Engineering polyhydroxyalkanoate content and monomer composition in the oleaginous yeast Yarrowia lipolytica by modifying the ß-oxidation multifunctional protein

Abstract

Recombinant strains of the oleaginous yeast Yarrowia lipolytica expressing the PHA synthase gene (PhaC) from Pseudomonas aeruginosa in the peroxisome were found able to produce polyhydroxyalkanoates (PHA). PHA production yield, but not the monomer composition, was dependent on POX genotype (POX genes encoding acyl-CoA oxidases) (Haddouche et al. FEMS Yeast Res 10:917-927, 2010). In this study of variants of the Y. lipolytica β-oxidation multifunctional enzyme, with deletions or inactivations of the R-3-hydroxyacyl-CoA dehydrogenase domain, we were able to produce hetero-polymers (functional MFE enzyme) or homo-polymers (with no 3-hydroxyacyl-CoA dehydrogenase activity) of PHA consisting principally of 3-hydroxyacid monomers (>80%) of the same length as the external fatty acid used for growth. The redirection of fatty acid flux towards β-oxidation, by deletion of the neutral lipid synthesis pathway (mutant strain Q4 devoid of the acyltransferases encoded by the LRO1, DGA1, DGA2 and ARE1 genes), in combination with variant expressing only the enoyl-CoA hydratase 2 domain, led to a significant increase in PHA levels, to 7.3% of cell dry weight. Finally, the presence of shorter monomers (up to 20% of the monomers) in a mutant strain lacking the peroxisomal 3-hydroxyacyl-CoA dehydrogenase domain provided evidence for the occurrence of partial mitochondrial β-oxidation in Y. lipolytica.

Single cell oil production on molasses by Yarrowia lipolytica strains overexpressing DGA2 in multicopy

“…This work (Beopoulos et al 2012) and JMY1915 (Q4-Δmfe, Leu − Ura − ) (Haddouche et al 2011). Schematic representation of successive strain constructions is depicted in Fig.…”

“…Auxotrophic Leu − strains were transformed with LEU2 fragment when necessary to obtain prototrophic strains. Dotted arrows indicate hidden steps, which are described in detail elsewhere(Haddouche et al (2011),Lazar et al (2013)) …”

Yarrowia lipolytica: Safety assessment of an oleaginous yeast with a great industrial potential

“…However, based on available evidence, the developments in these fields seem to be at an exploratory and/or more premature stage of commercialization. Yarrowia lipolytica itself, or enzyme preparations derived from it, have been investigated for the production of polyhydroxyalkanoates and other (biodegradable) polymers, single-cell oil and specialty lipids (Barrera-Rivera et al, 2012;Haddouche et al, 2011;Papanikolaou & Aggelis, 2011;Sabirova et al, 2011). Yarrowia lipids have also been explored as a cocoa butter substitute (Papanikolaou & Aggelis, 2011).…”

Exploring medium-chain-length polyhydroxyalkanoates production in the engineered yeast Yarrowia lipolytica

“…Heterologous expression of the key enzymes for mcl-PHA synthesis in a proper host provides a main solution for mcl-PHA synthesis by changing the flux of intermediates of fatty acid metabolism [11,13,14,26,28,35]. Heterologous production of mcl-PHA in recombinant Escherichia coli and Saccharomyces cerevisiae were studied extensively [17,25,26,32].…”