Engineering oleaginous yeast Yarrowia lipolytica for violacein production: extraction, quantitative measurement and culture optimization

Abstract

1Violacein is a naturally occurring anticancer therapeutic with deep purple color. 2 Yeast fermentation represents an alternative approach to efficiently manufacturing 3 violacein from inexpensive feedstocks. In this work, we optimized the extraction 4 protocol to improve violacein recovery ratio and purity from yeast culture, including 5 the variations of organic solvents, the choice of mechanical shear stress, incubation 6 time and the use of cell wall-degrading enzymes. We also established the quantitative 7 correlation between HPLC and microplate reader method. We demonstrated that both 8 HPLC and microplate reader are technically equivalent to measure violacein from 9 yeast culture. Furthermore, we optimized the yeast cultivation conditions, including 10 carbon/nitrogen ratio and pH conditions. Our results indicated that ethyl acetate is the 11 best extraction solvent with glass beads grinding the cell pellets, the maximum 12 violacein and deoxyviolacein production was 70.04 mg/L and 5.28 mg/L in shake 13 flasks, respectively. Violacein purity reaches 86.92% at C/N ratio of 60, with addition 14 of 10 g/L CaCO 3 to control the media pH. Taken together, the development of 15 efficient extraction protocol, quantitative correlation between HPLC and microplate 16 reader, and the optimization of culture conditions set a new stage for engineering 17 violacein production in Y. lipolytica. This information should be valuable for us to 18 build a renewable and scalable violacein production platform from the novel host 19 oleaginous yeast species. 20 21

EngineeringYarrowia lipolyticaas a chassis forde novosynthesis of five aromatic-derived natural products and chemicals

“…Surprisingly, overexpression of genes ylTRP2, ylTRP3, VioA , VioB, VioC, VioD, and VioE in strain YL33 led to the accumulation of black pigment in the fermentation broth (Supplementary Figure S8). Violacein and deoxyviolacein reached 366.30 mg/L and 55.12 mg/L (Figure 5d), which were 2.88-fold and 2.51-fold increase over the control strain ( po1fkV , 126.99 mg/L of violacein and 21.98 mg/L of deoxyviolacein) and 5.23-fold and 10.44-fold higher than the production reported in our previous work 65 . To further improve the violacein and deoxyviolacein production, we enhanced tryptophan availability by overexpressing genes ylTRP5, ylTRP4, ylTRP3, ylTRP2, and ylTRP1.…”

“…Both violacein and its derivate deoxyviolacein have demonstrated a broad range of biological activities 63 . By introducing violacein biosynthetic genes (VioA, VioB, VioC, VioD, and VioE, Figure 5c), we previously obtained violacein production around 31 mg/L in Y. lipolytica 28, 65 . Surprisingly, overexpression of genes ylTRP2, ylTRP3, VioA , VioB, VioC, VioD, and VioE in strain YL33 led to the accumulation of black pigment in the fermentation broth (Supplementary Figure S8).…”

Engineering Yarrowia lipolytica as a Chassis for De Novo Synthesis of Five Aromatic-Derived Natural Products and Chemicals

“… 60 By introducing violacein biosynthetic genes (VioA, VioB, VioC, VioD, and VioE, Figure 5 c), we previously obtained violacein production around 31 mg/L in Y. lipolytica . 30 , 62 Surprisingly, overexpression of genes ylTRP2 , ylTRP3 , VioA , VioB , VioC , VioD , and VioE in strain YL33 led to the accumulation of black pigment in the fermentation broth ( Supplementary Figure S8 ). Violacein and deoxyviolacein reached 366.30 ± 28.99 mg/L and 55.12 ± 2.81 mg/L ( Figure 5 d), which were 2.88-fold and 2.51-fold increase over the control strain ( po1fkV , 126.99 ± 9.08 mg/L of violacein and 21.98 ± 3.75 mg/L of deoxyviolacein) and 5.23-fold and 10.44-fold higher than the production reported in our previous work.…”

“…Violacein and deoxyviolacein reached 366.30 ± 28.99 mg/L and 55.12 ± 2.81 mg/L ( Figure 5 d), which were 2.88-fold and 2.51-fold increase over the control strain ( po1fkV , 126.99 ± 9.08 mg/L of violacein and 21.98 ± 3.75 mg/L of deoxyviolacein) and 5.23-fold and 10.44-fold higher than the production reported in our previous work. 62 To further improve the violacein and deoxyviolacein production, we enhanced tryptophan availability by overexpressing genes ylTRP5 , ylTRP4 , ylTRP3 , ylTRP2 , and ylTRP1 . However, no significant improvements of violacein (348.43 ± 27.21 mg/L) and deoxyviolacein (58.21 ± 4.62 mg/L) titer were observed in strain YL42 , suggesting that the supply of tryptophan was sufficient in strain YL41 and there might be other limiting steps in the violacein pathway.…”

Recent Advances in Synthetic, Industrial and Biological Applications of Violacein and Its Heterologous Production

“…Saccharomyces cerevisiae , a eukaryotic model organism, is also a well-established cellular factory and has been employed for the production of many industrially important metabolites and alcohols [ 111 ]. This gene cluster vioABCDE has been expressed in S. cerevisiae in several studies, but the violacein produced was not economically feasible as compared to the bacterial systems.…”