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The tyl genes of S. fradiae include the richest collection of regulators yet encountered in a single antibiotic biosynthetic gene cluster. Control of tylosin biosynthesis is now amenable to detailed study, and manipulation of these various regulatory genes is likely to influence yields in tylosin-production fermentations.

We have expressed a synthetic gene that encodes an antigen-binding single-chain FV protein (scFV) in transgenic tobacco plants. The scFV gene was created by polymerase chain reaction (PCR) amplification of the variable domain coding regions from a mouse monoclonal hybridoma cell line. The monoclonal cell line secretes an IgG1 antibody that binds to the plant regulatory photoreceptor protein, phytochrome. The cloned scFV gene was expressed initially in Escherichia coli and shown to produce a 28 kD, phytochrome-binding binding scFV protein. Transgenic tobacco plants expressing the scFV gene were also found to produce a functional scFV protein, and seeds from transgenic R1 progeny displayed aberrant phytochrome-dependent germination. The scFV from transgenic tobacco could be isolated, to near homogeneity, by a single phytochrome-Sepharose affinity chromatography step.

Regulation of tylosin production and morphological differentiation in Streptomyces fradiae by TylP, a deduced g-butyrolactone receptor activator, adpA (Ohnishi et al., 1999). A variation on this theme is seen in Streptomyces virginiae, in which gbutyrolactones ('butanolides') bind to BarA and derepress virginiamycin biosynthesis with no concurrent effect on sporulation (Nakano et al., 1998). In the latter case, BarA controls the expression of various genes, including barB. Because the tylosin biosynthetic (tyl ) gene cluster of Streptomyces fradiae (Fig. 1) contains obvious orthologues of barA and barB (namely tylP and tylQ respectively; Bate et al., 1999), it seemed likely a priori that one or more g-butyrolactone(s) regulate tylosin production and that the TylP protein (deduced to be a g-butyrolactone receptor) might be a repressor involved in such regulation. The aim of the present work was to test that hypothesis and (if substantiated) to identify targets for TylP. In doing so, we were mindful that g-butyrolactones and their receptors typically operate at relatively 'high' levels in regulatory cascades that control antibiotic production in actinomycetes. That being so and given that the tyl cluster is remarkable in containing at least five regulatory genes (including tylR, tylS and tylT, in addition to tylP and tylQ; Bate et al., 1999;, we were also interested in the possibility of establishing a hierarchical order among the tyl regulators. Accordingly, gene expression analysis (involving reverse transcription-polymerase chain reaction, RT-PCR) was applied to the entire tyl cluster before and after the onset of tylosin production in S. fradiae wild type and, concurrently, in engineered strains in which tylP was constitutively overexpressed or had been specifically disrupted. Such studies were complemented by fermentation analysis and by characterization of tyl promoters transplanted into S. lividans. Results Gene expression analysisWhen transcript analysis was first applied to the tyl gene cluster , it became clear that the TylQ protein is a repressor that controls tylR (a global activator of the tyl cluster; Bate et al., 1999), and that tylQ must be repressed before tylosin production can begin. Thus, in S. fradiae wild type, although tylQ was expressed before the onset of tylosin biosynthesis (i.e. at 18 h; Fig. 2), it was the only silent gene in the cluster after tylosin production had commenced (i.e. at 40 h; Fig. 2). In Molecular Microbiology (2002) 45(3), [735][736][737][738][739][740][741][742][743][744] George Stratigopoulos, Atul R. Gandecha and Eric Cundliffe* Department of Biochemistry, University of Leicester, Leicester LE1 7RH, UK. SummaryDuring promoter-probe analysis carried out in Streptomyces lividans, the TylP protein powerfully inhibited reporter gene expression from the tylP promoter, raising the likelihood that tylP is autoregulated in its native host, Streptomyces fradiae. Also in S. lividans, TylP negatively controlled the tylQ promoter, even though tylQ could still be switched off in ...

A synthetic gene encoding an anti-phytochrome single-chain Fv (scFv) antibody bearing an N-terminal signal peptide has been used to transform tobacco plants. Immunoblot analysis showed that transformed plants accumulate high levels of scFv protein, accounting for up to 0.5% of the total soluble protein fraction, which could be extracted by simple infiltration and centrifugation of leaf tissue. A substantial proportion of the scFv protein extracted in this way was found to possess antigen-binding activity. Callus cell suspension cultures derived from transformed plants secrete functional scFv protein into the surrounding medium. Compared with the levels of scFv protein observed in plants expressing the native scFv gene, the incorporation of an N-terminal signal peptide, to target the scFv to the apoplast, results in elevated accumulation of the protein.

The tylosin-biosynthetic (tyl) gene cluster occupies about 1% of the genome of Streptomycesfradiae and includes at least 43 open reading frames. In addition to structural genes required for tylosin production, the tyl cluster contains three resistance determinants and several regulatory genes. Tylosin production is evidently controlled by pathway-specific and pleiotropic regulators with the likely involvement of y-butyrolactone signalling factors. Accumulation of the polyketide aglycone is controlled by glycosylated macrolides and optimal performance of the complex polyketide synthase enzyme requires the activity of an editing thioesterase.

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