Construction and Characterization of 3rd Generation Saccharomyces cerevisiae Strains for PHB production form Xylose

University essay from Lunds universitet/Teknisk mikrobiologi; Lunds universitet/Bioteknik

Abstract: Poly-3-D-hydroxybutyrate (PHB) is a biopolymer naturally produced by a range of bacterial species. PHB production both from glucose and xylose has also been enabled by engineering the robust baker’s yeast Saccharomyces cerevisiae, however yields remain low. In the present study, additional modifications were carried out in S. cerevisiae strains already expressing functional enzymatic pathways for PHB production from xylose. The first approach was to improve the efficiency of acetyl-CoA conversion to acetoacetyl-CoA in the PHB pathway by introducing enzymes possessing lower Km for acetyl-CoA than the original expressed enzyme from Cupriavidus necator. Two distinct acetyl-CoA acetyltransferases were introduced, Acat1 from Rattus norvegicus and Erg10p from yeast using the CRISPR-Cas9 system; however no improvement in PHB production was obtained. The second approach was to increase the acetaldehyde pool so that the introduced heterologous acetylating acetaldehyde dehydrogenases (EutE) encoded by eutE would enhance the flux from acetaldehyde towards acetyl-CoA in one step using NAD+, which should increase PHB production, when combined with deletion of the main acetaldehyde dehydrogenase (ALD6) gene. Obtained results showed no PHB production in any strains expressing the EutE and therefore it was assumed that EutE was catalysing the reaction in the reverse direction resulting in increased acetaldehyde, which led to further investigations of the enzyme involving activity and overall functionality. Deletion of the eutE gene was also carried out in (EutE carrying) non-PHB producing strains to see if the PHB production would be restored. The eutE was also re-integrated in PHB producing strains. However in both cases no PHB was produced. These results indicated that the EutE gene integration was inactivating PHB production since removal of eutE did not restore PHB production. Therefore, the negative effect of EutE might be at the genetic level on either one or several of the PHB genes phaA, phaB or phaC.

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