Comparison of two cytochromes P-450 from Candida maltosa: primary structures, substrate specificities and effects of their expression in Saccharomyces cerevisiae on the proliferation of the endoplasmic reticulum

cDNAs were cloned, sequenced and expressed which encode two different cytochrome P-450 forms of the alkane-assimilating yeast Candida maltosa, designated as P-450Cm1 and P-450Cm2. The amino acid sequences deduced were about 55% identical. Expression in Saccharomyces cerevisiae resulted in the formation of intact microsomal P-450 systems catalyzing the hydroxylation of n-hexadecane and lauric acid with significantly different substrate preferences. A massive proliferation of the endoplasmic reticulum was observed in the S. cerevisiae cells which produced P-450. Depending on the P-450 form expressed, distinctly organized stacks of paired membranes appeared and occupied considerable areas of the cytoplasm. As shown by immunoelectron microscopy for P-450Cm1, the protein expressed was highly concentrated within these newly formed membrane structures.     

Citric acid production from sucrose using a recombinant strain of the yeast Yarrowia lipolytica

The yeast Yarrowia lipolytica is able to secrete high amounts of several organic acids under conditions of growth limitation and carbon source excess. Here we report the production of citric acid (CA) in a fed-batch cultivation process on sucrose using the recombinant Y. lipolytica strain H222-S4(p67ICL1) T5, harbouring the invertase encoding ScSUC2 gene of Saccharomyces cerevisiae under the inducible XPR2 promoter control and multiple ICL1 copies (10–15). The pH-dependent expression of invertase was low at pH 5.0 and was identified as limiting factor of the CA-production bioprocess. The invertase expression was sufficiently enhanced at pH 6.0–6.8 and resulted in production of 127–140 g l⁻¹ CA with a yield Y _CA of 0.75–0.82 g g⁻¹, whereas at pH 5.0, 87 g l ⁻¹ with a yield Y _CA of 0.51 gg⁻¹ were produced. The CA-productivity Q _CA increased from 0.40 g l ⁻¹ h⁻¹ at pH 5.0 up to 0.73 g l ⁻¹ h⁻¹ at pH 6.8. Accumulation of glucose and fructose at high invertase expression level at pH 6.8 indicated a limitation of CA production by sugar uptake. The strain H222-S4(p67ICL1) T5 also exhibited a gene–dose-dependent high isocitrate lyase expression resulting in strong reduction (<5%) of isocitric acid, a by-product during CA production.     

Relaxed predation results in reduced phenotypic integration in a suite of dragonflies

Although changes in magnitude of single traits responding to selective agents have been studied intensively, little is known about selection shaping networks of traits and their patterns of covariation. However, this is central for our understanding of phenotypic evolution as traits are embedded in a multivariate environment with selection affecting a multitude of traits simultaneously rather than individually. Here, we investigate inter‐ and intraspecific patterns of trait integration (trait correlations) in the larval abdomen of dragonflies as a response to a change in predator selection. Species of the dragonfly genus Leucorrhinia underwent a larval habitat shift from predatory fish to predatory dragonfly‐dominated lakes with an associated relaxation in selection pressure from fish predation. Our results indicate that the habitat‐shift‐induced relaxed selection pressure caused phenotypic integration of abdominal traits to be reduced. Intraspecific findings matched patterns comparing species from both habitats with higher abdominal integration in response to predatory fish. This higher integration is probably a result of faster burst swimming speed. The abdomen holds the necessary morphological machinery to successfully evade predatory fish via burst swimming. Hence, abdominal traits have to function in a tight coordinated manner, as maladaptive variation and consequently nonoptimal burst swimming would cause increased mortality. In predatory dragonfly‐dominated lakes, no such strong link between burst swimming and mortality is present. Our findings highlight the importance of studying multivariate trait relationships as a response to selection for understanding patterns of phenotypic diversification.     

Abhandlungen

Ohne Zusammenfassung     

Function and regulation of cytochrome P-450 in alkane-assimilating yeast

Transition of n-hexadecane utilizing cultures of Candida maltosa to oxygen-limited growth caused an up to 6-fold increase of the cellular cytochrome P-450 content. Enhanced cytochrome P-450 formation required protein de novo synthesis and was not due to a change of the apo/holo-enzyme ratio as demonstrated by cycloheximide inhibition and immunological quantitation. The effect of low oxygen concentration (pO₂=3–5%) was simulated by selective inhibition of alkane hydroxylation with carbon monoxide (at a pO₂ of 70–75%). Enhanced cytochrome P-450 formation occurred even when a constant growth rate was maintained through utilization of a second non-repressive growth substrate. However, the presence of n-alkanes was an essential precondition. It was concluded, that the cytochrome P-450 formation was mainly regulated by the intracellular inducer concentration which depends on the relative rates of alkane transport into the cell and the actual alkane hydroxylating activity of the enzyme system.Abbreviation cyt cytochrome     

Influence of the growth substrate and the oxygen concentration in the medium on the cytochrome P-450 content in Candida guilliermondii

Summary The induction of cytochrome P-450 in Candida guilliermondii by different growth substrates was investigated. Hexadecane is a strong inducer, whereas its oxidation products induce only weakly. It could be shown that the oxygen concentration in the medium influences the cytochrome P-450 content in cells growing on hexadecane. At oxygen concentrations between 100% and 15% of saturation the cytochrome P-450 content reaches 25–35 nmol/g dry weight. Reducing the oxygen to lower concentrations increase the cytochrome P-450 content up to approximately 100 nmol/g dry weight.     

Effect of steroid biosynthesis modifiers on progesterone biotransformation by recombinant yeasts expressing cytochrome P450cl7

Using recombinant microorganisms S. cerevisiae GRF18/YEp 5117α, expressing bovine adrenocortical cytochrome P450cl7, we have studied the effect of various modifiers of steroid biosynthesis on the relationship between reactions of the 17α-hydroxylation and 20α-reduction of progesterone. Dexamethasone and metyrapone had no effect on the reaction of progesterone 17α-hydroxylation and 20α-reduction of 17α-hydroxyprogesterone. Mifepriston and danazol did not covalently modify amino acid residues of the cytochrome P450cl7 or its heme group under the conditions of progesterone biotransformation by recombinant yeasts. Ketokonazole, mifepriston and danazol were found to be low-affinity competitive inhibitors, but the 20-dihydroderivatives of progesterone were mixed type inhibitors of the cytochrome P450cl7. All modifiers used did not affect the functional properties of the yeast analog of 20α-hydroxysteroid dehydrogenase. Based on the effect on catalytic parameters of the cytochrome P450cl7, the all modifiers used can be arranged in the following order: 20β-dihydroprogesterone (maximal effect) > mifepriston = ketokonazole > 20α-dihydroprogesterone > danazol > dexamethasone, metyrapone (without effect).     

Aconitase overexpression changes the product ratio of citric acid production by <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis>

The yeast Yarrowia lipolytica secretes high amounts of various organic acids, like citric acid (CA) and isocitric acid (ICA) under an excess of carbon source and several conditions of growth limitation. Depending on the carbon source used, Y. lipolytica strains produce a mixture of CA and ICA in a characteristic ratio. To examine whether this CA/ICA product ratio can be influenced by gene–dose-dependent overexpression of aconitase (ACO)-encoding gene ACO1, a recombinant Y. lipolytica strain was constructed containing multiple copies of ACO1. The high-level expression of ACO in the ACO1 multicopy integrative transformant resulted in a shift of the CA/ICA product pattern into the direction of ICA. On sunflower oil, a striking increase of the ICA proportion from 35–49% to 66–71% was observed compared to wild-type strains without influencing the total amount of acids (CA and ICA) produced. On glycerol, glucose or sucrose, the ICA proportion increased only moderately from 10–12% to 13–17%. This moderate shift into the direction of ICA was also observed in an icl1-defective strain.