Efficient whole-cell-catalyzing cellulose saccharification using engineered Clostridium thermocellum

Abstract

BackgroundCost-efficient saccharification is one of the main bottlenecks for industrial lignocellulose conversion. Clostridium thermocellum naturally degrades lignocellulose efficiently using the cellulosome, a multiprotein supermolecular complex, and thus can be potentially used as a low-cost catalyst for lignocellulose saccharification. The industrial use of C. thermocellum is restrained due largely to the inhibition of the hydrolysate cellobiose to its cellulosome. Although the supplementation of beta-glucosidase may solve the problem, the production of the enzymes greatly complicates the process and may also increase the cost of saccharification.ResultsTo conquer the feedback inhibition and establish an efficient whole-cell catalyst for highly efficient cellulose saccharification, we constructed a recombinant strain of C. thermocellum ∆pyrF::CaBglA which produced a secretory exoglucanase CelS-bearing heterologous BGL using a newly developed seamless genome editing system. Without the extra addition of enzymes, the relative saccharification level of ∆pyrF::CaBglA was stimulated by over twofolds compared to its parent strain ∆pyrF through a two-stage saccharification process with 100 g/L Avicel as the carbon source. The production of reducing sugars and the relative saccharification level were further enhanced to 490 mM and 79.4%, respectively, with increased cell density.ConclusionsThe high cellulose-degrading ability and sugar productivity suggested that the whole-cell-catalysis strategy for cellulose saccharification is promising, and the C. thermocellum strain ∆pyrF::CaBglA could be potentially used as an efficient whole-cell catalyst for industrial cellulose saccharification.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-017-0796-y) contains supplementary material, which is available to authorized users.

Insights into Thermophilic Plant Biomass Hydrolysis from Caldicellulosiruptor Systems Biology

“…Recently, a β-glucosidase from Caldicellulosiruptor sp. F32 was produced heterologously in H. thermocellum as a fusion to the cellulosomal cellobiohydrolase, CelS [103] where it increased cellulose hydrolysis best when expressed as a chromosomal knock-in rather than from a replicating plasmid. Interestingly, in a follow-up study, when the same β-glucosidase was fused to a cohesin type II domain and incorporated into the cellulosome, it continued to act synergistically in the hydrolysis of microcrystalline cellulose, and hydrolyzed wheat straw at better rates than non-supplemented cultures [104].…”

“…Aside from metabolic engineering, there also has been a great deal of interest in supplementing the CAZyme repertoire of C. bescii with additional, synergistic enzymes. Initially, modular Caldicellulosiruptor β-glucosidases were introduced in H. thermocellum to relieve cellobiose inhibition of cellulases [103][104][105], and in turn, two different enzymes, a β-glucosidase from A. cellulolyticus [135] and cellobiose phosphorylase [136], were produced in C. bescii, which increased the efficiency of cellulose hydrolysis. An important first step in increasing cellulolytic activity in C. bescii was demonstrated by chromosomal expression of a β-glucanase and/or endoglucanse from A. cellulolyticus, which resulted in synergistic increases in cellulose hydrolysis, and over a 17-fold improvement of C. bescii growth on cellulose [137].…”

Inducing effects of cellulosic hydrolysate components of lignocellulose on cellulosome synthesis in Clostridium thermocellum

“…The cellulosome can interact with cellulosic substrates by virtue of the cellulose-binding modules (CBM), and the complex is anchored to the cell surface via an S-layer homology (SLH) module on an anchoring scaffoldin (Fontes and Gilbert, 2010;Artzi et al, 2017). The resulted multilevel synergy effects endow the cellulosome advantages of cellulose hydrolysis compared to fungus-derived free cellulases (Johnson et al, 1982;Lu et al, 2006;Hirano et al, 2015;Zhang et al, 2017). In addition, the cellulosome of C. thermocellum has the superiority for its substrate-coupled dynamic regulation mechanism (Raman et al, 2011;Smith and Bayer, 2013;Wilson et al, 2013;Wei et al, 2014).…”

Firmicutes-enriched IS1447 represents a group of IS3-family insertion sequences exhibiting unique + 1 transcriptional slippage

“…The importance of thermophilic bacteria in biorefineries has recently been proposed [ 25 ]. For example, Clostridium thermocellum (also named Ruminiclostridium thermocellum or Hungateiclostridium thermocellum ) is considered a promising biocatalyst in industrial biorefineries for lignocellulosic biomass utilization, and targeted genetic engineering has been widely performed on this cellulolytic and anaerobic thermophile [ 26 – 31 ]. Hence, the detection and functional analysis of thermophilic IS elements may promote greater understanding of the physiology of thermophiles and support the development of thermostable genetic tools.…”

“…We discovered an active IS element, IS 1447, that could mutate a thymidine kinase (Tdk) gene by insertion when Tdk was used as the counterselection marker during the genomic editing in the C. thermocellum DSM1313 strain [ 26 ]. IS 1447 was also detected in a mutated cipA gene of another C. thermocellum strain ATCC27405 through previous chemical mutagenesis [ 24 ].…”