Trichodiene Production in a Trichoderma harzianum erg1-Silenced Strain Provides Evidence of the Importance of the Sterol Biosynthetic Pathway in Inducing Plant Defense-Related Gene Expression.

Trichoderma species are often used as biocontrol agents against plant-pathogenic fungi. A complex molecular interaction occurs among the biocontrol agent, the antagonistic fungus, and the plant. Terpenes and sterols produced by the biocontrol fungus have been found to affect gene expression in both the antagonistic fungus and the plant. The terpene trichodiene (TD) elicits the expression of genes related to tomato defense and to Botrytis virulence. We show here that TD itself is able to induce the expression of Botrytis genes involved in the synthesis of botrydial (BOT) and also induces terpene gene expression in Trichoderma spp. The terpene ergosterol, in addition to its role as a structural component of the fungal cell membranes, acts as an elicitor of defense response in plants. In the present work, using a transformant of T. harzianum, which is silenced in the erg1 gene and accumulates high levels of squalene, we show that this ergosterol precursor also acts as an important elicitor molecule of tomato defense-related genes and induces Botrytis genes involved in BOT biosynthesis, in both cases, in a concentration-dependent manner. Our data emphasize the importance of a balance of squalene and ergosterol in fungal interactions as well as in the biocontrol activity of Trichoderma spp.

Effects of Trichothecene Production on the Plant Defense Response and Fungal Physiology: Overexpression of the Trichoderma arundinaceum tri4 Gene in T. harzianum.

Trichothecenes are fungal sesquiterpenoid compounds, the majority of which have phytotoxic activity. They contaminate food and feed stocks, resulting in potential harm to animals and human beings. Trichoderma brevicompactum and T. arundinaceum produce trichodermin and harzianum A (HA), respectively, two trichothecenes that show different bioactive properties. Both compounds have remarkable antibiotic and cytotoxic activities, but in addition, trichodermin is highly phytotoxic, while HA lacks this activity when analyzed in vivo. Analysis of Fusarium trichothecene intermediates led to the conclusion that most of them, with the exception of the hydrocarbon precursor trichodiene (TD), have a detectable phytotoxic activity which is not directly related to the structural complexity of the intermediate. In the present work, the HA intermediate 12,13-epoxytrichothec-9-ene (EPT) was produced by expression of the T. arundinaceum tri4 gene in a transgenic T. harzianum strain that already produces TD after transformation with the T. arundinaceum tri5 gene. Purified EPT did not show antifungal or phytotoxic activity, while purified HA showed both antifungal and phytotoxic activities. However, the use of the transgenic T. harzianum tri4 strain induced a downregulation of defense-related genes in tomato plants and also downregulated plant genes involved in fungal root colonization. The production of EPT by the transgenic tri4 strain raised levels of erg1 expression and reduced squalene accumulation while not affecting levels of ergosterol. Together, these results indicate the complex interactions among trichothecene intermediates, fungal antagonists, and host plants.

Overexpression of erg1 gene in Trichoderma harzianum CECT 2413: effect on the induction of tomato defence-related genes.

AIMS: To investigate the effect of the overexpression of erg1 gene of Trichoderma harzianum CECT 2413 (T34) on the Trichoderma-plant interactions and in the biocontrol ability of this fungus. METHODS AND RESULTS: Transformants of T34 strain overexpressing erg1 gene did not show effect on the ergosterol level, although a drastic decrease in the squalene level was observed in the transformants at 96 h of growth. During interaction with plants, the erg1 overexpression resulted in a reduction of the priming ability of several tomato defence-related genes belonging to the salicylate pathway, and also of the TomLoxA gene, which is related to the jasmonate pathway. Interestingly, other jasmonate-related genes, such as PINI and PINII, were slightly induced. The erg1 overexpressed transformants also showed a reduced ability to colonize tomato roots. CONCLUSIONS: The ergosterol biosynthetic pathway might play an important role in regulating Trichoderma-plant interactions, although this role does not seem to be restricted to the final product; instead, other intermediates such as squalene, whose role in the Trichoderma-plant interaction has not been characterized, would also play an important role. SIGNIFICANCE AND IMPACT OF THE STUDY: The functional analysis of genes involved in the synthesis of ergosterol could provide additional strategies to improve the ability of biocontrol of the Trichoderma strains and their interaction with plants.

Involvement of Trichoderma trichothecenes in the biocontrol activity and induction of plant defense-related genes.

Trichoderma species produce trichothecenes, most notably trichodermin and harzianum A (HA), by a biosynthetic pathway in which several of the involved proteins have significant differences in functionality compared to their Fusarium orthologues. In addition, the genes encoding these proteins show a genomic organization differing from that of the Fusarium tri clusters. Here we describe the isolation of Trichoderma arundinaceum IBT 40837 transformants which have a disrupted or silenced tri4 gene, a gene encoding a cytochrome P450 monooxygenase that oxygenates trichodiene to give rise to isotrichodiol, and the effect of tri4 gene disruption and silencing on the expression of other tri genes. Our results indicate that the tri4 gene disruption resulted in a reduced antifungal activity against Botrytis cinerea and Rhizoctonia solani and also in a reduced ability to induce the expression of tomato plant defense-related genes belonging to the salicylic acid (SA) and jasmonate (JA) pathways against B. cinerea, in comparison to the wild-type strain, indicating that HA plays an important function in the sensitization of Trichoderma-pretreated plants against this fungal pathogen. Additionally, the effect of the interaction of T. arundinaceum with B. cinerea or R. solani and with tomato seedlings on the expressions of the tri genes was studied.

Identification of loci and functional characterization of trichothecene biosynthesis genes in filamentous fungi of the genus Trichoderma.

Trichothecenes are mycotoxins produced by Trichoderma, Fusarium, and at least four other genera in the fungal order Hypocreales. Fusarium has a trichothecene biosynthetic gene (TRI) cluster that encodes transport and regulatory proteins as well as most enzymes required for the formation of the mycotoxins. However, little is known about trichothecene biosynthesis in the other genera. Here, we identify and characterize TRI gene orthologues (tri) in Trichoderma arundinaceum and Trichoderma brevicompactum. Our results indicate that both Trichoderma species have a tri cluster that consists of orthologues of seven genes present in the Fusarium TRI cluster. Organization of genes in the cluster is the same in the two Trichoderma species but differs from the organization in Fusarium. Sequence and functional analysis revealed that the gene (tri5) responsible for the first committed step in trichothecene biosynthesis is located outside the cluster in both Trichoderma species rather than inside the cluster as it is in Fusarium. Heterologous expression analysis revealed that two T. arundinaceum cluster genes (tri4 and tri11) differ in function from their Fusarium orthologues. The Tatri4-encoded enzyme catalyzes only three of the four oxygenation reactions catalyzed by the orthologous enzyme in Fusarium. The Tatri11-encoded enzyme catalyzes a completely different reaction (trichothecene C-4 hydroxylation) than the Fusarium orthologue (trichothecene C-15 hydroxylation). The results of this study indicate that although some characteristics of the tri/TRI cluster have been conserved during evolution of Trichoderma and Fusarium, the cluster has undergone marked changes, including gene loss and/or gain, gene rearrangement, and divergence of gene function.

Functional analysis of the Trichoderma harzianum nox1 gene, encoding an NADPH oxidase, relates production of reactive oxygen species to specific biocontrol activity against Pythium ultimum.

The synthesis of reactive oxygen species (ROS) is one of the first events following pathogenic interactions in eukaryotic cells, and NADPH oxidases are involved in the formation of such ROS. The nox1 gene of Trichoderma harzianum was cloned, and its role in antagonism against phytopathogens was analyzed in nox1-overexpressed transformants. The increased levels of nox1 expression in these transformants were accompanied by an increase in ROS production during their direct confrontation with Pythium ultimum. The transformants displayed an increased hydrolytic pattern, as determined by comparing protease, cellulase, and chitinase activities with those for the wild type. In confrontation assays against P. ultimum the nox1-overexpressed transformants were more effective than the wild type, but not in assays against Botrytis cinerea or Rhizoctonia solani. A transcriptomic analysis using a Trichoderma high-density oligonucleotide (HDO) microarray also showed that, compared to gene expression for the interaction of wild-type T. harzianum and P. ultimum, genes related to protease, cellulase, and chitinase activities were differentially upregulated in the interaction of a nox1-overexpressed transformant with this pathogen. Our results show that nox1 is involved in T. harzianum ROS production and antagonism against P. ultimum.

Detection of peptaibols and partial cloning of a putative peptaibol synthetase gene from T. harzianum CECT 2413.

The characterization of 11- and 18-residue peptaibols (peptides synthesized by peptide synthetases) at Trichoderma harzianum CECT 2413 (a filamentous fungus) was performed. Using a heterologous probe from tex1, the only peptaibol synthetase cloned and characterized so far in Trichoderma species, was cloned; a region that comprised 11676 bp of a second peptide synthetase gene detected in these strain (called salps2) and sequenced. The deduced sequence of Salps2 (3891 amino acids) contained three complete and a fourth incomplete module of a peptide synthetase, in which the typical adenylation, thiolation and condensation domains were found, but also an additional dehydrogenase/reductase domain in the C-terminus of the last module. Based on sequence similarity and analysis of its modular structure, it is proposed that Salps2 is a peptaibol synthetase. Additionally, analysis of =4.4-kb sequence downstream of salps2 was done and the signature sequences of Salps2 were identified and compared with those of available sequences of the other Trichoderma peptaibol synthetases.

ThPTR2, a di/tri-peptide transporter gene from Trichoderma harzianum.

The generation of a wide ESTs library and database from Trichoderma harzianum CECT 2413 was the base for identifying the gene ThPTR2, coding for a PTR family di/tri-peptide transporter. The deduced protein sequence of the ThPTR2 gene showed the conserved motifs and also the 12 transmembrane domains typical of the PTR transporters. The highest level of ThPTR2 expression was found when the fungus was grown in chitin as sole carbon source. We also found that ThPTR2 expression was increased when Trichoderma interacted directly in solid medium with the plant-pathogenic fungus Botrytis cinerea, showing that ThPTR2 is involved in the mycoparasitic process. Additionally, its expression was triggered by nitrogen starvation and a higher level of expression was also found when Trichoderma was grown in secondary nitrogen sources like allantoin, yeast extract, and urea. However, no difference was found when Trichoderma was grown in presence or absence of glucose as carbon source. Strain T34-15, a transformant that overexpressed the ThPTR2 gene, showed about a 2-fold increase in the uptake of the dipeptide Leu-Leu. Additionally, two transformants from the strain Trichoderma longibrachiatum T52 that overexpressed ThPTR2 were also studied, confirming the role of this gene in peptide transport. Other homologous genes to ThPTR2 were identified in other Trichoderma strains. ThPTR2 is the first experimentally confirmed PTR family transporter gene from filamentous fungi.

Cloning and characterization of the erg1 gene of Trichoderma harzianum: effect of the erg1 silencing on ergosterol biosynthesis and resistance to terbinafine.

Trichoderma species are commonly used as biocontrol agents of different plant-pathogenic fungi. Terpene compounds are involved in the biocontrol process due to their antifungal properties (e.g., ergokonins and viridins) but additionally their structural function in the cell membranes (ergosterol) is essential. We report here the characterization of the T. harzianum erg1 gene, encoding a squalene epoxidase, a key enzyme in the biosynthesis of triterpene derivatives such as ergosterol. In T. harzianum the partial silencing of the erg1 gene gave rise to transformants with a higher level of sensitivity to terbinafine, an antifungal compound that acts specifically over the squalene epoxidase activity. In addition, these silenced transformants produced lower levels of ergosterol than the wild type strain. Finally, the silencing of the erg1 gene resulted in an increase in the expression level of the erg7 gene that encodes the oxidosqualene lanosterol-cyclase, another enzyme of the terpene biosynthesis pathway.

Detection of putative peptide synthetase genes in Trichoderma species: application of this method to the cloning of a gene from T. harzianum CECT 2413.

Some of the secondary metabolites produced by Trichoderma, such as the peptaibols and other antibiotics, have a peptide structure and in their biosynthesis are involved proteins belonging to the Non-Ribosomal Peptide Synthetase family. In the present work, a PCR-mediated strategy was used to clone a region corresponding to an adenylation domain of a peptide synthetase (PS) gene from 10 different strains of Trichoderma. In addition, and using the fragment isolated by PCR from T. harzianum CECT 2413 as a probe, a fragment of 19.0 kb corresponding to a PS-encoding gene named salps1, including a 1.5 kb fragment of the promoter, was cloned and sequenced. The cloned region of salps1 contains four complete, and a fifth incomplete, modules, in which are found the adenylation, thiolation and condensation domains, but also an additional epimerization domain at the C-terminal end of the first module. The analysis of the Salps1 protein sequence, taking into consideration published data, suggests that it is neither a peptaibol synthetase nor a protein involved in siderophore biosynthesis. The presence of two breaks in the open reading frame and the expression of this gene under nitrogen starvation conditions suggest that salps1 could be a pseudogene.

Expression of a synthetic copy of the bovine chymosin gene in Aspergillus awamori from constitutive and pH-regulated promoters and secretion using two different pre-pro sequences.

A copy of the bovine chymosin gene (chy) with a codon usage optimized for its expression in Aspergillus awamori was constructed starting from synthetic oligonucleotides. To study the ability of this filamentous fungus to secrete bovine prochymosin, two plasmids were constructed in which the transcriptional, translational, and secretory control regions of the A. nidulans gpdA gene and pepB genes were coupled to either preprochymosin or prochymosin genes. Secretion of a protein enzymatically and immunologically indistinguishable from bovine chymosin was achieved in A. awamori transformants with each of these constructions. In all cases, the primary translation product (40.5 kDa) was self-processed to a mature chymosin polypeptide having a molecular weight of 35.6 kDa. Immunological assays indicated that most of the chymosin was secreted to the extracellular medium. Hybridization analysis of genomic DNA from chymosin transformants showed chromosomal integration of prochymosin sequences and, in some transformants, multiple copies of the expression cassettes were observed. Expression from the gpdA promoter was constitutive, whereas expression from the pepB promoter was strongly influenced by pH. A very high expression from the pepB promoter was observed during the growth phase. The A. awamori pepB gene terminator was more favorable for chymosin production than the S. cerevisiae CYC1 terminator.

Characterization of the gdhA gene from the phytopathogen Botrytis cinerea.

A 3.48-kb DNA region containing the gdhA gene, which codifies the NADP-dependent glutamate dehydrogenase enzyme from Botrytis cinerea, has been cloned and characterized. A fragment of 2351 nucleotides was sequenced and found to contain an ORF of 1350 bp that encodes a protein of 450 amino acids. The gene, containing two introns that showed polymorphic size between them, was located by pulsed-field gel electrophoresis in chromosome X in seven strains, which were isolated from several hosts and had different levels of pathogenesis. The protein was similar to the gdhA of various other organisms, with nine highly conserved motifs that included the known active site sequence. The cloned gene was proven to be functional since it complemented two different Aspergillus nidulans gdhA mutants, restoring high levels of NADP-dependent glutamate dehydrogenase activity to the transformants. gdhA was transcribed as a monocistronic transcript of 1.7 kb starting at an A or a T, located 40 or 47 bp, respectively, upstream from the initial ATG codon of the ORF. Transcription levels of the gdhA gene were high during the rapid growth phase. Very high expression levels of the gdhA gene were observed in media with asparagine as the nitrogen source, whereas glutamic acid repressed transcription of the gdhA gene. Similarly high levels of gdhA gene transcription were observed in media with acetate as the carbon source, while glycerol strongly repressed gdhA gene transcription. These results indicate that expression of the gdhA gene is subject to strong nitrogen and carbon regulation at the transcriptional level.

Characterization of the reverse transsulfuration gene mecB of Acremonium chrysogenum, which encodes a functional cystathionine-gamma-lyase.

In Acremonium chrysogenum, biosynthesis of cysteine for the formation of cephalosporin has been proposed to occur through the reverse transsulfuration pathway. A gene, named mecB, has been cloned from an A. chrysogenum C10 genomic library in lambdaEMBL3-ble. The cloned DNA fragment encodes a protein of 423 amino acids with a deduced molecular mass of 45 kDa that shows great similarity to cystathionine-gamma-lyases from Saccharomyces cerevisiae and other eukaryotic organisms. The protein was shown to be functional because it restores growth on methionine to A. nidulans C47 (mecB10), a mutant that is known to be defective in cystathionine-gamma-lyase. The cloned gene did not complement A. nidulans mecA or metG mutants. Enzyme activity assays confirmed that the cloned mecB gene encodes a cystathionine-gamma-lyase activity. The mecB gene is present in a single copy in the wild-type A. chrysogenum (Brotzu's strain) and also in the A. chrysogenum strain C10, a high cephalosporin producer. The gene is localized on chromosome VIII (5.3 Mb), as shown by hybridization to A. chrysogenum chromosomes resolved by pulsed-field gel electrophoresis. Transcription of the mecB gene gives rise to a major transcript of 1.5 kb and a minor one of 1.7 kb. The transcript levels were not significantly affected by addition of DL-methionine to the culture, indicating that expression of this gene is not regulated by methionine. The availability of this gene provides a very useful tool for understanding the proposed role of cystathionine-gamma-lyase in splitting cystathionine to supply cysteine for cephalosporin biosynthesis.

Targeted inactivation of the mecB gene, encoding cystathionine-gamma-lyase, shows that the reverse transsulfuration pathway is required for high-level cephalosporin biosynthesis in Acremonium chrysogenum C10 but not for methionine induction of the cephalosporin genes.

Targeted gene disruption efficiency in Acremonium chrysogenum was increased 10-fold by applying the double-marker enrichment technique to this filamentous fungus. Disruption of the mecB gene by the double-marker technique was achieved in 5% of the transformants screened. Mutants T6 and T24, obtained by gene replacement, showed an inactive mecB gene by Southern blot analysis and no cystathionine-gamma-lyase activity. These mutants exhibited lower cephalosporin production than that of the control strain, A. chrysogenum C10, in MDFA medium supplemented with methionine. However, there was no difference in cephalosporin production between parental strain A. chrysogenum C10 and the mutants T6 and T24 in Shen's defined fermentation medium (MDFA) without methionine. These results indicate that the supply of cysteine through the transsulfuration pathway is required for high-level cephalosporin biosynthesis but not for low-level production of this antibiotic in methionine-unsupplemented medium. Therefore, cysteine for cephalosporin biosynthesis in A. chrysogenum derives from the autotrophic (SH(2)) and the reverse transsulfuration pathways. Levels of methionine induction of the cephalosporin biosynthesis gene pcbC were identical in the parental strain and the mecB mutants, indicating that the induction effect is not mediated by cystathionine-gamma-lyase.

A cephalosporin C acetylhydrolase is present in the cultures of Nocardia lactamdurans.

Two protein bands with strong esterase activity are present in broths of Nocardia lactamidurans MA4213 cultures. One of them shows cephalosporin C acetylhydrolase (CAH) activity. This activity is maximal at 48 h of growth and shows a pattern of regulation slightly different from that of cephamycin production in medium supplemented with glucose (166 mM), glycerol (326 mM) or ammonium chloride (60 mM). The CAH activity was purified to homogeneity by DEAE-Sepharose ion-exchange, Sephadex G-75 gel filtration, and phenyl-Sepharose hydrophobic interaction chromatography. It showed a molecular mass of 72,100 Da. The N-terminus of the protein was determined and showed the amino acid sequence GGAAPGGPGAHPLWLPAGKD. The enzyme showed Km values of 7.0 mM and 8.3 mM for cephalosporin C and 7-aminocephalosporanic acid respectively but was not active on cephamycin C.

Overexpression and lack of degradation of thaumatin in an aspergillopepsin A-defective mutant of Aspergillus awamori containing an insertion in the pepA gene.

A gene encoding the sweet-tasting protein thaumatin (tha) with optimized codon usage was expressed in Aspergillus awamori. Mutants of A. awamori with reduced proteolytic activity were isolated. One of these mutants, named lpr66, contained an insertion of about 200 bp in the pepA gene, resulting in an inactive aspergillopepsin A. In vitro thaumatin degradation tests confirmed that culture broths of mutant lpr66 showed only a small thaumatin-degrading activity. A. awamori lpr66 has been used as host strain for thaumatin expression cassettes containing the tha gene under the control of either the cahB (cephalosporin acetylhydrolase) promoter of Acremonium chrysogenum or the gdhA (glutamate dehydrogenase) promoter of Aspergillus awamori. Residual proteolytic activities were repressed by using a mixture of glucose and sucrose as carbon sources and L-asparagine as nitrogen source. Degradation of thaumatin by acidic proteases was prevented by maintaining the pH value at 6.2 in the fermentor. Expression of cassettes containing the gdhA promoter was optimal in ammonium sulfate as nitrogen source, whereas transformants expressing the tha gene from the cahB promoter yielded higher thaumatin levels using L-asparagine as nitrogen source. Under optimal fermentation conditions, yields of 105 mg thaumatin/l were obtained, thus making this fermentation a process of industrial interest.

Thaumatin production in Aspergillus awamori by use of expression cassettes with strong fungal promoters and high gene dosage.

Four expression cassettes containing strong fungal promoters, a signal sequence for protein translocation, a KEX protease cleavage site, and a synthetic gene (tha) encoding the sweet protein thaumatin II were used to overexpress this protein in Aspergillus awamori lpr66, a PepA protease-deficient strain. The best expression results were obtained with the gdhA promoter of A. awamori or with the gpdA promoter of Aspergillus nidulans. There was good correlation of tha gene dosage, transcript levels, and thaumatin secretion. The thaumatin gene was expressed as a transcript of the expected size in each construction (1.9 or 1.4 kb), and the transcript levels and thaumatin production rate decayed at the end of the growth phase, except in the double transformant TB2b1-44-GD5, in which secretion of thaumatin continued until 96 h. The recombinant thaumatin secreted by a high-production transformant was purified to homogeneity, giving one major component and two minor components. In all cases, cleavage of the fused protein occurred at the KEX recognition sequence. This work provides new expression systems in A. awamori that result in very high levels of thaumatin production.

Organization of the gene cluster for biosynthesis of penicillin in Penicillium nalgiovense and antibiotic production in cured dry sausages.

Several fungal isolates obtained from two cured meat products from Spain were identified as Penicillium nalgiovense by their morphological features and by DNA fingerprinting. All P. nalgiovense isolates showed antibiotic activity in agar diffusion assays, and their penicillin production in liquid complex medium ranged from 6 to 38 microgram. ml-1. We constructed a restriction map of the penicillin gene cluster of P. nalgiovense and found that the organization of the penicillin biosynthetic genes (pcbAB, pcbC, and penDE) is the same as in Penicillium chrysogenum and Aspergillus nidulans. The pcbAB gene is located in an orientation opposite that of the pcbC and penDE genes in all three species. Significant amounts of penicillin were found in situ in the casing and the outer layer of salami meat during early stages of the curing process, coinciding with fungal colonization, but no penicillin was detected in the cured salami. The antibiotic produced in situ was sensitive to penicillinase.

Characterization and nitrogen-source regulation at the transcriptional level of the gdhA gene of Aspergillus awamori encoding an NADP-dependent glutamate dehydrogenase.

A 28.7-kb DNA region containing the gdhA gene of Aspergillus awamori was cloned from a genomic DNA library. A fragment of 2570 nucleotides was sequenced that contained ORF1, of 1380 bp, encoding a protein of 460 amino acids (Mr 49.4 kDa). The encoded protein showed high similarity to the NADP-dependent glutamate dehydrogenases of different organisms. The cloned gene was functional since it complemented two different Aspergillus nidulans gdhA mutants, restoring high levels of NADP-dependent glutamate dehydrogenase to the transformants. The A. awamori gdhA gene was located by pulsed-field gel electrophoresis in a 5.5-Mb band (corresponding to a doublet of chromosomes II and III), and was transcribed as a monocistronic transcript of 1.7 kb. Transcript levels of the gdhA gene were very high during the rapid growth phase and decreased drastically after 48 h of cultivation. Very high expression levels of the gdhA gene were observed in media with ammonium or asparagine as the nitrogen source, whereas glutamic acid repressed transcription of the gdhA gene. These results indicate that expression of the gdhA gene is subject to a strong nitrogen regulation at the transcriptional level.

Characterization of the cefF gene of Nocardia lactamdurans encoding a 3'-methylcephem hydroxylase different from the 7-cephem hydroxylase.

The cefF gene of Nocardia lactamdurans, encoding a functional 2-oxoglutarate-dependent 3'-methylcephem hydroxylase (deacetoxycephalosporin C hydroxylase) has been found to be closely linked to the pcbC gene in the cephamycin C gene cluster. The open-reading frame is 933 bp long and could encode a protein of M(r) 34,366. Introduction of cefF in the cephamycin-non-producer Streptomyces lividans conferred 3'-methylcephem-hydroxylating activity to the transformants but did not result in hydroxylation at carbon 7 of cephamycin. No 3'-methylcephem hydroxylase activity was observed when the cefF gene was introduced in S. lividans in pIJ699 (a vector containing two transcriptional terminators that prevent read-through expression), which suggests that this gene lacks an independent promoter.