Non-conventional yeasts as producers of polyhydroxyalkanoates?genetic engineering of Arxula adeninivorans

Abstract

The non-conventional yeast Arxula adeninivorans was equipped with the genes phbA, phbB and phbC of the polyhydroxyalkanoate (PHA) biosynthetic pathway of Ralstonia eutropha, which encode beta-ketothiolase, NADPH-linked acetoacetyl-CoA reductase and PHA synthase, respectively. Arxula strains transformed solely with the PHA synthase gene (phbC) were able to produce PHA. However, the maximum content of the polymer detected in these strains was just 0.003% poly-3-hydroxybutyrate (PHB) and 0.112% poly-3-hydroxyvalerate (PHV). The expression of all three genes (phbA, phbB, phbC) resulted in small increases in the PHA content of the transgenic Arxula cells. However, under controlled cultivation conditions with minimal medium and ethanol as the carbon source, the recombinant yeast was able to accumulate up to 2.2% PHV and 0.019% PHB. Possible reasons for these differences are discussed.

Biosynthesis of biodegradable polyhydroxyalkanotes biopolymers in genetically modified yeasts

“…It is known that the Nile-red stain emitted strongly positive red fluorescence signals only with hydrophobic compounds like PHAs and lipids. Nile-red intended to show any lipid particles inside the cells and it did not react with any tissue constituent except by solution and could be detected by fluorescence spectroscopy or flow cytometery (Terentiev, et al, 2004). The results of the Nile-Red staining assay were compatible with both restriction mapping and the PCR detection assays of PHB genes.…”

“…Previously, Leaf et al, (1996), Breuer, et al, (2002 and Terentiev et al, (2004) reported that no increase in PHA levels produced by expressing the entire threegene PHA pathway compared with those found in yeast transformants where only the phbC gene was expressed. They suggested that competition for the precursors from pathways may lead to the synthesis of other storage compounds.…”

“…Therefore, engineering of novel pathways in eukaryotic cell systems seems to be a beneficial alternative to the production of PHAs in bacteria. Yeast cells can be used as models to gain information on PHAs synthesis in eukaryotes (Leaf, et al, 1996, Carlson, et al, 2002, Breuer, et al, 2002and Terentiev, et al, 2004. Seebach, et al, (1994) describes evidence of PHB existing in yeast and many other eukaryotic cells, which contain small amounts of low molecular mass PHBs.…”

“…First, yeasts have been studied intensively from physiology, molecular biology and biotechnology points of views. Second, yeasts are physiologically flexible and they are larger than bacteria (Carlson, et al 2002 andTerentiev, et al, 2004). Moreover, yeast like, Sac charo myces cer evisi ae, Kl yver omyc es marxianus, Candida utilis and others, has been approved as a GRAS microorganism by Food and Drug Administration (Boze, et al, 1994).…”

Characterization and expression analysis of a gene cluster for nitrate assimilation from the yeast Arxula adeninivorans

“…An expression platform based on A. adeninivorans has been developed in recent years, and has been used to synthesize recombinant proteins such as human serum albumin (HSA), lipase, invertase, xylitol dehydrogenase, transaldolase, interleukin-6, α-amylase, acid phosphatase and phytase, and for production of polyhydroxyalkanoates (Wartmann et al, 2002(Wartmann et al, , 2003a(Wartmann et al, , 2003bBöer et al, 2004bBöer et al, , 2005aBöer et al, , 2005bBöer et al, , 2005cBöer et al, , 2007Terentiev et al, 2004;Steinborn et al, 2005Steinborn et al, , 2007El Fiki et al, 2007;Kaur et al, 2007). The system involves the use of vectors with multiple cloning sites for the integration of selection markers, expression cassettes bearing strong constitutive or inducible promoters, autonomously replicating sequences and chaperone cassettes, which are inserted between rDNA-targeting sequences (Steinborn et al, 2007).…”