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