Lipid production in Yarrowia lipolytica is maximized by engineering cytosolic redox metabolism

Journal name:
Nature Biotechnology
Volume:
35,
Pages:
173–177
Year published:
DOI:
doi:10.1038/nbt.3763
Received
Accepted
Published online

Microbial factories have been engineered to produce lipids from carbohydrate feedstocks for production of biofuels and oleochemicals. However, even the best yields obtained to date are insufficient for commercial lipid production. To maximize the capture of electrons generated from substrate catabolism and thus increase substrate-to-product yields, we engineered 13 strains of Yarrowia lipolytica with synthetic pathways converting glycolytic NADH into the lipid biosynthetic precursors NADPH or acetyl-CoA. A quantitative model was established and identified the yield of the lipid pathway as a crucial determinant of overall process yield. The best engineered strain achieved a productivity of 1.2 g/L/h and a process yield of 0.27 g–fatty acid methyl esters/g-glucose, which constitutes a 25% improvement over previously engineered yeast strains. Oxygen requirements of our highest producer were reduced owing to decreased NADH oxidization by aerobic respiration. We show that redox engineering could enable commercialization of microbial carbohydrate-based lipid production.

At a glance

Figures

  1. Improving the lipid yields of engineered Y. lipolytica by introducing synthetic pathways that recycles NADH.c to NADPH.c or acetyl-CoA.
    Figure 1: Improving the lipid yields of engineered Y. lipolytica by introducing synthetic pathways that recycles NADH.c to NADPH.c or acetyl-CoA.

    (a) Illustration of the three synthetic pathways, namely, NADP+-dependent GPD (red line), POM cycle (purple line), and NOG pathway (blue line). Different color shadings indicate different pathways including EMP pathway (light purple), oxidative pentose phosphate pathway (oxiPPP, blue), POM cycle (orange) and NOG pathway (gray color). (b,c) Lipid titer and dry cell weight (b), and lipid yield of control and engineered Y. lipolytica strains (c) in shake flask experiments. (d) Time-course profiles of lipid accumulation (lipid titer) and cell growth (dry cell weight) of engineered Y. lipolytica strains including ADgapc, ADme and ADpp compared to those of control strain AD in fed-batch fermentations. For columns 1 –7 and 11–14 in b, c, n = 3; for column 8–10 in b and c, n = 2; for d, n = 2. Error bars, mean ± s.d. Statistically significant differences between each engineered Y. lipolytica strain and the baseline strain AD were denoted *P < 0.05, **P < 0.01 (two-tailed Student's t-test). DCW, dry cell weight.

  2. Yield model-directed optimization of strain engineering and the resultant fermentation profiles of engineered Y. lipolytica contains combination of synthetic pathways.
    Figure 2: Yield model-directed optimization of strain engineering and the resultant fermentation profiles of engineered Y. lipolytica contains combination of synthetic pathways.

    (a) Time courses of cell growth and lipid production of ADgy (red line) and ADgm (blue line) in comparison to those of AD (gray line) under the same fermentation conditions. (b) Optimization of the fed-batch fermentation by increasing nitrogen. ADgm-hi, ADgm cultured in a high-density fed-batch fermentation in which the starting ammonium concentration doubles. (c) Yield model-directed optimization of process yield. The green colored line represents the maximum possible yield, while the two red lines define, respectively, the upper and lower bounds of the lipid pathway yield for a particular strain harboring a specific synthetic pathway for NADH recycling. The blue lines represent the upper and lower bounds of the overall process yield for strains harboring the same synthetic pathway. For ac, n = 2. Error bars, mean ± s. d.

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Affiliations

  1. Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

    • Kangjian Qiao,
    • Thomas M Wasylenko,
    • Kang Zhou,
    • Peng Xu &
    • Gregory Stephanopoulos

Contributions

K.Q. and G.S. conceived the project and wrote the manuscript. K.Q., T.M.W., K.Z. and P. X. designed and performed all the experiments. K.Q., T.M.W., K.Z., P.X. and G.S. analyzed the results.

Competing financial interests

The authors have filed a patent (US Provisional Application No.: 62/243,824) on the process yield optimization methods.

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