We cloned a polyketide synthase gene (pks12) from Fusarium graminearum, a devastating fungal pathogen of cereals. Transformation-mediated gene disruption led to an easily detectable albino phenotype of the disruptants. We used the disruption of the pks12 gene as a visible marker for transformation-mediated homologous recombination and optimized the transformation procedure to achieve a high rate of homologous recombination. In combination with the published genomic sequence data and the generation of expressed sequence tags (ESTs) for F. graminearum, this is a useful tool to investigate this important plant pathogen on a molecular level. Optimized transformation of F. graminearum resulted in at least 93% homologous recombination events when the homologous genomic DNA fragment in the vector had a size of approximately 800bp and was linearized in the middle. Using a genomic sequence of approximately 500bp in the transformation vector, 70% of the transformants still exhibited homologous recombination. On the contrary, no more than 10% homologous recombination events were observed when less than 400bp DNA fragments were used. We co-transformed F. graminearum with two different vectors. One vector harboured a DNA insert homologous to the pks12 gene, while the other vector consisted of the same vector backbone carrying the selection marker specific for F. graminearum. About 70% of the transformants had a disrupted pks12 gene, and all of these showed an integration of the second vector into the pks disruption vector. Therefore, the time-consuming construction of a single transformation vector can be avoided; furthermore, it is now easily feasible to express a gene construct at a defined and mutated genomic site.

In mammals, the final 1 IF-hydroxylation step of the hydrocortisone biosynthesis pathway is performed by a mitochondrial enzyme, namely cytochrome P-450, Ip, together with the electron carriers adrenodoxin and NADPH adrenodoxin oxidoreductase. Successful production of a functional steroid 1 1p-hydroxylase activity was obtained in recombinant yeast in vivo. This conversion was achieved by coexpression of a mitochondrially targeted adrenodoxin and a modified bovine P-450, ,/, whose natural presequence was replaced by a yeast presequence, together with an unexpected yeast endogenous NADPHadrenodoxin-reductase-like activity. Adrenodoxin and P-450, behave as a mitochondrial matrix and membrane protein, respectively. Saccharomyces cerevisiae apparently produces a mitochondrial protein which is capable of transferring electrons to bovine adrenodoxin, which in turn transfers the electrons to P-450, , , + The endogenous adrenodoxin oxidoreductase gains electrons specifically from NADPH. The notion that a yeast microsomal NADPH P-450 oxidoreductase can transfer electrons to mammalian microsomal P-450s can be extended to mitochondria, where an NADPH adrenodoxin oxidoreductase protein transfers electrons to adrenodoxin and renders a mitochondrial mammalian P-450 functional in vivo. The physiological function of this yeast NADPH adrenodoxin oxidoreductase activity is not known.Keywords: cytochrome P-450; mitochondria; yeast; bioconversion ; electron transport.In the adrenal cortex of vertebrates, hydrocortisone is synthesized from cholesterol in a transformation that requires five enzymic steps. Four out of the five reactions are carried out by enzymes belonging to the superfamily of cytochrome P-450 proteins (i.e. encoded by the CYP gene family; for a review, see [I] and [2]). Almost all the P-450 proteins from eukaryotes are anchored to the endoplasmic reticulum membrane facing the cytosol [3]. Only a few members of this family are localized to the inner mitochondrial membrane; among them are the sidechain-cleaving P-450 (P-450,,,) and the 1 1p-hydroxylase (P-450,,,); these enzymes catalyze the first and the last steps, respectively, in hydrocortisone biosynthesis. In a complex reaction sequence, P-450," removes the side chain of cholesterol, generating pregnenolone as the first step of the pathway, whereas P-450,,, hydroxylates I I-deoxycortisol at position C1 I as the ultimate step in hydrocortisone biosynthesis. The latter enzyme can hydroxylate various corticosteroids in positions C18 or C19, it also has an inherent aldosterone synthase activity with 1 %hydro-Correspondence to T. Achstetter, TRANSGENE, 11 rue de MolsFax: +33 88 22 58 07. Abbreviations. P-450, , , $, P-450,,, and P-450,,,,,, cytochromes P-450 110-18 hydroxylase, P-450 cholesterol side chain cleavage and P-450 vitamin D, 25-hydroxylase; ADR, NADPH-adrenodoxin oxidoreductase; matADX, mature form of ADX; CoxVI,,,, cytochrome oxidase subunit VI prepeptide; TEFl, transcription elongation factor 1 ; CYCl isocytochrome C,.(ECl.14.15.4); NADPH-adrenodoxin oxido...

The mammalian electron transfer chain of mitochondrial cytochrome P450 forms involved in steroidogenesis includes very specific proteins, namely adrenodoxin reductase and adrenodoxin. Adrenodoxin reductase transfers electrons from NADPH to adrenodoxin, which subsequently donates them to the cytochrome P450 forms. The Saccharomyces cerevisiae ARH1 gene product (Arh1p) presents homology to mammalian adrenodoxin reductase. We demonstrate the capacity of recombinant Arh1p, made in Escherichia coli, to substitute for its mammalian homologue in ferricyanide, cytochrome c reduction, and, more importantly, in vitro 11␤-hydroxylase assays. Electrons could be transferred from NADPH and NADH as measured in the cytochrome c reduction assay. Apparent K m values were determined to be 0.5, 0.6, and 0.1 M for NADPH, NADH, and bovine adrenodoxin, respectively. These values differ slightly from those of mammalian adrenodoxin reductase, except for NADH, which is a very poor electron donor to the mammalian protein. Subcellular fractionation studies have localized Arh1p to the inner membrane of yeast mitochondria. The biological function of Arh1p remains unknown, and to date, no mitochondrial cytochrome P450 has been identified. ARH1 is, however, essential for yeast viability because an ARH1 gene disruption is lethal not only in aerobic growth conditions but also, surprisingly enough, during fermentation.In mammalian cells, cytochrome P450-containing monooxygenase systems are usually classified in two main categories: the mitochondrial and the microsomal types (1, 2). In both cases, electron transfer chains are involved in which electrons flow from an electron donor, NADPH, to a terminal electron acceptor, cytochrome P450. In the microsomal system, NADPH-cytochrome P450 reductase transfers electrons from NADPH as the reducing equivalent of microsomal cytochrome P450 forms. This protein of about 80 kDa contains FAD 1 and flavin mononucleotide prosthetic groups (3, 4). The electron transport machinery of mitochondrial cytochrome P450 forms is different from that of microsomal cytochrome P450 because it consists of two proteins, NADPH-ferredoxin reductase, a 50-kDa flavoprotein attached to the inner membrane of mitochondria, and a ferredoxin, a 12-14-kDa soluble protein of the mitochondrial matrix. These two proteins contain one prosthetic group per molecule, FAD and an iron-sulfur cluster (2Fe-2S), respectively (2, 5). Electrons flow from NADPH to NADPH-ferredoxin reductase and are transferred to the ferredoxin. The latter donates electrons to the cytochrome P450 that catalyzes the enzymatic reaction.Mitochondrial cytochrome P450-containing monooxygenase systems are primarily involved in the hydroxylation of bile acids and in vitamin D and steroid biosynthesis. In the latter case, NADPH-adrenodoxin reductase (ADR) and adrenodoxin (ADX), corresponding to NADPH-ferredoxin reductase and ferredoxin, respectively, form the electron transfer chain of mitochondrial cytochrome P450 forms. Unlike the microsomal NADPH-cytochrome P450 reductase,...

No abstract

No abstract