A Golden-Gate Based Cloning Toolkit to Build Violacein Pathway Libraries in .

Violacein is a naturally occurring anticancer therapeutic compound with deep purple color. In this work, we harnessed the modular and combinatorial feature of a Golden Gate assembly method to construct a library of violacein producing strains in the oleaginous yeast , where each gene in the violacein pathway was controlled by three different promoters with varying transcriptional strength. After optimizing the linker sequence and the Golden Gate reaction, we achieved high transformation efficiency and obtained a panel of representative recombinant strains. By evaluating the gene expression profile of 21 yeast strains, we obtained three colorful compounds in the violacein pathway: green (proviolacein), purple (violacein), and pink (deoxyviolacein). Our results indicated that strong expression of , , and favors violacein production with minimal byproduct deoxyvioalcein in , and high deoxyviolacein production was found strongly associated with the weak expression of . By further optimizing the carbon to nitrogen ratio and cultivation pH, the maximum violacein reached 70.04 mg/L with 5.28 mg/L of deoxyviolacein in shake flasks. Taken together, the development of Golden Gate cloning protocols to build combinatorial pathway libraries, and the optimization of culture conditions set a new stage for accessing the violacein pathway intermediates and engineering violacein production in . This work further expands the toolbox to engineering as an industrially relevant host for plant or marine natural product biosynthesis.