Production of a functional cell wall-anchored minicellulosome by recombinant Clostridium acetobutylicum ATCC 824

Abstract

BackgroundThe use of fossil fuels is no longer tenable. Not only are they a finite resource, their use is damaging the environment through pollution and global warming. Alternative, environmentally friendly, renewable sources of chemicals and fuels are required. To date, the focus has been on using lignocellulose as a feedstock for microbial fermentation. However, its recalcitrance to deconstruction is making the development of economic processes extremely challenging. One solution is the generation of an organism suitable for use in consolidated bioprocessing (CBP), i.e. one able to both hydrolyse lignocellulose and ferment the released sugars, and this represents an important goal for synthetic biology. We aim to use synthetic biology to develop the solventogenic bacterium C. acetobutylicum as a CBP organism through the introduction of a cellulosome, a complex of cellulolytic enzymes bound to a scaffold protein called a scaffoldin. In previous work, we were able to demonstrate the in vivo production of a C. thermocellum-derived minicellulosome by recombinant strains of C. acetobutylicum, and aim to develop on this success, addressing potential issues with the previous strategy.ResultsThe genes for the cellulosomal enzymes Cel9G, Cel48F, and Xyn10A from C. cellulolyticum were integrated into the C. acetobutylicum genome using Allele-Coupled Exchange (ACE) technology, along with a miniscaffoldin derived from C. cellulolyticum CipC. The possibility of anchoring the recombinant cellulosome to the cell surface using the native sortase system was assessed, and the cellulolytic properties of the recombinant strains were assayed via plate growth, batch fermentation and sugar release assays.ConclusionsWe have been able to demonstrate the synthesis and in vivo assembly of a four-component minicellulosome by recombinant C. acetobutylicum strains. Furthermore, we have been able to anchor a minicellulosome to the C. acetobutylicum cell wall by the use of the native sortase system. The recombinant strains display an improved growth phenotype on xylan and an increase in released reducing sugar from several substrates including untreated powdered wheat straw. This constitutes an important milestone towards the development of a truly cellulolytic strain suitable for CBP.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0526-x) contains supplementary material, which is available to authorized users.

Consolidated bioprocessing for butanol production of cellulolytic Clostridia: development and optimization

“…Subsequently, a number of key issues have been addressed towards the secretion of heterologous cellulosome and cellulases, including the investigation and optimization of leader peptide, promoter strength and molecular chaperones (Mingardon et al, 2011;Fierobe et al, 2012;Hyeon et al, 2013). Recently, allele-coupled exchange (ACE) technology was adapted to stably integrate a hybrid cellulosome operon composed of cellulosomal enzymes and miniscaffolding genes into chromosomes (Kov acs et al, 2013;Willson et al, 2016). Furthermore, the secreted mini-cellulosome was successfully anchored to the C. acetobutylicum cell wall via the native sortase system (Willson et al, 2016).…”

“…Recently, allele-coupled exchange (ACE) technology was adapted to stably integrate a hybrid cellulosome operon composed of cellulosomal enzymes and miniscaffolding genes into chromosomes (Kov acs et al, 2013;Willson et al, 2016). Furthermore, the secreted mini-cellulosome was successfully anchored to the C. acetobutylicum cell wall via the native sortase system (Willson et al, 2016). The thus generated recombinant strains displayed an improved growth phenotype on lignocellulosic biomass (Willson et al, 2016).…”

“…Furthermore, the secreted mini-cellulosome was successfully anchored to the C. acetobutylicum cell wall via the native sortase system (Willson et al, 2016). The thus generated recombinant strains displayed an improved growth phenotype on lignocellulosic biomass (Willson et al, 2016). This represents a milestone towards enabling solventogenic Clostridia to be cellulolytic.…”

Part by Part: Synthetic Biology Parts Used in Solventogenic Clostridia

“…The majority of promoters used in the genetic engineering of clostridia have been identified from the transcriptional units of important metabolic genes and most have been shown to be able to drive strong constitutive expression of a gene of interest 87 . The most commonly used constitutive promoter in C. acetobutylicum (Table 1) is that of the native thiolase gene (thlA) 88,89 , which has been used for the expression of genes involved in production of solvents such as butanol 90 and isopropanol 26 , the transcriptional regulator gene tetR 91 (used a minimal promoter variant-miniPthl), and, in a modified form, for the expression of cellulosomal scaffoldins 92 and glycoside hydrolases 25,93 Thiolase promoters have also been used for expression in other clostridia such as C.…”

“…91 . Activity analyzed using GusA 63 and several FbFP 48 reporters thl (C. perfringens) Used for expression of srtA genes from C. acetobutylicum, L. monocytogenes, and B. cereus 25 .…”