Engineering the oleaginous yeast Yarrowia lipolytica for production of α-farnesene

Abstract

BackgroundYarrowia lipolytica, a non-traditional oil yeast, has been widely used as a platform for lipid production. However, the production of other chemicals such as terpenoids in engineered Y. lipolytica is still low. α-Farnesene, a sesquiterpene, can be used in medicine, bioenergy and other fields, and has very high economic value. Here, we used α-farnesene as an example to explore the potential of Y. lipolytica for terpenoid production.ResultsWe constructed libraries of strains overexpressing mevalonate pathway and α-farnesene synthase genes by non-homologous end-joining (NHEJ) mediated integration into the Y. lipolytica chromosome. First, a mevalonate overproduction strain was selected by overexpressing relevant genes and changing the cofactor specificity. Based on this strain, the downstream α-farnesene synthesis pathway was overexpressed by iterative integration. Culture conditions were also optimized. A strain that produced 25.55 g/L α-farnesene was obtained. This is the highest terpenoid titer reported in Y. lipolytica.ConclusionsYarrowia lipolytica is a potentially valuable species for terpenoid production, and NHEJ-mediated modular integration is effective for expression library construction and screening of high-producer strains.

Metabolic engineering for increased lipid accumulation in Yarrowia lipolytica – A Review

“…Moreover, Y. lipolytica can utilize a variety of inexpensive renewable substrates as carbon sources and can accommodate high flux of acetyl-CoA (Magdouli et al, 2017; Ledesma-Amaro and Nicaud, 2016a; Nambou et al, 2014;Poli et al, 2014). These characteristics make Y. lipolytica a remarkable industrial host for the production of many products, including lipid-derived biodiesel (Ledesma-Amaro and Nicaud, 2016b; Ledesma-Amaro, 2015); alkanes (Bruder et al, 2019); odd-chain fatty acids (Park et al, 2020;Park et al, 2018); plant-derived terpenoids, such as α-farnesene (Liu et al, 2019c;Yang et al, 2016), carotenoids (Jacobsen et al, 2020;Larroude et al, 2018a), linalool (Cao et al, 2017), and others (Ma et al, 2019); organic acids, such as citric acid (Kamzolova and Morgunov, 2017), isocitric acid (Rzechonek et al, 2019), succinic acid (Li et al, 2018;Li et al, 2017), α-ketoglutarate (Lei et al, 2019;Zeng et al, 2017), pyruvic acid (Kamzolova and Morgunov, 2018) and crotonic acid (Wang et al, 2019); as well as sugar alcohols, such as erythritol (Liu et al, 2019b;Liu et al, 2017c), erythrulose (Carly et al, 2018), isomaltulose (Zhang et al, 2018). Even more value-added functional polyunsaturated fatty acids (PUFAs), such as γ-linolenic acid (Sun et al, 2017), arachidonic acid (Liu et al, 2019a;Liu et al, 2017a;Liu et al, 2017b), eicosapentaenoic acid (Xue et al, 2013), and docosahexaenoic acid (Gemperlein et al, 2019) were produced in engineered Y. lipolytica strains.…”

Yarrowia lipolytica Strains Engineered for the Production of Terpenoids

“…β-farnesene has been produced at titers of 130 g/L by highly modified S. cerevisiae in 200,000-liter bioreactors and therefore is an excellent terpene to demonstrate the effect of metabolic engineering strategies (Meadows et al, 2016). While β-farnesene has not been produced previously in Y. lipolytica, the isomer α-farnesene has been produced by engineered Y. lipolytica at 25.55 g/L in 1-liter bioreactors (Liu et al, 2019). Interestingly, switching the endogenous squalene promoter with the weak pERG11 promoter did not benefit β-farnesene production, although the same strategy was shown to boost β-carotene production in Y. lipolytica (Kildegaard et al, 2017).…”

New frontiers: harnessing pivotal advances in microbial engineering for the biosynthesis of plant-derived terpenoids

“…Lipid engineering in yeast was accomplished through the overproduction of triacylglycerol and a knock-out of FLD1, which regulates lipid droplet size, resulting in oversized lipid droplets that accumulate and store lycopene, an acyclic tetraterpene, resulting in record titers of 2.37 g/L [44]. These challenges have brought recent attention to Yarrowia as a production host for plantderived terpenes due to its capacity to accumulate lipophilic compounds and the potential to utilize technology developed for S. cerevisiae in this new host [45,46]. A recent pivotal study harnessed peroxisomes to produce squalene at an unprecedented titer through dual cytoplasmic-peroxisomal engineering [47].…”