Biotransformation of alkanes, alkylbenzenes and their derivatives by genetically engineered Yarrowia lipolytica strains
Van Rooyen, Newlandé
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A variety of microorganisms, including yeasts, are capable of utilizing n- alkanes as carbon source (Schmitz et al., 2000; Watkinson & Morgan, 1990). The over expression of P450 genes such as the CYP52 family coding for the alkane hydroxylases may lead to an increase in activity and increased formation of possible useful products from hydrocarbon metabolism (Iida et al., 2000). Disruption of the -oxidation pathway by deleting the genes coding for acyl CoA-oxidases, also leads to the accumulation of products that would normally be broken down (Picataggio et al., 1991). The genetic engineering of these two points of control opens up many possibilities for the accumulation of different products from hydrocarbons. Although some work was done concerning these systems in Candida tropicalis very little work has been done in Yarrowia lipolytica. It was the aim of the project to investigate the biotransformation of alkanes, alkylbenzenes and their derivatives by different groups of genetically engineered Y. lipolytica strains in order to investigate a number of questions. The possible accumulation of monocarboxylic acids in Yarrowia lipolytica was inestigated by using substrates such as undecene and hexylbenzene. Y. lipolytica MTLY37 a -oxidation disrupted strain with POX2, POX3, POX4 and POX5 genes deleted could not accumulate any monocarboxylic acid from undecene. The undecene was however fully utilized indicating that this strain still had some -oxidation activity. Little phenylacetic acid was formed (0.4 mM) from hexylbenzene. Another product that could not be positively identified at the time, but which might have been phenylhexanoic acid accumulated (4mM). No monocarboxylic acids other than phenylacetic acid could also be accumulated from alkylbenzenes in strains with blocked - oxidation expressing CPR and CYP genes, leading to the conclusion that Y. lipolytica can not accumulate monocarboxylic acids. Y. lipolytica strains with disrupted -oxidation as well as a strain with functional -oxidation expressing additional YlCPR and CYP52F1 genes accumulated the full-length dioic acid from 5-methylundecane. All these strains also sequentially broke down the 5-methylundecanedioic acid to 5- methylnonanedioic acid, 3-methylheptanedioic acid and 3-methylpentanedioic acid. Y. lipolytica MTLY76 was the only strain that did not degrade the 5- methylundecanedioic acid completely. Using hexylbenzene as substrate it was possible to establish that ethanol delayed the induction of both the native ALK genes as well as the inserted CYP genes. However, the cloned genes were later induced quite strongly (probably by the phenylalkanoic acids formed from hexylbenzene) for an extended period, while the native genes were only weekly induced. The maximum activity of Y. lipolytica was slightly lower when ethanol was used as inducer (13µmol.min -1 l -1 ) than when oleic acid was used as inducer (19µmol.min -1 l -1 ). The alkane hydroxylase activity was however maintained for a longer time when ethanol was used as inducer. When dodecane was used as inducer native genes were strongly induced for a relatively long period, but not as long as the cloned genes after ethanol. Alkylbenzenes as substrate was also useful to distinguish between alkane hydroxylase activity of native and cloned monooxygenases. A significant difference in the activity of Y. lipolytica TVN356 expressing CPR together with CYP557A1 (putative fatty acid hydroxylase from Rhodotorula retinophila) and Y. lipolytica TVN91 expressing CPR together with CYP53 (benzoate para- hydroxylase from R. minuta) could be observed (14µmol.min -1 l -1 and 8µmol.min -1 l -1 respectively) when decylbenzene was used as substrate. To better study the hydroxylase activity of inserted P450s, it may be better to use the ICL1 promoter to drive the expression of the inserted CYP genes and use ethanol as inducer.