Screening a genomic library for genes involved in propionate tolerance in Yarrowia lipolytica

Abstract

Microbial oils are regarded as promising alternatives to fossil fuels. For bio‐oil production to be sustainable over the long term, utilizing low‐cost substrates like volatile fatty acids (VFAs) is crucial. Increasing attention is being paid to one of the most common VFAs: propionate, a substrate that could be used to produce the odd‐chain FAs of industrial interest. However, little is known about microbial responses to propionate‐induced stress and the genes involved. Using genomic library screening, we identified two genes involved in propionate tolerance in Yarrowia lipolytica—MFS1 and RTS1. Strains containing each of the genes displayed enhanced tolerance to propionate even when the genes were expressed in truncated form via a replicative plasmid. Compared with the control strain, the strain overexpressing MFS1 under a constitutive promoter displayed greater tolerance to propionate: It had a shorter lag phase and higher growth rate in propionate medium (0.047 hr−1 versus 0.030 hr−1 for the control in 40 g/L propionate); it also accumulated more total lipids and more odd‐chain lipids (10% and 3.3%, respectively) than the control. The strain overexpressing RTS1 showed less tolerance for propionate than the strains harboring the truncated form (0.057 hr−1 versus 0.065 hr−1 in 40 g/L propionate medium) but still had higher tolerance than the control strain. Furthermore, the overexpression of RTS1 seemed to confer tolerance to other weak acids such as lactate, formic acid, malic acid, and succinic acid. This work provides a basis for better understanding the response to propionate‐induced stress in Y. lipolytica.

One major facilitator superfamily transporter is responsible for propionic acid tolerance in Pseudomonas putida KT2440

“…1), such as the genes of methylcitrate synthase (PP_2335, prpC), propionyl-CoA synthase (PP_2351, prpE) and L-threonine aldolase (PP_0321, ltaE; Ma et al, 2020). Knock-out of enzymes responsible for the transformation of intermediates in the PA production pathway led to an increased sensitivity to PA, most likely because the cells accumulate higher intracellular concentrations of PA. A recent study also reported that in Yarrowia lipolytica, the deletion of a gene in the methylcitrate pathway causes a severe growth defect (Park and Nicaud, 2020). A useful chassis for production should be endowed with suitable production properties but also the required determinants for tolerating the product.…”

“…As expected, we found a large number of upregulated genes involved in cell metabolism and stress regulation in the cells cultured with PA (data not shown). A recent study showed that overexpression of the MFS1 gene could improve PA tolerance in Yarrowia lipolytica , a lipid producer that uses PA as the sole substrate (Park and Nicaud, 2020). Thus, we searched for MFS transporter genes differentially expressed in P. putida in the presence of PA. We found 7 MFS genes annotated in the KT2440 genome showing differential expression (Table 1).…”

Engineering precursor pools for increasing production of odd-chain fatty acids inYarrowia lipolytica

“…We previously engineered Y. lipolytica to accumulate a high amount of OCFAs, 41.9% of total lipids [Park et al 2018; Park and Nicaud, 2019]. One of the main modifications was the deletion of the PHD1 gene preventing propionyl-CoA consumption through the methyl citrate pathway.…”

“…Through further modifications, deletion of MFE1 to inhibit β-oxidation and TGL4 to inhibit triacylglycerols (TAG) remobilization, and overexpression of GPD1 and DGA2 to push and pull TAG biosynthesis, we constructed obese strain (JMY3776). In addition, we overexpressed the lipid droplet protein LDP1 to enhance the storage of TAG and named the strain as obese-L strain (JMY7228) [Bhutada et al 2018; Park and Nicaud, 2019]. All these modifications are described in Figure 1(b) together with newly introduced modifications in this study.…”

De novo Biosynthesis of Odd-Chain Fatty Acids in Yarrowia lipolytica Enabled by Modular Pathway Engineering

“…Therefore, to date, most studies have been focused on processes that involve propionate supplementation. However, due to the high cost (Poirier et al, 1995;Aldor et al, 2002) and toxic effects of propionate (Fontanille et al, 2012;Park and Nicaud, 2019), it is crucial to find alternative pathways for generating propionyl-CoA to be able to produce odd-chain FAs on large scales. There have been a few studies in which odd-chain FAs have been produced using glucose.…”