(254d) Metabolic Regulation and Engineering in Red Yeast, a Basidiomycete of Biotechnological Interest (Industry Candidate)

Basidiomycetes, one of the two major divisions of fungi, have ecological roles of biotechnological interest. As endophytes, they increase the health of forests and agricultural crops. As decomposers, they facilitate turnover of biomass and are essential components of carbon, nitrogen, and phosphorous cycles. To fulfill these roles, they have developed unique biosynthetic capabilities that could be useful starting points for metabolic engineering. Basidiomycetes are still underinvestigated compared to model organisms, and their cell factory potential is underexplored. To this end, we have identified the basidiomycete Xanthophyllomyces dendrorhous, or red yeast, as a potential platform host because it natively produces high titers of the carotenoid astaxanthin. Yet, a dearth of genetic tools and a limited understanding of basidiomycete metabolic regulation has fundamentally limited it as a platform host. Thus, we have engaged in genomics-driven characterization and engineering of carotenoid biosynthesis in two strains of red yeast, one wild type (CBS 6938) and an evolved astaxanthin overproducer (UBV-AX4).

Our initial experiments revealed that red yeast carotenogenesis is regulated by light, but only in wild type X. dendrorhous. Further investigation with an LED apparatus identified UV and blue light as key activators of carotenogenesis, while UBV-AX4 was relatively unaffected. This difference in phenotypic behavior provides an ideal scenario to elucidate natural basidiomycete optogenetic circuits. To elucidate this mechanism, we sequenced the genomes of both the wild type and UBV-AX4 strains and collected transcriptomic data for strains exposed to different wavelengths of light. This data reveals differentially regulated genes for further investigation.

We next sought to isolate and characterize basidiomycete transcriptional parts for pathway engineering. However, red yeast produces a variety of compounds that are fluorescent, limiting the use of certain fluorescent reporters. Specifically, X. dendrorhous naturally has high fluorescence from carotenoids. We thus evaluated various reporters for optimal characterization, including NanoLuc, GFP, BFP, and three RFPs - mCherry, mKate, and mCardinal. We determined that the RFP mCardinal is the ideal reporter for this organism, as it can be measured with flow cytometry, it exhibited little background fluorescence at the RFP excitation/emission channel, and was the brightest among the RFPs tested. Using mCardinal, we characterized 10 promoters and 10 terminators. These results point to a need to determine the ideal reporter in nonconventional yeasts, especially those that produce secondary metabolites that fluoresce and absorb at wavelengths that can interfere with flow cytometry.

In sum, this work represents a genomics-driven strain engineering effort of a nonconventional basidiomycete yeast. With high quality genomes, transcriptomic elucidation of metabolic pathway regulation, and expression part characterization capabilities, we have built a foundation for future metabolic engineering of red yeast for production of industrially relevant compounds.