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Engineering global transcription to tune lipophilic properties in Yarrowia lipolytica

Author:
Wang, Man, Liu, Guan-Nan, Liu, Hong, Zhang, Lu, Li, Bing-Zhi, Li, Xia, Liu, Duo, Yuan, Ying-Jin
Source:
Biotechnology for biofuels 2018 v.11 no.1 pp. 115
ISSN:
1754-6834
Subject:
DNA assembly, DNA-directed RNA polymerase, Yarrowia lipolytica, beta-carotene, biochemical pathways, engineering, fatty acids, free fatty acids, genes, genotype, hydrophobicity, ketone bodies, metabolism, mutagenesis, mutants, phenotype, polymerase chain reaction, transcription (genetics), transcription factors, transcriptomics, yeasts
Abstract:
BACKGROUND: Evolution of complex phenotypes in cells requires simultaneously tuning expression of large amounts of genes, which can be achieved by reprograming global transcription. Lipophilicity is an important complex trait in oleaginous yeast Yarrowia lipolytica. It is necessary to explore the changes of which genes’ expression levels will tune cellular lipophilic properties via the strategy of global transcription engineering. RESULTS: We achieved a strategy of global transcription engineering in Y. lipolytica by modifying the sequences of a key transcriptional factor (TF), SPT15-like (Yl-SPT15). The combinatorial mutagenesis of this gene was achieved by DNA assembly of up to five expression cassettes of its error-prone PCR libraries. A heterologous beta-carotene biosynthetic pathway was constructed to research the effects of combined Yl-SPT15 mutants on carotene and lipid production. As a result, we obtained both an “enhanced” strain with 4.7-fold carotene production and a “weakened” strain with 0.13-fold carotene production relative to the initial strain, nearly 40-fold changing range. Genotype verification, comparative transcriptome analysis, and detection of the amounts of total and free fatty acids were made for the selected strains, indicating effective tuning of cells’ lipophilic properties. We exploited the key pathways including RNA polymerase, ketone body metabolism, fatty acid synthesis, and degradation that drastically determined cells’ variable lipophilicity. CONCLUSIONS: We have examined the effects of combinatorial mutagenesis of Yl-SPT15 on cells’ capacity of producing beta-carotene and lipids. The lipophilic properties in Y. lipolytica could be effectively tuned by simultaneously regulating genome-wide multi-gene expression levels. The exploited gene targets and pathways could guide design and reconstruction of yeast cells for tunable and optimal production of other lipophilic products.
Agid:
5932618