Abstract
BackgroundHigh supply of raw, residual glycerol from biodiesel production plants promote the search for novel biotechnological methods of its utilization. In this study we attempted modification of glycerol catabolism in a nonconventional yeast species Yarrowia lipolytica through genetic engineering approach.ResultsTo address this, we developed a novel genetic construct which allows transferring three heterologous genes, encoding glycerol dehydratase, its reactivator and a wide-spectrum alcohol oxidoreductase under the control of glycerol-induced promoter. The three genes, tandemly arrayed in an expression cassette with a marker gene ura3, regulatory and targeting sequences (G3P dh promoter and XPR-like terminator, 28S rDNA as a target locus), were transferred into Yarrowia lipolytica cells. The obtained recombinant strain NCYC3825 was characterized at the molecular level and with respect to its biotechnological potential. Our experiments indicated that the novel recombinant strain stably borne one copy of the expression cassette and efficiently expressed heterologous alcohol oxidoreductase, while glycerol dehydratase and its reactivator were expressed at lower level. Comparative shake flask cultivations in glucose- and glycerol-based media demonstrated higher biomass production by the recombinant strain when glycerol was the main carbon source. During bioreactor (5 L) fed-batch cultivation in glycerol-based medium, the recombinant strain was characterized by relatively high biomass and lipids accumulation (up to 42 gDCW L-1, and a peak value of 38%LIPIDS of DCW, respectively), and production of high titers of citric acid (59 g L-1) and 2-phenylethanol (up to 1 g L-1 in shake flask cultivation), which are industrially attractive bioproducts.ConclusionsDue to heterogeneous nature of the observed alterations, we postulate that the main driving force of the modified phenotype was faster growth in glycerol-based media, triggered by modifications in the red-ox balance brought by the wide spectrum oxidoreductase. Our results demonstrate the potential multidirectional use of a novel Yarrowia lipolytica strain as a microbial cell factory.
Highlights
High supply of raw, residual glycerol from biodiesel production plants promote the search for novel biotechnological methods of its utilization
The pYLG1 vector presented in this work bears three heterologous genes, natively involved in glycerol metabolism
Our preliminary experiments indicated that Y. lipolytica is unable to produce vitamin B12, and no activities involved in its production and transportation have been identified, to date (KEGG database)
Summary
Residual glycerol from biodiesel production plants promote the search for novel biotechnological methods of its utilization. Yarrowia lipolytica is a dimorphic, nonconventional yeast species with unique metabolic properties, known for its efficient growth on raw glycerol from biodiesel production plants. Interest in this species stems from its metabolic potential expressed in exceptional ability to utilize and accumulate hydrophobic substances [12,13,14] as well as to produce high amounts of valuable metabolites, such as: citric and isocitric acid [15,16], succinic acid [17], erythritol [18], γ-decalactone [19] and biosurfactants [20]. This fact, together with its exceptional performance in utilization of different raw biomaterials and their bioconversion into high-valueadded bioproducts stimulates its frequent application in industrial processes [27]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
