A novel multigene expression construct for modification of glycerol metabolism in Yarrowia lipolytica

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.

Citric Acid Production by Yarrowia lipolytica Yeast on Different Renewable Raw Materials

“…Later, the authors of the last article improved the process of CA production up to 52.0 g L −1 with the product yield (Y CA ) of 0.64 g g −1 using strain Y. lipolytica ACA-YC 5033, which was also able to remove harmful phenolic compounds from olive mill wastewaters [29]. The glycerol-grown yeast Y. lipolytica NRRL YB-423 produced 21.6 g L −1 of CA with mass yield of 0.54 g g −1 [12]; strain Y. lipolytica ACA-DC 50109 synthesized 62.5 g L −1 of CA with mass yield of 0.56 g g −1 from raw glycerol [20], while the recombinant strain Y. lipolytica NCYC3825 was able to produce 58 g L −1 of CA [30]. Earlier, we indicated that the other mutant Y. lipolytica NG40/UV7 synthesized, CA (122.2 g L −1 ) with the yield of 0.95 g g −1 .…”

Biotechnological applications of Yarrowia lipolytica: Past, present and future

“…high biomass of 42 g/L and a peak lipid accumulation of 38% of total lipids in the dry cellular weight (DCW) were accumulated (Celińska and Grajek, 2013a).…”

“…2-Phenylethanol, a rose-like aroma compound, has many potential applications.It can be naturally synthesized by different microorganisms. Recently, it was demonstrated that Y. lipolytica is deemed a novel 2-phenylethanol producer Celińska and Grajek (2013a). demonstrated that 2-phenylethanol is produced as a by-product together with citric acid production with glycerol as a carbon source.…”

Yarrowia lipolytica as an Oleaginous Cell Factory Platform for Production of Fatty Acid-Based Biofuel and Bioproducts

“…Recently, lipid accumulation has been found in the Y. lipolytica strains grown on glycerol in the range of 37.1-54.4% of cell dry weight (Sriwongchai et al, 2013). The introduction of heterologous glycerol dehydratase genes and several alcohol oxidoreductases under the control of glycerol inducible promoter, G3P dh, gave rise to a higher growth rate and a lipid yield at13 g/L of culture volume (40% of DCW) (Celinska and Grajek, 2013). This could be enhanced by the codon optimization of the heterologous genes and the prevention of further degradation of accumulated lipid.…”