Metabolic Engineering of Ethanol Production in Thermoanaerobacter mathranii BG1

Shuo Yao

    Research output: Book/ReportPh.D. thesis

    339 Downloads (Pure)

    Abstract

    Strain BG1 is a xylanolytic, thermophilic, anaerobic, Gram-positive bacterium originally isolated from an Icelandic hot spring. The strain belongs to the species Thermoanaerobacter mathranii. The strain ferments glucose, xylose, arabinose, galactose and mannose simultaneously and produces ethanol, acetate, lactate, CO2, and H2 as fermentation end-products. As a potential ethanol producer from lignocellulosic biomass, tailor-made BG1 strain with the metabolism redirected to produce ethanol is needed. Metabolic engineering of T. mathranii BG1 is therefore necessary to improve ethanol production. Strain BG1 contains four alcohol dehydrogenase (ADH) encoding genes. They are adhA, adhB, bdhA and adhE encoding primary alcohol dehydrogenase, secondary alcohol dehydrogenase, butanol dehydrogenase and bifunctional alcohol/acetaldehyde dehydrogenase, respectively. The presence in an organism of multiple alcohol dehydrogenases with overlapping specificities makes the determination of the specific role of each ADH difficult. Deletion of each individual adh gene in the strain revealed that the adhE deficient mutant strain fails to produce ethanol as the fermentation product. The bifunctional alcohol/acetaldehyde dehydrogenase, AdhE, is therefore proposed responsible for ethanol production in T. mathranii BG1, by catalyzing sequential NADHdependent reductions of acetyl-CoA to acetaldehyde and then to ethanol under fermentative conditions. Moreover, AdhE was conditionally expressed from a xylose-induced promoter in a recombinant strain (BG1E1) with a concomitant deletion of a lactate dehydrogenase. Over-expression of AdhE in strain BG1E1 with xylose as a substrate facilitates the production of ethanol at an increased yield. With a cofactor-dependent ethanol production pathway in T. mathranii BG1, it may become crucial to regenerate cofactor to increase the ethanol yield. Feeding the cells with a more reduced carbon source, such as mannitol, was shown to increase ethanol yield beyond that obtained with glucose and xylose. The ldh gene coding for lactate dehydrogenase was deleted from strain BG1 to eliminate an NADH oxidation pathway (BG1L1). To further facilitate NADH regeneration used for ethanol formation, a heterologous gene gldA encoding an NAD+ dependent glycerol dehydrogenase (GLDH) was expressed in T. mathranii with or without concomitant deletion of a lactate dehydrogenase. With a functional lactate formation pathway, expression of GLDH in a recombinant T. mathranii strain (BG1G2) leads to a significantly decreased ethanol yield accompanied by an increased lactate formation, which was shown to be the preferred route for the regeneration of NAD+. However, with an inactivated lactate formation pathway, expression of GLDH in another recombinant T. mathranii strain (BG1G1) leads to an increased carbon flux channelled towards the production of ethanol over acetate, hence restoring the redox balance. Finally, it was shown that strain BG1G1 acquired the capability to utilize glycerol as an extra carbon source in the presence of xylose, and utilization of the more reuduced substrate glycerol resulted in a higher ethanol yield.
    Original languageEnglish
    Place of PublicationRoskilde
    PublisherDanmarks Tekniske Universitet, Risø Nationallaboratoriet for Bæredygtig Energi
    Number of pages105
    ISBN (Print)978-87-550-3858-5
    Publication statusPublished - Apr 2009
    SeriesRisø-PhD
    Number68(EN)

    Keywords

    • Bio energy
    • Microbial energy technology
    • Risø-PhD-68(EN)
    • Risø-PhD-68
    • Risø-PhD-0068

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