UC Irvine

The need for sustainable practices while minimizing harmful carbon emissions is driving a research shift towards bio-based manufacturing initiatives. Exploiting yeast metabolism has proven to be advantageous for biosynthesis of products across multiple industrial sectors. The yeast Saccharomyces cerevisiae is the most widely studied eukaryote and the yeast of choice in biomanufacturing. An alternative emerging yeast Kluyveromyces marxianus boasts a rapid doubling time, thermal and acid tolerance, and known assimilation of a broad range of low-cost feedstocks, attributes all suited for industrial applications. Current engineering approaches for both yeasts focus on traditional metabolic engineering (i.e. combinations of gene knock outs/ins, up/down regulations, etc.), which can compromise cell health and reduce production capabilities. In this work, we develop spatially and temporally controlled synthetic tools to be used in conjunction with traditional metabolic engineering in these yeasts, for more efficient utilization of metabolism and enhanced production of select societally relevant products. A common complication in biosynthesis is product toxicity to the microbial host. Separating growth and production phases can be beneficial for improving toxic compound synthesis. To demonstrate this and further develop K. marxianus for production of toxic products, we developed a set of hybrid carbon-responsive promoters for stationary phase product synthesis. Two hybrids, PIT350 and PIN450, demonstrated exceptional strength, partial repression during growth, and strong stationary phase activation in glucose- and lactose-based medium, respectively. PIN450 was then used to produce four compounds of varying toxicity (triacetic acid lactone, 6-methylsalicylic acid, indole-acetic acid, and sabinene) from the waste-derived sugar lactose, demonstrating greater production (ranging from 1.5- to 6.6-fold) relative to growth-associated production. Spatial organization of metabolic pathways is also important to consider for high yield and titer production. Colocalization of pathway enzymes allows for substrates to be shuttled between enzymes more efficiently, increasing desired product synthesis. We introduce two transformative methods for protein colocalization in the cytosols of both S. cerevisiae and K. marxianus: (1) peroxisomal surface display, a technique for displaying proteins on the peroxisome membrane via an anchoring motif from the peroxin Pex15, and (2) an RNA-driven protein assembly technique that exploits the highly specific interactions of orthogonal Cas6 proteins and the predictability of RNA base-pairing. We highlight the advantages of peroxisomal surface display by displaying enzymes from different pathways (indole-acetic acid, proviolacein, and TAL) and demonstrate production increases ranging from 2.5- to 11-fold for all three compounds compared to when individual pathway enzymes were left freely diffusing in the cytosol. We used our RNA-driven protein assembly technique to reconstitute a split luminescence reporter, and for metabolon formation and improved TAL synthesis in S. cerevisiae. Finally, we reconfigure our RNA-based technique towards using split RNA aptamer fragments to create an on-demand chemically triggerable protein assembly system for S. cerevisiae, which we successfully used to reconstitute two split reporter systems. Yeasts are important microbes and modern cell factories for production of commodity and high-value chemicals and the development and implementation of control systems in yeast will only further propel these microbes forward for industrial biomanufacturing.