Production of abscisic acid in the oleaginous yeast Yarrowia lipolytica.

Abscisic acid (ABA) is a phytohormone with applications in agriculture and human health. ABA can be produced by Botrytis cinerea, a plant pathogenic filamentous fungus. However, the cultivation process is lengthy and strain improvement by genetic engineering is difficult. Therefore, we engineered the oleaginous yeast Yarrowia lipolytica as an alternative host for ABA production. First, we expressed five B. cinerea genes involved in ABA biosynthesis (BcABA1,BcABA2,BcABA3,BcABA4 and BcCPR1) in a Y. lipolytica chassis with optimized mevalonate flux. The strain produced 59.2 mg/L of ABA in small-scale cultivation. Next, we expressed an additional copy of each gene in the strain, but only expression of additional copy of BcABA1 gene increased the ABA titer to 168.5 mg/L. We then integrated additional copies of the mevalonate pathway and ABA biosynthesis encoding genes, and we expressed plant ABA transporters resulting in an improved strain producing 263.5 mg/L and 9.1 mg/g dry cell weight (DCW) ABA. Bioreactor cultivation resulted in a specific yield of 12.8 mg/g DCW ABA; however, surprisingly, the biomass level obtained in bioreactors was only 10.5 g DCW/L, with a lower ABA titer of 133.6 mg/L. While further optimization is needed, this study confirms Y. lipolytica as a potential alternative host for the ABA production.

Engineering of Yarrowia lipolytica for terpenoid production.

Terpenoids are a group of chemicals of great importance for human health and prosperity. Terpenoids can be used for human and animal nutrition, treating diseases, enhancing agricultural output, biofuels, fragrances, cosmetics, and flavouring. However, due to the rapid depletion of global natural resources and manufacturing practices relying on unsustainable petrochemical synthesis, there is a need for economic alternatives to supply the world's demand for these essential chemicals. Microbial biosynthesis offers the means to develop scalable and sustainable bioprocesses for terpenoid production. In particular, the non-conventional yeast Yarrowia lipolytica demonstrates excellent potential as a chassis for terpenoid production due to its amenability to industrial production scale-up, genetic engineering, and high accumulation of terpenoid precursors. This review aims to illustrate the scientific progress in developing Y. lipolytica terpenoid cell factories, focusing on metabolic engineering approaches for strain improvement and cultivation optimization.

Engineering of Yarrowia lipolytica for the production of plant triterpenoids: Asiatic, madecassic, and arjunolic acids.

Several plant triterpenoids have valuable pharmaceutical properties, but their production and usage is limited since extraction from plants can burden natural resources, and result in low yields and purity. Here, we engineered oleaginous yeast Yarrowia lipolytica to produce three valuable plant triterpenoids (asiatic, madecassic, and arjunolic acids) by fermentation. First, we established the recombinant production of precursors, ursolic and oleanolic acids, by expressing plant enzymes in free or fused versions in a Y. lipolytica strain previously optimized for squalene production. Engineered strains produced up to 11.6 mg/g DCW ursolic acid or 10.2 mg/g DCW oleanolic acid. The biosynthetic pathway from ursolic acid was extended by expressing the Centella asiatica cytochrome P450 monoxygenases CaCYP716C11p, CaCYP714E19p, and CaCYP716E41p, resulting in the production of trace amounts of asiatic acid and 0.12 mg/g DCW madecassic acid. Expressing the same C. asiatica cytochromes P450 in oleanolic acid-producing strain resulted in the production of oleanane triterpenoids. Expression of CaCYP716C11p in the oleanolic acid-producing strain yielded 8.9 mg/g DCW maslinic acid. Further expression of a codon-optimized CaCYP714E19p resulted in 4.4 mg/g DCW arjunolic acid. Lastly, arjunolic acid production was increased to 9.1 mg/g DCW by swapping the N-terminal domain of CaCYP714E19p with the N-terminal domain from a Kalopanax septemlobus cytochrome P450. In summary, we have demonstrated the production of asiatic, madecassic, and arjunolic acids in a microbial cell factory. The strains and fermentation processes need to be further improved before the production of these molecules by fermentation can be industrialized.

Yarrowia lipolytica Strains Engineered for the Production of Terpenoids.

Terpenoids are a diverse group of over 55,000 compounds with potential applications as advanced fuels, bulk and fine chemicals, pharmaceutical ingredients, agricultural chemicals, etc. To facilitate their bio-based production, there is a need for plug-and-play hosts, capable of high-level production of different terpenoids. Here we engineer Yarrowia lipolytica platform strains for the overproduction of mono-, sesqui-, di-, tri-, and tetraterpenoids. The monoterpene platform strain was evaluated by expressing Perilla frutescens limonene synthase, which resulted in limonene titer of 35.9 mg/L and was 100-fold higher than when the same enzyme was expressed in the strain without mevalonate pathway improvement. Expression of Callitropsis nootkatensis valencene synthase in the sesquiterpene platform strain resulted in 113.9 mg/L valencene, an 8.4-fold increase over the control strain. Platform strains for production of squalene, complex triterpenes, or diterpenes and carotenoids were also constructed and resulted in the production of 402.4 mg/L squalene, 22 mg/L 2,3-oxidosqualene, or 164 mg/L ß-carotene, respectively. The presented terpenoid platform strains can facilitate the evaluation of terpenoid biosynthetic pathways and are a convenient starting point for constructing efficient cell factories for the production of various terpenoids. The platform strains and exemplary terpenoid strains can be obtained from Euroscarf.