We report on the production of hydrocortisone, the major adrenal glucocorticoid of mammals and an important intermediate of steroidal drug synthesis, from a simple carbon source by recombinant Saccharomyces cerevisiae strains. An artificial and fully self-sufficient biosynthetic pathway involving 13 engineered genes was assembled and expressed in a single yeast strain. Endogenous sterol biosynthesis was rerouted to produce compatible sterols to serve as substrates for the heterologous part of the pathway. Biosynthesis involves eight mammalian proteins (mature forms of CYP11A1, adrenodoxin (ADX), and adrenodoxin reductase (ADR); mitochondrial forms of ADX and CYP11B1; 3beta-HSD, CYP17A1, and CYP21A1). Optimization involved modulating the two mitochondrial systems and disrupting of unwanted side reactions associated with ATF2, GCY1, and YPR1 gene products. Hydrocortisone was the major steroid produced. This work demonstrates the feasibility of transfering a complex biosynthetic pathway from higher eukaryotes into microorganisms.

Ferrocene (Fc)-labeled peptides are end-grafted onto gold electrodes via a flexible polyethylene glycol (PEG) linker, and their ability to act as substrates for proteolytic enzymes trypsin and alpha-thrombin is investigated by cyclic voltammetry. It is shown that whereas a short Fc-tetrapeptide substrate is rapidly cleaved by trypsin, a longer Fc-heptapeptide substrate is required for alpha-thrombin detection. However, in both cases it is observed that not all of the Fc-peptide chains present on the electrode surface are cleavable by the proteases and that the cleavage yield is actually controlled by the surface coverage in the Fc-peptide. Surface dilution of the Fc-peptide using a backfilling molecule such as MCH (6-mercapto-1-hexanol) was required to obtain a cleavage yield larger than 80%. The kinetics of Fc-peptide cleavage by trypsin or alpha-thrombin is then shown to be adequately described by Michaelis Menten kinetics, allowing enzymatic constants k(cat) and K(M) to be determined. The obtained rate constant values showed that the affinity of the enzymes for their respective Fc-peptide substrates is very high (i.e., low K(M) values) whereas that for the cleavage step itself is relatively low (low k(cat) values). Partial compensation of these parameters yields a fast response of the Fc-peptide electrodes to the proteases in solution in the 1-1000 nM range. The type of molecule used to backfill the Fc-peptide layers, either MCH or PEG(6) chains, is shown to modulate the activity of the proteases versus the Fc-peptide layers: in particular, the PEG(6) diluent is specifically shown to decrease the ability of alpha-thrombin to cleave its Fc-peptide substrate whereas trypsin activity is unaffected by the presence of PEG chains.

While studying the effect of steroids on the growth of the yeast Saccharomyces cerevisiae, we found that pregnenolone was converted into the acetate ester. This reaction was identified as a transfer of the acetyl group from acetyl-CoA to the 3b-hydroxyl group of pregnenolone. The corresponding enzyme, acetyl-CoA:pregnenolone acetyltransferase (APAT) is specific for D 5 -or D 4 -3b-hydroxysteroids and short-chain acyl-CoAs. The apparent K m for pregnenolone is <0.5 mm. The protein associated with APAT activity was partially purified and finally isolated from an SDS/polyacrylamide gel. Tryptic peptides were generated and N-terminally sequenced. Two peptide sequences allowed the identification of an open reading frame (YGR177c, in the S. cerevisiae genome database) translating into a 62-kDa protein of hitherto unknown function. This protein encoded by a gene known as ATF2 displays 37% identity with an alcohol acetyltransferase encoded by the yeast gene ATF1. Disruption of ATF2 led to the complete elimination of APAT activity and consequently abolished the esterification of pregnenolone. In addition, a toxic effect of pregnenolone linked to the disruption of ATF2 was observed. Pregnenolone toxicity is more pronounced when the atf 2-D mutation is introduced in a yeast strain devoid of the ATP-binding cassette transporters, PDR5 and SNQ2. Our results suggest that Atf 2p (APAT) plays an active role in the detoxification of 3b-hydroxysteroids in association with the efflux pumps Pdr5p and Snq2p.

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 production of recombinant human basic fibroblast growth factor (rhbFGF) in Escherichia coli cells yielded active forms of this polypeptide which, however, displayed a high degree of instability towards oxidative processes. Biochemical studies in our laboratory and those of others indicated that the reactivity of the four cysteine residues was the main cause of the observed instability. Several attempts to obtain more stable derivatives of rhbFGF were carried out by modification of the sulfhydryl groups. Among these, treatment of rhbFGF with iodoacetic acid led to the isolation of a partially carboxymethylated form (Cm-FGF). Peptide mapping analysis of the modified protein showed that two cysteines (78 and 96) were blocked by a carboxymethyl group. The remaining cysteines (34 and 101) were not modified under the conditions used and were found to be in the reduced form. Cm-FGF and unmodified rhbFGF showed similar affinity both for heparin and for high-affinity receptors. Cm-FGF was more stable than the unmodified molecule as measured by HPLC and SDSjPAGE analysis. Interestingly, Cm-FGF was more active than unmodified rhbFGF in stimulating proliferation of endothelial cells and DNA synthesis in 3T3 fibroblasts. This new derivative could represent a desirable complementation to rhbFGF for the development of more stable pharmaceutical formulations in wound healing applications.

Natural hirudin variant 2 with a lysine residue in position 47 (rHV2-Lys47) was produced in a genetically engineered strain of Saccharomyces cerevisiae as a secreted protein of 65 amino acids and purified to greater than 99% homogeneity. Only reversed-phase high-performance liquid chromatography (RP-HPLC) using very shallow acetonitrile gradients indicated the presence of a component in the final product (approximately 1% of total protein) with a slightly increased retention time. Using successive RP-HPLC purification steps, this hydrophobic impurity was isolated and separated into two constituents defined as components A1 and A2 which differed from the parent molecule by mass reductions of 17.2 Da (A1) and 17.6 Da (A2), respectively, as determined by electrospray mass spectrometry (ESMS). Proteolytic digestion with endoprotease Glu-C from Staphylococcus aureus (V8 protease) and analysis of the peptide mixture by ESMS showed that the mass difference between rHV2-Lys47 and component A1 was due to a modification between amino acids 1 and 43, while the corresponding mass difference with component A2 was the result of a modification within the peptide fragment comprising residues 50-61. Further analyses using amino acid sequencing and ESMS in combination with collision-activated dissociation (CAD) detected modifications at residues Asn33-Gly34 in component A1 and at Asn53-Gly54 in component A2. Both of these sites were previously shown to be susceptible to spontaneous deamidation under slightly basic pH conditions. Thus, the mass reductions of approximately 17 Da and the fact that both asparagines, Asn33 in component A1 and Asn53 in component A2, proved to be resistant to Edman degradation provided strong support for them being stable succinimide intermediates of the corresponding deamidation reactions. Both intermediates were shown to have inhibition constants for human alpha-thrombin on the order of 1 pM, identical to that of rHV2-Lys47. The isoelectric point of component A2 was determined to be within 0.01 pH unit of that of the parent molecule by isoelectric focusing in an immobilized pH gradient.

Based on the observation that the growth of solid tumors is dependent on the formation of new blood vessels, therapeutic strategies aimed at inhibiting angiogenesis have been proposed. A number of proteins with angiostatic activity have been described, but their development as therapeutic agents has been hampered by difficulties in their production and their poor pharmacokinetics. These limitations may be resolved using a gene therapy approach whereby the genes are delivered and expressed in vivo. Here we compared adenoviral delivery of endostatin, proliferin-related protein (PRP), and interferon-inducible protein 10 (IP10) genes. Recombinant adenoviruses carrying the three angiostatic genes express biologically active gene products as determined in vitro in endothelial cell proliferation and migration assays, and in vivo by inhibition of neoangiogenesis in rat chambers. Eradication of established tumors in vivo, in the murine B16F10 melanoma model in immunocompetent mice, was not achieved by intratumoral injection of the different vectors. However, the combination of intravenous plus intratumoral injections allowed rejection of tumors. Ad-PRP or Ad-IP10 were significantly more efficient than Ad-endostatin, leading to complete tumor rejection and prolonged survival in a high proportion of treated animals. These data support the use of in vivo gene delivery approaches to produce high-circulating and local levels of antiangiogenic agents for the therapy of local and metastatic human tumors.

Prourokinase is a plasminogen activator of 431 amino acids which displays a clot-lysis activity through a fibrin-dependent mechanism, and which seems to be a promising agent for the treatment of acute myocardial infarction.The preparation of recombinant prourokinase in bacteria has been hampered by its insolubility and by difficulty in refolding the polypeptide chain.In this paper we describe the renaturation process of two recombinant proteins expressed in Escherichiu coli as inclusion bodies: prourokinase and a deletion derivative (Al25-prourokinase) in which 125 amino acids of the N-terminal region have been removed. Deletion of this sequence brings to higher refolding yields and faster kinetics (first-order rate constant of renaturation of 0.57 h-l for A 125-prourokinase and 0.25 h-l for prourokinase).Our process involves sequential steps of denaturation, reduction and controlled refolding of the polypeptide chain. When applied to pure, non-glycosylated and active prourokinase, it gives a refolding yield of about 80%, demonstrating the efficiency of the renaturation procedure. Lower yields (15% and 30%, respectively, for prourokinase and Al25-prourokinase) were obtained when the same refolding protocol was applied to inclusion bodies from bacteria.After purification to homogeneity (as shown by HPLC and SDS/PAGE) specific activities were 160 000 and 250000 IU/mg protein, respectively, for prourokinase and Al25-prourokinase.As with prourokinase, the deletion mutant A 125-prourokinase displays a zymogenic nature, being activated by plasmin to the active two-chain form; however, this mutant is approximately fourfold more resistant than prourokinase to plasmin activation, and consequently shows a different fibrinolytic profile.Prourokinase (proUK) is the single-chain precursor of urokinase-type plasminogen activator, a serine protease that plays a key role in the fibrinolytic system by converting plasminogen to plasmin [l]. The proUK molecule consists of 411 amino acids and 12 disulfide bonds and can be subdivided into three domains homologous with known serine protease structures, namely epidermal growth factor (EGF)-like, kringle and serine-protease domains [2]. ProUK is specifically converted to two-chain urokinase after limited proteolysis by plasmin at the Lys158-Ile159 bond; an additional site located at Lys135-Lys136 can also be cleaved by plasmin to generate a low-molecular-mass urokinase which displays the same activity as the high-molecular-mass form [3].ProUK was found to be an effective thrombolytic agent for the treatment of acute myocardial infarction; however, due to its short half-life in circulation, high therapeutic doses Currespondence to G.