Abstract
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 Yarrowia lipolytica, 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 Y. lipolytica 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 VioB, VioC, and VioD favors violacein production with minimal byproduct deoxyvioalcein in Y. lipolytica, and high deoxyviolacein production was found strongly associated with the weak expression of VioD. 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 Y. lipolytica. This work further expands the toolbox to engineering Y. lipolytica as an industrially relevant host for plant or marine natural product biosynthesis.
Highlights
Violacein and deoxyviolacein belong to bisindol pigments with deep purple color, which are derived from the tryptophan biosynthetic pathway and naturally produced by a number of marine bacteria, such as Janthinobacterium lividum,[1,2] Chromobacterium violaceum,[3−5] and Pseudoalteromonas luteoviolacea.[6]
L-tryptophan pathway, the violacein biosynthetic pathway involves five steps, encoded by vioA, vioB, vioC, vioD, and vioE,[15] which were organized in an operon form containing all five genes
Upon characterizing the strains from a representative collection of library, we found that the optimal violacein production in Y. lipolytica requires strong expression for all the five genes
Summary
Clinical trials and biomedical studies indicate both compounds possess strong antibacterial,[3,7] anticancer,[8] antiviral,[9] trypanocidal,[10] and antiprotozoal[11] properties. These characteristics make violacein a superior chemical scaffold and drug candidate for the development of clinically active antimicrobial, antiviral, and chemotherapeutic agents. L-tryptophan pathway, the violacein biosynthetic pathway involves five steps, encoded by vioA, vioB, vioC, vioD, and vioE,[15] which were organized in an operon form containing all five genes. Two molecules of L-tryptophan are oxidatively condensed by vioA and vioB to form indole-3-pyruvic acid (IPA). Without involving the first reduction step of vioD, deoxyviolacein is formed as the major byproduct.[15−17]
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