Biomimetic strategy for constructing Clostridium thermocellum cellulosomal operons in Bacillus subtilis

Abstract

BackgroundEnzymatic conversion of lignocellulosic biomass into soluble sugars is a major bottleneck in the plant biomass utilization. Several anaerobic organisms cope these issues via multiple-enzyme complex system so called ‘cellulosome’. Hence, we proposed a “biomimic operon” concept for making an artificial cellulosome which can be used as a promising tool for the expression of cellulosomal enzymes in Bacillus subtilis.ResultsAccording to the proteomic analysis of Clostridium thermocellum ATCC27405 induced by Avicel or cellobiose, we selected eight highly expressed cellulosomal genes including a scaffoldin protein gene (cipA), a cell-surface anchor gene (sdbA), two exoglucanase genes (celK and celS), two endoglucanase genes (celA and celR), and two xylanase genes (xynC and xynZ). Arranging these eight genes in two different orders, we constructed two different polycistronic operons using the ordered gene assembly in Bacillus method. This is the first study to express the whole CipA along with cellulolytic enzymes in B. subtilis. Each operon was successfully expressed in B. subtilis RM125, and the protein complex assembly, cellulose-binding ability, thermostability, and cellulolytic activity were demonstrated. The operon with a higher xylanase activity showed greater saccharification on complex cellulosic substrates such as Napier grass than the other operon.ConclusionsIn this study, a strategy for constructing an efficient cellulosome system was developed and two different artificial cellulosomal operons were constructed. Both operons could efficiently express the cellulosomal enzymes and exhibited cellulose saccharification. This strategy can be applied to different industries with cellulose-containing materials, such as papermaking, biofuel, agricultural compost, mushroom cultivation, and waste processing industries.Electronic supplementary materialThe online version of this article (10.1186/s13068-018-1151-7) contains supplementary material, which is available to authorized users.

Kluyveromyces marxianus: Current State of Omics Studies, Strain Improvement Strategy and Potential Industrial Implementation

“…Since the anchoring protein OlpB has seven type II cohesins, the interaction between CipA-OlpB can accommodate up to 9 × 7 = 63 cellolosomal enzymes in a single cellulosome complex. Up to now, several research groups have been sought to design cellulosome microbes that can express a full size of cellulosome structure instead of some individual cellulosomal genes called mini-cellulosomes [31][32][33][34][35][36]. Recently, the group of Anandharaj et al [37] succeeded in developing an engineered K. marxianus host that can express a full size of H. thermocellum cellulosome on its cell surface.…”

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

“…(B) Eight‐component cellulosome engineered on the surface of a single B. subtilis strain through introduction of artificial operons (adapted from ref. ). The engineered cellulosome consists of the cell surface anchor SdbA, the adaptor scaffoldin CipA (comprising nine cohesins, coh, and one CBM), two exoglucanases (CelK, CelS), two endoglucanases (CelA, CelR), and two xylanases (XynC, XynZ) derived from C. thermocellum .…”

“…Remarkably, assembly of minicellulosomes in a single B. subtilis strain dates back to 2004 . Recently, artificial operons encoding eight cellulosomal subunits of C. thermocellum have been assembled and transformed in B. subtilis . Operons included genes for the full‐length adaptor scaffoldin CipA (featuring nine cohesin domains), the anchoring scaffoldin SdbA, and six enzymatic subunits featuring exoglucanase, endoglucanase, and xylanase activity (Fig.…”

“…This allowed secretion and partial surface display of large engineered cellulosomes in a single recombinant strain. Improved saccharification of raw cellulosic materials by recombinant B. subtilis was reported, although it was not mentioned that this improved phenotype was able to support B. subtilis growth on these substrates . However, no examples of RCSs have targeted LA‐producing Bacillus strains.…”

Engineering of industrially important microorganisms for assimilation of cellulosic biomass: towards consolidated bioprocessing

“…In particular, being relatives of Clostridium spp., they are an interesting option for reconstruction of cellulosomes. For example, a recent report describes use of OGAB to assemble a reduced cellulosome system with intact scaffoldin and its cell surface anchor protein, together with 6 assorted cellulosomal biomass degradation enzymes, and demonstrated correct assembly, enzyme activity, and degradation of grass biomass [33]. While B. subtilis is not well suited to production of cellulosic ethanol, it seems a good candidate for engineering of processes for manufacture of other products from cellulosic materials.…”