Rapid optimisation of cellulolytic enzymes ratios in Saccharomyces cerevisiae using in vitro SCRaMbLE

Abstract

Background For the economic production of biofuels and other valuable products from lignocellulosic waste material, a consolidated bioprocessing (CBP) organism is required. With efficient fermentation capability and attractive industrial qualities, Saccharomyces cerevisiae is a preferred candidate and has been engineered to produce enzymes that hydrolyze cellulosic biomass. Efficient cellulose hydrolysis requires the synergistic action of several enzymes, with the optimum combined activity ratio dependent on the composition of the substrate. Results In vitro SCRaMbLE generated a library of plasmids containing different ratios of a β-glucosidase gene (CEL3A) from Saccharomycopsis fibuligera and an endoglucanase gene (CEL5A) from Trichoderma reesei. S. cerevisiae, transformed with the plasmid library, displayed a range of individual enzyme activities and synergistic capabilities. Furthermore, we show for the first time that 4,6-O-(3-ketobutylidene)-4-nitrophenyl-β-d-cellopentaoside (BPNPG5) is a suitable substrate to determine synergistic Cel3A and Cel5A action and an accurate predictive model for this synergistic action was devised. Strains with highest BPNPG5 activity had an average CEL3A and CEL5A gene cassette copy number of 1.3 ± 0.6 and 0.8 ± 0.2, respectively (ratio of 1.6:1). Conclusions Here, we describe a synthetic biology approach to rapidly optimise gene copy numbers to achieve efficient synergistic substrate hydrolysis. This study demonstrates how in vitro SCRaMbLE can be applied to rapidly combine gene constructs in various ratios to allow screening of synergistic enzyme activities for efficient substrate hydrolysis.

Rapid Colorimetric Detection of Genome Evolution in SCRaMbLEd Synthetic Saccharomyces cerevisiae Strains

“…SCRaMbLE is a novel genome evolution system associated with the synthetic Sc2.0 strains, allowing the combinatorial deletion, duplication and translocation of multiple genes at a time. Not only is SCRaMbLE a valuable tool for studying epistatic interactions between genes, it has also been demonstrated as a useful approach to generate large libraries of novel strains with improved industrial phenotypes [7,12,15,29]. However, this indiscriminatory rearrangement of functional genomic units frequently results in inviable or unfit phenotypes due to the loss of essential genes or the disruption of fitness-related metabolic pathways.…”

“…These SCRaMbLEd strains contained only one synthetic chromosome in a haploid genome context; therefore, it is conceivable that strains harboring more synthetic DNA, with more loxP recombinase recognition sites, could produce greater genomic diversity with associated novel phenotypes of interest. Thus far, SCRaMbLE has also been explored for a variety of fundamental and applied applications, including genome minimization [2], biosynthetic pathway assembly [8,11] and for the optimization of hydrolytic enzyme ratios for biofuel applications [12].…”