SCRaMbLEing to Generate Saccharomyces cerevisiae with novel gene combinations
Creators: name: Wightman, Elizabeth Louise Izolde
Publisher: name: Macquarie University
Year: 2024
Subjects: classificationCode: 399999
subject: Other education not elsewhere classified

description: Owing to their diverse genetics and attractive qualities, yeasts are harnessed by industry for a multitude of specialised purposes in the production of food, beverages, pharmaceuticals, chemical building blocks and fuel. Better strains are constantly demanded by industry with improvements such as enhanced enzyme production and/or secretion and increased tolerance to harsh growth conditions. While a myriad of strains has been developed using tried and tested genetic engineering strategies, new synthetic biology tools are now available for gene recombination on a genome-wide scale.  
The Yeast 2.0 project aims to design and construct the world’s first synthetic eukaryotic organism. As well as the complete reconstruction of the 16 chromosomes of <em>Saccharomyces cerevisiae</em>, SCRaMbLE was developed as a powerful system to facilitate the generation of large libraries of novel strains. Synthetic Chromosome Rearrangement and Modification by LoxPSym mediated Evolution enables the rearrangement of <em>S. cerevisiae </em>genes on demand to produce millions of strains with unique genomes that can be screened for complex phenotypes. As of 2020, work is currently underway within the international Yeast 2.0 consortium, and as this study focuses on, a contribution to the construction was performed by the assembly and integration of three sections (megachunks) of synthetic chromosome XVI into the genome (Chapter 2). The integration of each megachunk (replacing corresponding native DNA) was rigorously confirmed by the presence of PCRTags (watermarks of synthetic DNA), and the resulting strain was evaluated for robust fitness in different growth conditions.  
While SCRaMbLE has been applied successfully in the optimisation of pathways and the enhancement of desirable traits in <em>S. cerevisiae</em>, there are limitations associated with this strategy, especially when estimating the level of SCRaMbLE that has occurred in a population. Different levels of SCRaMbLE may be required for specific applications such as genome minimisation or for the SCRaMbLE-in of heterologous pathways. As such, a system named Red-White SCRaMbLE was developed as a simple, visual strategy to assess the level of SCRaMbLE among diploid <em>S. cerevisiae </em>populations. Red-White SCRaMbLE was then optimised and applied in heterologous semi-synthetic diploid strains containing DNA from industrially-relevant haploids. This work was facilitated by a custom bioinformatics pipeline that enabled the elucidation of SCRaMbLE ‘hot spots’ in synthetic chromosomes, where certain genes were more (or less) likely to be deleted via SCRaMbLE than others.  
The development of a consolidated bioprocessing (CBP) organism is required for the biofuel industry that produces and secretes enzymes for the hydrolysis of biomass, and efficiently ferments the resulting sugars into fuel. <em>Saccharomyces cerevisiae </em>is a promising CBP candidate as it fulfils the latter requirement; heterologous genes encoding hydrolytic enzymes are now required, however the ideal gene ratios to confer efficient hydrolysis activity are unknown. <em>In vitro </em>SCRaMbLE can generate large libraries of plasmids containing different copy numbers of chosen genes for direct screening in <em>S. cerevisiae</em>. As a proof of concept, genes encoding the hydrolytic enzymes, Cel3A (β-glucosidase I) from <em>Saccharomycopsis fibuligera </em>and Cel5A (endoglucanase II) from <em>Trichoderma reesei </em>were <em>in vitro </em>SCRaMbLEd to enable hydrolysis of the synthetic substrate BPNPG5 (4,6-O-(3-Ketobutylidene)-4-nitrophenyl-β-D-cellopentaoside). This substrate was evaluated for the first time for the optimisation of Cel3A and Cel5A enzyme ratios and has significant advantages over alternatives. This system provided convenient means to screen hundreds of strains with various gene combination for the selection of beneficial gene ratios, and as such, <em>in vitro </em>SCRaMbLE could be applied to the development of other hydrolysis systems for more recalcitrant substrates.