining its lignocellulose degradation ability in controlled anaerobic bioreactors. Results:We enriched a termite gut-derived consortium able to transform lignocellulose into carboxylates under anaerobic conditions. To assess the impact of substrate natural microbiome on the enrichment and the maintenance of termite gut microbiome, the enrichment process was performed using both sterilized and non-sterilized straw. The enrichment process was carried out in bioreactors operating under industrially relevant aseptic conditions. Two termite gut-derived microbial consortia were obtained from Nasutitermes ephratae by sequential batch culture on raw wheat straw as the sole carbon source. Analysis of substrate loss, carboxylate production and microbial diversity showed that regardless of the substrate sterility, the diversity of communities selected by the enrichment process strongly changed compared to that observed in the termite gut. Nevertheless, the community obtained on sterile straw displayed higher lignocellulose degradation capacity; it showed a high xylanase activity and an initial preference for hemicellulose. Conclusions:This study demonstrates that it is possible to enrich and maintain a microbial consortium derived from termite gut microbiome in controlled anaerobic bioreactors, producing useful carboxylates from raw biomass. Our results suggest that the microbial community is shaped both by the substrate and the conditions that prevail during enrichment. However, when aseptic conditions are applied, it is also affected by the biotic pressure exerted by microorganisms naturally present in the substrate and in the surrounding environment. Besides the efficient lignocellulolytic consortium enriched in this study, our results revealed high levels of xylanase activity that can now be further explored for enzyme identification and overexpression for biorefinery purposes.

Project description:Lignocellulose degradation by microbial consortia is multifactorial; hence, it must be analyzed from a holistic perspective. In this study, the temporal transcriptional activity of consortium PM-06, a nixtamalized maize pericarp (NMP) degrader, was determined and related to structural and physicochemical data to give insights into the mechanism used to degrade this substrate. Transcripts were described in terms of metabolic profile, carbohydrate-active enzyme (CAZyme) annotation, and taxonomic affiliation. The PM-06 gene expression pattern was closely related to the differential rates of degradation. The environmental and physiological conditions preceding high-degradation periods were crucial for CAZyme expression. The onset of degradation preceded the period with the highest degradation rate in the whole process, and in this time, several CAZymes were upregulated. Functional analysis of expressed CAZymes indicated that PM-06 overcomes NMP recalcitrance through modular enzymes operating at the proximity of the insoluble substrate. Increments in the diversity of expressed modular CAZymes occurred in the last stages of degradation where the substrate is more recalcitrant and environmental conditions are stressing. Taxonomic affiliation of CAZyme transcripts indicated that Paenibacillus macerans was fundamental for degradation. This microorganism established synergistic relationships with Bacillus thuringiensis for the degradation of cellulose and hemicellulose and with Microbacterium, Leifsonia, and Nocardia for the saccharification of oligosaccharides. IMPORTANCE Nixtamalized maize pericarp is an abundant residue of the tortilla industry. Consortium PM-06 efficiently degraded this substrate in 192 h. In this work, the temporal transcriptional profile of PM-06 was determined. Findings indicated that differential degradation rates are important sample selection criteria since they were closely related to the expression of carbohydrate-active enzymes (CAZymes). The initial times of degradation were crucial for the consumption of nixtamalized pericarp. A transcriptional profile at the onset of degradation is reported for the first time. Diverse CAZyme genes were rapidly transcribed after inoculation to produce different enzymes that participated in the stage with the highest degradation rate in the whole process. This study provides information about the regulation of gene expression and mechanisms used by PM-06 to overcome recalcitrance. These findings are useful in the design of processes and enzyme cocktails for the degradation of this abundant substrate.

Dataset Information

A combined cell-consortium approach for lignocellulose degradation by specialized Lactobacillus plantarum cells.

Background

Results

Conclusions

Publications

A combined cell-consortium approach for lignocellulose degradation by specialized Lactobacillus plantarum cells.

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