Utilising in vitro SCRaMbLE to generate cellulolytic saccharomyces cerevisiae for the biofuel Industry.

Biofuels are an attractive alternative to fossil fuels because the raw materials are abundant, cheap and renewable. Cellulosic biomass is converted to fuel in a two-step process: the degradation of cellulose to sugars, followed by the fermentation of those sugars to fuel. The development of a consolidated bioprocessing (CBP) organism that efficiently performs both processes is highly economically desirable. Therefore, there has been a lot of focus on the development of a cellulolytic Saccharomyces cerevisiae. Cellulose degradation requires the synergistic action of three classes of enzymes: EGs, CBHs, and BGLs. To engineer high degradation activity, simple overexpression of each enzyme is ineffective. High activity requires an optimised ratio of enzyme expression, the absolute optimum being unknown and unique to each different substrate. As a proof of concept, we utilised in vitro SCRaMbLE to optimise the cellulolytic activity of two foreign cellulases, S. fibuligera bgl1 and T. reesei egl2 in S. cerevisiae. This SCRaMbLE system, based on cre-loxP recombination, generates a library of constructs containing different copy numbers and ratios of the two genes. The library was screened for degradation activity on a substrate that required the synergy of both enzymes. The optimal gene ratio was uncovered using real-time PCR. In vitro SCRaMbLE is therefore suitable to be used with more cellulolytic genes with a view of reaching optimal cellulase activity on specific substrates.