Microbial production is an attractive approach to synthesizing terpenoids, enabling scalable access to high-value bioactive compounds. In this work, we report engineering of the basidiomycete red yeast Xanthophyllomyces dendrorhous to produce the sesquiterpene β-elemene, a compound with reported antitumor activity. X. dendrorhous is a promising host due to high native flux through the mevalonate pathway and its growth on diverse substrates, which have led to its use in commercial production of the carotenoid astaxanthin. We recently reported strong constitutive promoters derived from X. dendrorhous transcriptomic data, and incorporated them into a modular DNA assembly standard. We used this synthetic biology parts library to construct terpenoid pathway variants. Specifically, we used the high-strength UspA promoter, Pxduspa, to drive expression of the Lactuca sativa LsLTC2 gene, achieving stable production of β-elemene via homozygous genomic integration into the red yeast genome. We first modified the type strain, X. dendrorhous CBS 6938, and measured 45 mg/L of β-elemene in the engineered strain. We then performed the same engineering in an industrial astaxanthin overproducing strain, UBV-AX4, which produced nearly 450 mg/L. To understand the differences between CBS 6938 and UBV-AX4 that might explain the tenfold difference in titer, we conducted differential transcriptomics. We found evidence that the stress response is deregulated in UBV-AX4, which forces the terpenoid pathway to be on even in ideal growth conditions. These results indicate that the transcriptional regulatory network of stress is a key factor controlling final titer in red yeast. In summary, this work highlights the importance of strain selection in microbial engineering, and underscores the potential of X. dendrorhous as a nonconventional yeast chassis for natural product biosynthesis.
