SaCas9-Mediated CrtYB Disruption Coupled with Heterologous α-Bisabolol Production in Rhodotorula mucilaginosa JY1105

Rhodotorula mucilaginosa is a promising host for terpene biosynthesis, but its rational engineering remains constrained by limited genome-editing tools. Here, we developed a Staphylococcus aureus Cas9 (SaCas9) workflow in R. mucilaginosa JY1105 for CrtYB disruption and demonstrated heterologous α-bisabolol production. Five sgRNAs targeted CrtYB, encoding bifunctional phytoene synthase/lycopene cyclase; its disruption provided visible depigmentation for preliminary screening. Among 25 sequenced transformants per guide, CrtYB3 and CrtYB5 yielded 10 (40%) and 4 (16%) white colonies, respectively; each white colony carried an on-target deletion of 11–26 bp, whereas all red colonies retained the wild-type target sequence. No white colonies or target-site deletions were detected for CrtYB1, CrtYB2, or CrtYB4. A codon-optimized chamomile TPS1 cassette encoding α-bisabolol synthase was integrated by random integration into wild-type JY1105 and the edited R1-ΔCrtYB derivative. The selected highest producers produced 37.52 ± 3.09 and 41.45 ± 2.08 mg L−1 α-bisabolol, respectively. To our knowledge, this is the first report of heterologous α-bisabolol production in R. mucilaginosa. This study demonstrates the potential of R. mucilaginosa as an editable chassis for heterologous sesquiterpene biosynthesis and provides a foundation for further development of SaCas9-assisted metabolic engineering in this non-conventional yeast.