Combining evolutionary and metabolic engineering in Rhodosporidium toruloides for lipid production with non-detoxified wheat straw hydrolysates
Improving the yield of carbohydrate to lipid conversion and lipid productivity are two critical goals to develop an economically feasible process to commercialize microbial oils. Lignocellulosic sugars are potential low-cost carbon sources for this process but their use is limited by the toxic compounds produced during biomass pretreatment at high solids loading, and by the pentose sugars (mainly xylose) which are not efficiently metabolized by many microorganisms. Adaptive laboratory evolution was used to select a Rhodosporidium toruloides strain with robust growth in non-detoxified wheat straw hydrolysates, produced at 20% solids loading, and better xylose consumption rate. An arabinose-inducible cre-lox recombination system was developed in this evolved strain that was further engineered to express a second copy of the native DGAT1 and SCD1 genes under control of the native xylose reductase (XYL1) promoter. Fed-batch cultivation of the engineered strain in 7-L bioreactors produced 39.5 g lipid/L at a rate of 0.334 g/Lh−1 and 0.179 g/g yield, the best results reported in R. toruloides with non-detoxified lignocellulosic hydrolysates to date.
KeywordsOleaginous yeasts Metabolic engineering Adaptive evolution Wheat straw hydrolysates Rhodosporidium toruloides
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
- Davis R, Tao L, Tan ECD, Biddy MJ, Beckham GT, Scarlata C, Jacobson J, Cafferty K, Ross J, Lucas J, Knorr D, Schoen P (2013) Process design and economics for the conversion of lignocellulosic biomass to hydrocarbons: dilute acid and enzymatic deconstruction of biomass to sugars and biological conversion of sugars to hydrocarbons NREL/TP-5100-60223. NREL, GoldenCrossRefGoogle Scholar
- Fei Q, O’Brien M, Nelso R, Chen X, Lowell A, Dowe N (2016) Enhanced lipid production by Rhodosporidium toruloides using different fed-batch feeding strategies with lignocellulosic hydrolysate as the sole carbon source. Biotechnol Biofuels 9(1):130. https://doi.org/10.1186/s13068-016-0542-x CrossRefPubMedPubMedCentralGoogle Scholar
- Goodharzi H, Bennet BD, Amini S, Reaves ML, Hottes AK, Rabinowitz JD, Tavazoie S (2010) Regulatory and metabolic rewiring during laboratory evolution of ethanol tolerance in Escherichia coli. Mol Syst Biol 6:378Google Scholar
- Koh CM, Liu Y, Moehninsi DM, Ji L (2014) Molecular characterization of KU70 and KU80 homologes and explotation of a KU70-deficient mutant for improving gene deletion frequency in Rhodosporidium toruloides. BMC Microbiol 14(1):50. https://doi.org/10.1186/1471-2180-14-50 CrossRefPubMedPubMedCentralGoogle Scholar
- Lin X, Wang Y, Zhang S, Zhu Z, Zhou YJ, Yang F, SunW, Yang X, Zhao ZK (2014) Functional integration of multiple genes into the genome of the oleagionous yeast Rhodosporidium toruloides. FEMS Yeast Res 14(4):547–555Google Scholar
- Liu Y, Koh CM, Sun L, Hlaing MM, Du M, Peng N, Li J (2013) Characterization of glyceraldehyde-3-phosphate dehydrogenase gene RtGPD1 and development of genetic transformation method by dominant selection in oleaginous yeast Rhodosporidium toruloides. Appl Microbiol Biotechnol 97(2):719–729. https://doi.org/10.1007/s00253-012-4223-9 CrossRefPubMedGoogle Scholar
- Liu H, Jiao X, Wang Y, Yang X, Sun W, Wang J, Zhang S, Zhao ZK (2017) Fast and efficient genetic transformation of oleaginous yeast Rhodosporidium toruloides by using electroporation. FEMS Yeast Res 17(2). https://doi.org/10.1093/femsyr/fox2017
- Matsakas L, Bonturi N, Miranda EA, Rova U, Christakopoulos P (2015) High concentrations of dried sorghum stalks as a biomass feedstock for single cell oil production by Rhodosporidium toruloides. Biotechnol Biofuels 8(1):6. https://doi.org/10.1186/s13068-014-0190-y CrossRefPubMedPubMedCentralGoogle Scholar
- Matsakas L, Novak K, Enman J, Christakopoulos P, Rova U (2017) Acetate-detoxification of wood hydrolysates with alkali tolerant Bacillus sp. as a strategy to enhance the lipid production from Rhodosporidium toruloides. Bioresour Technol. https://doi.org/10.1016/j.biortech.2017.04.002
- Minty J, Lesnefsky A, Lin F, Chen Y, Zaroff T, Veloso A, Xie B, McConnell C, Ward R, Schwartz D, Rouillard JM, Gao Y, Gulari E, Lin XN (2011) Evolution combined with genomic study elucidates genetic bases of isobutanol tolerance in Escherichia coli. Microb Cell Factories 10(1):18. https://doi.org/10.1186/1475-2859-10-18 CrossRefGoogle Scholar
- Park YK, Nicaud JM, Ledesma-Amaro R (2017) The engineering potential of Rhodosporidium toruloides as a workhorse for biotechnological applications. Trends Biotecnol. https://doi.org/10.1016/j.tibtech.2017.10.013
- Slininger PJ, Dien BS, Kurtzman CP, Moser BR, Bakota EL, Thompson SR, PJ O’B, Cotta MA, Balan V, Jin M, da Costa-Sousa L, Dale B (2016) Comparative lipid production by oleaginous yeasts in hydrolyzates of lignocellulosic biomass and process strategy for high titers. Biotechnol Bioeng 113(8):1676–1690. https://doi.org/10.1002/bit.25928 CrossRefPubMedGoogle Scholar
- Zhang S, Ito M, Skerker JM, Arkin AP, Rao CV (2016b) Metabolic engineering of the oleaginous yeast Rhodosporidium toruloides IFO0880 for lipid overproduction during high-density fermentation. Appl Microbiol Biotechnol 100(21):9393–9405. https://doi.org/10.1007/s00253-016-7815-y CrossRefPubMedGoogle Scholar
- Zhao X, Peng F, Du W, Liu C, Liu D (2012) Effects of some inhibitors on the growth and lipid accumulation of oleaginous yeast Rhodosporidium toruloides and preparation of biodiesel by enzymatic transesterification of the lipid. Bioprocess Biosyst Eng 35(6):993–1004. https://doi.org/10.1007/s00449-012-0684-6 CrossRefPubMedGoogle Scholar