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Fatty aldehyde dehydrogenase multigene family involved in the assimilation of n-alkanes in Yarrowia lipolytica.

ABSTRACT: In the n-alkane assimilating yeast Yarrowia lipolytica, n-alkanes are oxidized to fatty acids via fatty alcohols and fatty aldehydes, after which they are utilized as carbon sources. Here, we show that four genes (HFD1-HFD4) encoding fatty aldehyde dehydrogenases (FALDHs) are involved in the metabolism of n-alkanes in Y. lipolytica. A mutant, in which all of four HFD genes are deleted (?hfd1-4 strain), could not grow on n-alkanes of 12-18 carbons; however, the expression of one of those HFD genes restored its growth on n-alkanes. Production of Hfd2Ap or Hfd2Bp, translation products of transcript variants generated from HFD2 by the absence or presence of splicing, also supported the growth of the ?hfd1-4 strain on n-alkanes. The FALDH activity in the extract of the wild-type strain was increased when cells were incubated in the presence of n-decane, whereas this elevation in FALDH activity by n-decane was not observed in ?hfd1-4 strain extract. Substantial FALDH activities were detected in the extracts of Escherichia coli cells expressing the HFD genes. Fluorescent microscopic observation suggests that Hfd3p and Hfd2Bp are localized predominantly in the peroxisome, whereas Hfd1p and Hfd2Ap are localized in both the endoplasmic reticulum and the peroxisome. These results suggest that the HFD multigene family is responsible for the oxidation of fatty aldehydes to fatty acids in the metabolism of n-alkanes, and raise the possibility that Hfd proteins have diversified by gene multiplication and RNA splicing to efficiently assimilate or detoxify fatty aldehydes in Y. lipolytica.

SUBMITTER: Iwama R

PROVIDER: S-EPMC4246085 | biostudies-literature | 2014 Nov

REPOSITORIES: biostudies-literature

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Fatty aldehyde dehydrogenase multigene family involved in the assimilation of n-alkanes in Yarrowia lipolytica.

Iwama Ryo R Kobayashi Satoshi S Ohta Akinori A Horiuchi Hiroyuki H Fukuda Ryouichi R

The Journal of biological chemistry 20141014 48

In the n-alkane assimilating yeast Yarrowia lipolytica, n-alkanes are oxidized to fatty acids via fatty alcohols and fatty aldehydes, after which they are utilized as carbon sources. Here, we show that four genes (HFD1-HFD4) encoding fatty aldehyde dehydrogenases (FALDHs) are involved in the metabolism of n-alkanes in Y. lipolytica. A mutant, in which all of four HFD genes are deleted (Δhfd1-4 strain), could not grow on n-alkanes of 12-18 carbons; however, the expression of one of those HFD gene ...[more]

PMID: 25315778

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Project description: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.

Project description:The oleaginous yeast Yarrowia lipolytica has attracted much attention due to its ability to utilize a wide range of substrates to accumulate high lipid content and its flexibility for genetic manipulation. In this study, intracellular lipid metabolism in Y. lipolytica was tailored to produce fatty acid, a renewable oleochemical and precursor for production of advanced biofuels. Two main strategies, including blocking activation and peroxisomal uptake of fatty acids and elimination of biosynthesis of lipids, were employed to reduce fatty acid consumption by the native pathways in Y. lipolytica. Both genetic modifications improved fatty acid production. However, disruption of the genes responsible for assembly of nonpolar lipid molecules including triacylglycerols (TAGs) and steryl esters resulted in the deleterious effects on the cell growth. The gene tesA encoding thioesterase from Escherichia coli was expressed in the strain with disrupted faa genes encoding fatty acyl-CoA synthetases and pxa1 encoding peroxisomal acyl-CoA transporter, and the titer of fatty acids resulted in 2.3 g/L in shake flask culture, representing 11-fold improvement compared with the parent strain. Expressing the native genes encoding acetyl-CoA carboxylase (ACC) and hexokinase also increased fatty acid production, although the improvement was not as significant as that with tesA expression. Saturated fatty acids including palmitic acid (C16:0) and stearic acid (C18:0) increased remarkably in the fatty acid composition of the recombinant bearing tesA compared with the parent strain. The recombinant expressing tesA gene resulted in high lipid content, indicating the great fatty acid producing potential of Y. lipolytica. The results highlight the achievement of fatty acid overproduction without adverse effect on growth of the strain. Results of this study provided insight into the relationship between fatty acid and lipid metabolism in Y. lipolytica, confirming the avenue to reprogram lipid metabolism of this host for overproduction of renewable fatty acids.

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Fatty aldehyde dehydrogenase multigene family involved in the assimilation of n-alkanes in Yarrowia lipolytica.

Publications

Fatty aldehyde dehydrogenase multigene family involved in the assimilation of n-alkanes in Yarrowia lipolytica.

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