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Drop-In Biofuel production by using fatty acid photodecarboxylase from Chlorella variabilis in the oleaginous yeast Yarrowia lipolytica

Stefan Bruder, Eva Johanna Moldenhauer, Robert Denis Lemke, Rodrigo Ledesma-Amaro, Johannes Kabisch
doi: https://doi.org/10.1101/468876
Stefan Bruder
1Computer-aided Synthetic Biology, Technische Universität Darmstadt, Darmstadt, Germany
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Eva Johanna Moldenhauer
1Computer-aided Synthetic Biology, Technische Universität Darmstadt, Darmstadt, Germany
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Robert Denis Lemke
1Computer-aided Synthetic Biology, Technische Universität Darmstadt, Darmstadt, Germany
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Rodrigo Ledesma-Amaro
2Department of Bioengineering and Imperial College Centre for Synthetic Biology, Imperial College London, London, United Kingdom
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Johannes Kabisch
1Computer-aided Synthetic Biology, Technische Universität Darmstadt, Darmstadt, Germany
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  • For correspondence: johannes@kabisch-lab.de
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Abstract

Background Oleaginous yeasts are potent hosts for the renewable production of lipids and harbor great potential for derived products, such as biofuels. Several promising processes have been described that produce hydrocarbon drop-in biofuels based on fatty acid decarboxylation and fatty aldehyde decarbonylation. Unfortunately, besides fatty aldehyde toxicity and high reactivity, the most investigated enzyme, aldehyde-deformylating oxygenase, shows unfavorable catalytic properties which hindered high yields in previous metabolic engineering approaches.

Results To demonstrate an alternative alkane production pathway for oleaginous yeasts, we describe the production of diesel-like, odd-chain alkanes and alkenes, by heterologously expressing a recently discovered light-driven oxidase from Chlorella variabilis (CvFAP) in Yarrowia lipolytica. Initial experiments showed that only strains engineered to have an increased pool of free fatty acids showed to be susceptible to sufficient decarboxylation. Providing these strains with glucose and light in a synthetic medium resulted in titers of 10.9 mg/L of hydrocarbons. Using custom 3D printed labware for lighting bioreactors, and an automated pulsed glycerol fed-batch strategy, intracellular titers of 58.7 mg/L were achieved.

Conclusions Oleaginous yeasts such as Yarrowia lipolytica can transform renewable resources such as glycerol into fatty acids and lipids. By heterologously expressing a fatty acid photodecarboxylase from the algae Chlorella variabilis hydrocarbons were produced in several scales from microwell plate to 400 ml bioreactors. The developed bioprocess shows a route to the renewable production of hydrocarbons for a variety of applications ranging from representing a substrate for further enzymatic or chemical modification or as a drop-in biofuel blend.

Short abstract Oleaginous yeasts are potent hosts for the renewable production of lipids, fatty acids and derived products such as biofuels. Here, we describe, the production of odd-numbered alkanes and alkenes with a length of 17 and 15 carbons by expression of a fatty acid photodecarboxylase (CvFAP) from Chlorella variabilis in different Yarrowia lipolytica strains under different regimes of blue light exposure in several scales from microwell plate to 400 ml bioreactors.

Footnotes

  • The manuscript was extended by testing of CvFAP with custom 3D printed labware for LED illumination. Titer were increased to 58.7mg/l. Several bioprocesses were tested.

  • https://gitlab.com/kabischlab.de/led-labware-cvfap/

  • List of abbreviations
    Å
    Ångström
    AA
    amino acid
    AAR (FAR)
    (fatty) acyl-ACP reductase
    ADO
    aldehyde-deformylating oxygenase
    CAR
    carboxylic acid reductase
    Cdw
    cell dry weight
    Cv/r
    Chlorella variabilis/reinhardtii
    DO
    dissolved oxygen
    DOX
    α-dioxygenase
    FFA
    free fatty acids
    GMC
    glucose-methanol-choline
    SCO
    single cell oil
    FAP
    fatty acid photodecarboxylase
    FFA
    free fatty acids
    RT
    room temperature
    YaliTAR
    transformation-associated recombination assisted by Y. lipolytica
    WT
    wild type
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    Posted April 27, 2019.
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    Drop-In Biofuel production by using fatty acid photodecarboxylase from Chlorella variabilis in the oleaginous yeast Yarrowia lipolytica
    Stefan Bruder, Eva Johanna Moldenhauer, Robert Denis Lemke, Rodrigo Ledesma-Amaro, Johannes Kabisch
    bioRxiv 468876; doi: https://doi.org/10.1101/468876
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    Drop-In Biofuel production by using fatty acid photodecarboxylase from Chlorella variabilis in the oleaginous yeast Yarrowia lipolytica
    Stefan Bruder, Eva Johanna Moldenhauer, Robert Denis Lemke, Rodrigo Ledesma-Amaro, Johannes Kabisch
    bioRxiv 468876; doi: https://doi.org/10.1101/468876

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