Abstract
As a means to develop oleaginous biorefinery, Yarrowia lipolytica was utilized to produce ω-hydroxy palmitic acid from glucose using evolutionary metabolic engineering and synthetic FadR promoters for cytochrome P450 (CYP) expression. First, a base strain was constructed to produce free fatty acids (FFAs) from glucose using metabolic engineering strategies. Subsequently, through ethyl methanesulfonate (EMS)-induced random mutagenesis and fluorescence-activated cell sorting (FACS) screening, improved FFA overproducers were screened. Additionally, synthetic promoters containing bacterial FadR binding sequences for CYP expression were designed to respond to the surge of the concentration of FFAs to activate the ω-hydroxylating pathway, resulting in increased transcriptional activity by 14 times from the third day of culture compared to the first day. Then, endogenous alk5 was screened and expressed using the synthetic FadR promoter in the developed strain for the production of ω-hydroxy palmitic acid. By implementing the synthetic FadR promoter, cell growth and production phases could be efficiently decoupled. Finally, in batch fermentation, we demonstrated de novo production of 160 mg/L of ω-hydroxy palmitic acid using FmeN3-TR1-alk5 in nitrogen-limited media. This study presents an excellent example of the production of ω-hydroxy fatty acids using synthetic promoters with bacterial transcriptional regulator (i.e., FadR) binding sequences in oleaginous yeasts.
Highlights
Construction of biorefinery using microorganisms and biomass can be competitive to that of oil refinery, only when the cost of carbon resource substrates is economical enough to that of petroleum (Cherubini, 2010)
Among the non-conventional yeasts, Yarrowia lipolytica is recognized as a promising industrial strain due to its ability to convert a broad range of carbon substrates such as glucose and glycerol, fructose, fatty acids, and hydrophobic hydrocarbon compounds into high content of intracellular neutral lipids (Barth and Gaillardin, 1996; Mori et al, 2013; Workman et al, 2013; Abghari and Chen, 2014; Lazar et al, 2014) [substrate range of Y. lipolytica is well-reviewed (Ledesma-Amaro and Nicaud, 2016)]
We confirmed that activation of fatty acid synthesis to fatty acyl-CoA and channeling into other pathways consuming fatty acyl-CoA should be avoided to accumulate free fatty acids (FFAs) instead of triacylglyceride (Figure 1C)
Summary
Construction of biorefinery using microorganisms and biomass can be competitive to that of oil refinery, only when the cost of carbon resource substrates is economical enough to that of petroleum (Cherubini, 2010). Waste vegetable oils and glycerol from food industries are excellent and cheap enough carbon sources to compete with petroleum, since the price of the resources and sustainable society maintenance in terms of carbon recycling give sufficient legitimacy to develop various bioprocesses utilizing the cheap carbon sources (da Silva et al, 2009; Chen et al, 2018) In this regard, unlike Saccharomyces cerevisiae, which is characterized to efficiently produce bioethanol from simple sugars such as glucose and sugar cane/beet (Sarkar et al, 2012), non-conventional yeasts have drawn our attention as they have unique traits such as methylotroph or oleaginous yeast strains (Houard et al, 2002; Radecka et al, 2015; Rebello et al, 2018). Y. lipolytica is well-suited for the bioproduction of lipid-derived bulk chemicals, as it can assimilate cheap carbon sources, even industrial wastes, into intracellular lipids
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