Evaluation of Yarrowia Lipolytica as a Host for Cytochrome P450 Monooxygenase Expression
Biohydroxylation reactions are catalyzed by various types of hydroxylating enzymes (Ayala and Torres, 2004) which include dioxygenases, lipooxygenases as well as CYP450 monooxygenases. These particular hydroxylation reactions have several advantages over chemical synthesis. Several microorganisms including yeasts have the ability to hydroxylate various substrates. The exploitation of microbial hydroxylations for the production of industrially useful products such as pharmaceuticals is a more recent development (Holland et al., 2000). Yeasts from the genera Schizosaccharomyces, Pichia, Saccharomyces and Yarrowia have all been used to express foreign CYP450 genes (Mukarami et al., 1990; Nthangeni et al., 2004) since they offer an advantage especially when a eukaryotic environment is required for the functional expression of the heterologous gene (Blanquet et al., 2003). A recent evaluation of several yeasts revealed that Y. lipolytica is, a highly attractive alternative host for secretion and expression cloning (Muller et al., 1998; Juretzek et al., 2001). However, a literature search on successful expression of CYP450s in Y. lipolytica yielded only six cases. Three of these were done in our laboratory. In most of the reported cases, the recombinant CYP450 activities were never evaluated in terms of whole cell biotransformations. It was therefore the aim of this study to evaluate Y. lipolytica as a recombinant whole-cell biocatalyst for hydroxylation reactions by using available Y. lipolytica strains overexpressing different CYP450s which were (i) CYP1A1 coding for polyaromatic hydrocarbon hydroxylase (ii) CYP53B1 coding for benzoate para-hydroxylase (iii) CYP52F1 coding for alkane hydroxylase and (iv) CYP557A1 coding for putative fatty acid hydroxylase. Hydroxylase activities of the genetically engineered strains were compared with activities in wild type yeasts expressing the relevant CYP450s. A variety of substrates used for biotransformation reactions included, acetanilide, benzoic acid , phenylnonane, trans-cinnamic acid, 4-nitrophenyl octyl ether and 4-nonyloxybenzoic acid. Experiments using Y. lipolytica overexpressing CYP1A1 illustrated the limitation of using Y. lipolytica for the biotransformation of substrates such as AA since the endogenous enzymes degraded this substrate within only 12 h after substrate addition. In an attempt to distinguish the activities of the putative fatty acid hydroxylase and the alkane hydroxylase overexpressed in Y. lipolytica from the endogenous CYP450s, 4-nitrophenyl octyl ether, 4-nonyloxybenzoic acid and phenylnonane were used as substrates. 4-nitrophenyl octyl ether proved to be expensive and less sensitive to TLC, GC and GC-MS analyses. It has been used in other studies because it yielded p-nitrophenol which can be assayed colourimetrically by measuring absorbance at 420 nm. However, in our experiments, intermediates accumulated that were not completely transformed into p-nitrophenol. Further biotransformation experiments were carried out using 4- nonyloxybenzoic acid as the substrate. Biotransformation experiments were done using Y. lipolytica strains with intact and partially disrupted -oxidation pathway overexpressing CYP52F1 and CYP557A1. Additional experiments were carried out using wild type Y. lipolytica W29, R. minuta and R. retinophila strains. The results demonstrated that, the wild type Y. lipolytica W29 demonstrated the highest specific hydroxylase activity when 4-nonyloxybenzoic acid was used as the substrate. The main limitation here was the inability to selectively induce the overexpressed CYP450 genes alone without the background endogenous CYP450 activity. Due to the limitations above, the next strain used was Y. lipolytica TVN91 (overexpressing benzoate-para hydroxylase from R. minuta). In this case, the host strain lacked the specific CYP450 to perform the same hydroxylation reaction. The substrate used here was BA. Different growth and induction conditions were evaluated to optimize benzoate-para-hydroxylase activities. A comparison of the hydroxylase activities indicated that the activity of the recombinant Y. lipolytica strain overexpressing the CYP53B1 was about 30 times slower than that of the wild type R. minuta from which the gene was cloned. Continuous addition of stearic acid resulted in the best activity with Y. lipolytica TVN91, because the hydroxylase activity was maintained for a longer duration. When PN and CA were used to evaluate substrate transport limitation, the results demonstrated that substrate transport was not limiting and the specific hydroxylase activity was not increased. PN was initially converted to BA before hydroxylation to form pHBA. These results further demonstrated that hydroxylase activity of PN was much faster than that of BA. The results from the bioreactor study demonstrated that an improved aeration and mixing led to an increase in the benzoate para-hydroxylase activity. The possibility of using a chemically defined media (YNB) supplemented with yeast extract and casamino acid for biotransformation was also demonstrated. The results of this study also demonstrated that it is possible to use harvested recombinant cells for biotransformation without significant loss of activity. This makes it possible to study in detail the kinetics of the overexpressed CYP450s. It was, however apparent that the hydroxylase activities were significantly increased by both aeration and cell concentration.