lipolytica is abundant in acetyl-CoA due to its cytosolic ATP citrate lyase (ACL). lipolytica might be engineered as a novel production platform for violacein production due to its GRAS status.
violacein were isolated from marine environment with high GC content (up to 65% in the coding sequence), we argue that Y. lipolytica has been widely adopted as host for production of citric acid, β-carotenoids and β-ionone in food industry. Yarrowia lipolytica has been considered to be non-pathogenic and has been classified as ‘generally regarded as safe’ (GRAS) by the US Food and Drug Administration (FDA). Although the native host represents some advantage to produce violacein, the pathogenicity significantly limited their industrial application.
#Csm perferred engineering calculator skin#
lividum have been related with serious skin infection in immune-compromised people, and both strain have been classified as biosafety level II bacteria. Īt present, most of the reported violacein-producing host are gram-negative bacteria with human pathogenicity.
Without involving the first reduction step of vioD, deoxyviolacein is formed as the major byproduct. Subsequently, protodeoxyviolaceinate is reduced to violacein by vioD and VioC. Then, IPA is decarboxylated to form protodeoxyviolaceinic acid via vioE.
Two molecules of L-tryptophan are oxidatively condensed by vioA and vioB to form indole-3-pyruvic acid (IPA). Branched from the L-tryptophan pathway, the violacein biosynthetic pathway involves five steps, encoded by vioA, vioB, vioC, vioD and vioE, which were organized in an operon form containing all the five genes. in 1991 and was fully characterized by Balibar et al. Violacein biosynthetic pathway was first discovered by Pemberton et al. These characteristics make violacein a superior chemical scaffold and drug candidate for the development of clinically active agents. Clinical trials and biomedical studies indicate both compounds possess strong antibacterial, anticancer, antiviral, trypanocidal and antiprotozoal properties. Violacein and deoxyviolacein belong to bisindol pigments with deep purple color, which are derived from tryptophan biosynthetic pathway and naturally produced by a number of marine bacteria such as Janthinobacterium lividum, Chromobacterium violaceum, Pseudoalteromonas luteoviolacea, et al. This information should be valuable for us to build a renewable and scalable violacein production platform from the novel host oleaginous yeast species. Taken together, the development of efficient extraction protocol, quantitative correlation between HPLC and microplate reader, and the optimization of culture conditions set a new stage for engineering violacein production in Y. Violacein purity reaches 86.92% at C/N ratio of 60, with addition of 10 g/L CaCO 3 to control the media pH. Our results indicated that ethyl acetate is the best extraction solvent with glass beads grinding the cell pellets, the maximum violacein and deoxyviolacein production was 70.04 mg/L and 5.28 mg/L in shake flasks, respectively. Furthermore, we optimized the yeast cultivation conditions, including carbon/nitrogen ratio and pH conditions. We demonstrated that both HPLC and microplate reader are technically equivalent to measure violacein from yeast culture. We also established the quantitative correlation between HPLC and microplate reader method. In this work, we optimized the extraction protocol to improve violacein recovery ratio and purity from yeast culture, including the variations of organic solvents, the choice of mechanical shear stress, incubation time and the use of cell wall-degrading enzymes. Yeast fermentation represents an alternative approach to efficiently manufacturing violacein from inexpensive feedstocks. Violacein is a naturally occurring anticancer therapeutic with deep purple color.
0 kommentar(er)
