Fatty alcohol production in Lipomyces starkeyi and Yarrowia lipolytica

Abstract

Background: Current biological pathways to produce biofuel intermediates amenable to separations and catalytic upgrading to hydrocarbon fuels are not cost effective. Previously, oleaginous yeasts have been investigated primarily for lipid production. However, yeasts store neutral lipids intracellularly making recovery difficult and expensive. In addition, once recovered from the cells, lipids are difficult to blend directly with the existing fuels without upgrading. We have, therefore, begun to investigate secreted fatty acid-derived products which can be easily recovered and upgraded to fuels.

Results: In this study, we successfully demonstrate the production of fatty alcohols by the oleaginous yeasts, Yarrowia lipolytica and Lipomyces starkeyi, through expression of the fatty acyl-CoA reductase gene from Marinobactor aquaeolei VT8. This strategy resulted in the production of 167 and 770 mg/L of fatty alcohols in shake flask from Y. lipolytica and L starkeyi, respectively. When using a dodecane overlay during fermentation, 92 and 99% of total fatty alcohols produced by Y. lipolytica and L. starkeyi, respectively, were extracted into the dodecane phase, which compares favorably to the 3 and 50% recovered, respectively, without the dodecane layer. In both oleaginous yeasts, long chain length, saturated fatty alcohols, i.e., hexadecanol (C16:0) and octadecanol (C18:0), were predominant and accounted for more than 85% of the total fatty alcohols produced. To the best of our knowledge, this is the first report of fatty alcohol production in L. starkeyi.

Conclusion: This work demonstrates that the oleaginous yeasts, Y. lipolytica and L. starkeyi, can serve as platform organisms for the production of fatty acid-derived biofuels and bioproducts.

Keywords: Fatty alcohols; Lipomyces starkeyi; Metabolic engineering; Oleaginous yeasts; Yarrowia lipolytica.

Fig. 1

Biosynthetic pathway of fatty alcohol in Yarrowia lipolytica and Lipomyces starkeyi. The yellow-shaded pathway from fatty acyl-CoA to fatty alcohol indicates the engineered steps for the fatty alcohol synthesis described in this work. ACC acetyl-CoA carboxylase; ACL ATP citrate lyase; G3P glyceraldehyde 3-phosphate; DGA diacylglycerolacyltransferase; DHAP dihydroxyacetone phosphate; F-6-P fructose 6-phosphate; FAS fatty acid synthase; G-6-P glucose 6-phosphate; LPA lysophosphatidic acid; MaFAR fatty acid reductase of Marinobactor aquaeolei VT8; OAA oxaloacetate; PD pyruvate dehydrogenase; PL phospholipid; Pyr pyruvate; TAG triacylglycerol. The diagram is based on summary views of lipid biosynthesis in the recent literature described in the text of this paper

Fig. 2

Cell growth (a) and fatty alcohols production (b) by far-expressing Y. lipolytica growing on mineral medium. The control represents empty vector control

Fig. 3

Lipid and fatty alcohol compositions in Yl[FAR] growing on mineral medium at 5 days. Fermentation medium was overlaid with dodecane. a Lipid composition. b Fatty alcohol composition. The control represents empty vector control

Fig. 4

Fatty alcohol production by far-expressing L. starkeyi transformants on mineral medium at 5 days. Fermentation medium was overlaid with dodecane. C represents empty vector control

Fig. 5

Lipid compositions in Ls[FAR] transformant 10 growing on mineral medium at 5 days. The standard deviation of triplicates was <0.35%. The control represents empty vector control

Fig. 6

Fatty alcohol compositions in Ls[FAR] transformants 6 and 10 growing on mineral medium at 5 days

Fig. 7

Cell growth, fatty alcohols, and lipid production by Ls[FAR] transformant 10 on mineral medium. a Glucose as substrate. b Xylose as substrate

Fig. 8

Pattern of fatty alcohol composition during fatty alcohol production process. a Glucose as substrate. b Xylose as substrate