Metabolic engineering of oleaginous yeasts for fatty alcohol production Wei Wang, Hui Wei, Eric Knoshaug, Stefanie Van Wychen, Qi Xu, Michael E. Himmel, Min Zhang National Renewable Energy Laboratory, Golden, CO 80401 * [email protected] Abstract Production of fatty alcohol in Lipomyces Ls[FAR] 900 70.0 To develop pathways for advanced biological upgrading of sugars to C16 C18 800 hydrocarbons, we are seeking biological approaches to produce high 60.0 C18:1 C20 carbon efficiency intermediates amenable to separations and catalytic 700 50.0 upgrading to hydrocarbon fuels. In this study, we successfully 600 demonstrated fatty alcohols production by the oleaginous yeasts, Yarrowia 500 40.0 400 lipolytica and Lipomyces starkeyi, by expressing the bacterial fatty acyl- 30.0 CoA reductase (FAR) gene, resulting in the production of 167 and 770 mg/L 300 Fatty alcohol(%) 20.0 200 of fatty alcohols in shaking flasks, respectively. Moreover, we find much (mg/L) production alcohol Fatty higher extracellular distribution of fatty alcohols produced by FAR- 100 10.0 expressing L. starkeyi compared to Y. lipolytica. In both yeasts, long chain 0 0.0 C t1 t2 t3 t4 t5 t6 t7 t8 t9 t10 t11 t12 t13 t14 t15 t16 t17 t18 t19 t20 length saturated fatty alcohols, i.e., hexadecanol and octadecanol were t1 t2 t3 t4 t5 t6 t7 t8 t9 t10 t11 t12 t13 t14 t15 t16 t17 t18 t19 t20 predominant, accounting for more than 85% of total fatty alcohols 70 transformants were confirmed to produce fatty alcohols with varied levels, among which produced. Our work demonstrates that, in addition to Y. lipolytica, L. # 10 produced 770 mg/L. This production level is about 5 fold more than in Y. lipolytica, which starkeyi can also serve as a platform organism for production of fatty acid- could be related to the robust fatty acid synthesis in L. starkey as indicateda by high lipid content derived biofuels and bioproducts following metabolic engineering. in wild type cells; The patterns of fatty alcohol composition in L. starkeyi FAR transformants were similar to Engineered pathway for fatty alcohols that of Y. lipolytica. i.e. long chain fatty alcohols were the major constituents. production in oleaginous yeast Partition of fatty alcohols in FAR transformants 100.0 cell pellet The distributions of the fatty 90.0 b supernatant alcohols produced by Y. lipolytica 80.0 70.0 and L. starkeyi were quite different. 60.0 In L. starkeyi, more fatty alcohols, 50.0 at least 50% of the total, were 40.0 secreted out of the cells into the 30.0 medium. This difference could be Fatty alcohol distribution (%) alcohol distribution Fatty 20.0 related to the permeability of cell 10.0 membranes. 60µm 0.0 Lipomyces Yarrowia [hw1]New data Fatty alcohol production with glucose and Production of fatty alcohols in Yarrowia Yl[FAR] Cell growth xylose as substrate 5.0 180 glucose as substrate xylose as substrate 4.5 160 50.0 800 50.0 800 4.0 OD600 OD600 140 45.0 Glucose 700 45.0 3.5 Xylose 700 120 FAME FAME Control 40.0 40.0 3.0 Fatty alcohol Fatty alcohol 100 600 600 YI[FAR] 35.0 2.5 35.0 80 OD600 500 500 2.0 30.0 30.0 60 25.0 400 1.5 Fatty alcohol (mg/L) 25.0 400 40 1.0 20.0 300 20.0 20 300 0.5 Fatty alcohols (mg/L) 15.0 Fatty alcohol (mg/L) 15.0 0.0 0 200 (%) (g/L), FAME Xylose OD600, 200 10.0 10.0 0 20 40 60 80 100 120 140 with dodecane w/o dodecane overlay (%) (g/L), glucose FAME OD600, 100 100 Time (h) 5.0 5.0 0.0 0 0.0 0 we did not find that our Yl[FAR] transformant exhibited slower growth than the 0 20 40 60 80 100 120 140 160 0 20 40 60 80 100 120 140 160 parent strain; Expressing the FAR gene in Y. lipolytica resulted in production of fatty Time (h) Time (h) alcohol up to 167 mg/L in mineral medium. L. starkeyi can utilize both glucose and xylose for fatty alcohol productions; The profile of fatty alcohol production on xylose is similar to that on glucose, except cell Fatty alcohols composition in Yl[FAR] mass and the fatty alcohol titer were slightly lower than those on glucose. Over-expressing the FAR gene in Y. 60.0 lipolytica led to a decrease in lipid content, Conclusions and challenges 50.0 indicating the carbon flux originally to TAG synthesis was partially redirected towards fatty 40.0 This study demonstrates fatty alcohol production by oleaginous yeasts Y. lipolytica alcohol production; and L. starkeyi. Our work indicates that, in addition to Y. lipolytica, L. starkeyi could 30.0 The fatty alcohols with long chain length, i.e. hexadecanol (C16), octadecanols (C18), and also serve as a platform organism for production of fatty acid-derived biofuels and 20.0 Fatty alcohol (%) alcohol Fatty (C18:1) accounted for 53.1%, 36.4%, and bioproducts via metabolic engineering. 10.0 10.5% respectively of total alcohols. Saturated To achieve this goal, however, more efficient molecular biology tools have to be 0.0 fatty alcohols were predominant in all produced fatty alcohols which could be beneficial for developed for further genetic manipulation of L. starkeyi; in addition, more genomic downstream hydrotreating to hydrocarbons information has to be acquired. This work was supported by the U.S. Department of Energy Bioenergy The information contained in this poster is subject to a government license. Technology Office (DOE-BETO) under Contract No. DE-AC36-08-GO28308 The 38th Symposium on Biotechnology for Fuels and Chemicals, with the National Renewable Energy Laboratory Baitimore, MD, April 25 - 29, 2016 NREL/PO-2700-65466.