Confiabilidad y validez de constructo de la subescala de atribuciones causales (SEAT)

To further investigate the role of RedH in 2-undecylpyrrole biosynthesis, het- erologous expression of redHinStreptomyces venezuelaeATCC10712 was performed.

S. venezuelaewas chosen as a host because it has been fully sequenced and its genome does not contain any gene cluster similar to the red cluster (Mervin Bibb, personal communication). Moreover it also contains no gene similar to redH and it does not produce prodiginines.

Conjugational transfer of pOSV556redH from E. coli between

medium forS. venezuelae), overlaid with hygromycin. After growth, a single exconju- gant was selected to carry out feeding experiments on R5 agar medium.

Feeding of wild typeS. venezuelae with synthetic 2-UP and MBC did not result in visible red-pigment production. However LC-MS analysis of acidified organic mycelial extracts showed that a small amount of undecylprodiginine was present. This was consistent with a similar observation made for the M511/redH::oriT-apr mutant. WhenS. venezuelae/pOSV556redHwas fed with synthetic 2-UP and MBC, red-pigment production was visible and LC-MS/MS analysis of acidified organic extracts of the mycelia confirmed the presence of large quantities of undecylprodiginine (Figure 4.25) (Haynes et al., 2008). 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 Time [min] 0 2 4 7 x10 Intens. 2 m/z= 394 190.90 394.29 282.36 +MS, 5.60-5.89min #(347-365) 0 1 2 3 6 x10 Intens. 100 200 300 400 500 600 700 800 900 m/z

Figure 4.25 Top: EIC (m/z = 394) from LC-MS analyses of acidified organic extracts ofS. venezuelae+ 2-UP + MBC (bottom, blue trace) andS. venezuelae+ pOSV556redH+ 2-UP + MBC (top, red trace). Bottom: mass spectrum of peak with retention time of ~5.75 minutes of the upper (red) chromatogram.

Feeding of synthetic MBC and 2-UP to S. venezuelae expressing redH estab- lished the role of RedH as an enzyme responsible for the condensation of 2-UP and MBC to form undecylprodiginine in theS. coelicolorprodiginine biosynthetic pathway.

4.5 Conclusions

Among the genes within the redcluster that have not been previously character- ised, several were replaced on the S. coelicolor chromosome with an apramycin resistance cassette using a PCR targeting strategy (Gust et al., 2002).

An M511/redL(A domain)::oriT-aprmutant was constructed and no prodiginine antibiotics were produced in the mutant. A feeding experiment with dodecanoic acid, the proposed A domain substrate, did not restore production of prodiginines, suggesting that the A domain of RedL is required for loading this intermediate onto the first ACP domain of RedL. Production of prodiginine antibiotics in the mutant was only restored when 2-UP was fed. Genetic complementation was carried out to examine whether the RedL A and ACP domains could interactin trans, but in the complemented mutant no prodiginines were produced. Perhaps deletion of the RedL A domain caused inactivation of entire protein. To circumvent this problem the region ofredLencoding the A domain could be replaced with a in-frame “scar”. Point mutations ofredL A domain active site could be an even better approach, making RedL A domain unactive and at the same time RedL correctly folded.

A redK::oriT-apr mutant of S. coelicolor M511 was constructed and it was observed that it did not produce 2 or 3. A ring C hydroxylated analogue of undecyl-

prodiginine could be detected in the mutant as well as a hydroxylated analogue of 2-UP (probably 4-hydroxy-2-undecylpyrrole, the proposed product of RedL and the likely substrate of RedK). RedK is similar to putative NAD(P)H-dependent oxidoreductases and could catalyse reduction of the keto group in the ketone tautomer of 4-hydroxy-2- undcylpyrrole, followed by elimination of water to yield 2-UP.

The proposed role of RedJ as a specific hydrolase that cleaves the dodecanoyl chain from RedQ during 2-UP biosynthesis was investigated by constructing a redJ

mutant. Prodiginine production was not abolished in the mutant, but only reduced (by about 80 +/- 3% to wild type level). TheredJ gene encodes a type II thioesterase. Such genes are very often present in NRPS and type I PKS gene clusters. Although their role in secondary metabolite biosynthesis is not clear, deletions of them can be easily com- plemented with heterologous type II thioesterase genes. In S. coelicolor, another two type II thioesterases are encoded elsewhere in the genome and could perhaps comple- ment theredJdeletion. To elucidate the exact function of RedJ it could be overproduced inE. coli, purified and biochemically investigated. Kevin Reynolds group is investigat- ing this (Reynolds, personal communication).

No clear role for RedT in prodiginine biosynthesis has been proposed. Thus a M511/redT::oriT-aprmutant was constructed.2and 3were still produced in the mutant but in very low amounts. Prodiginine production was increased by addition of MBC and accumulation of 2-UP was detected in theredTmutant, indicating that RedT is involved in, but not required for MBC biosynthesis. As BLAST searches did not identify any proteins of known function with similarity to RedT and no new intermediates were accumulated in the mutant, the function of this enzyme in MBC biosynthesis remains

unclear. To elucidate function of RedT it could be overproduced inE. coli, purified and crystallised. An X-ray structure could give insight into its function.

Once 2-UP and MBC are biosynthesised, condensation of these two intermedi- ates, catalysed by RedH, was proposed to occur to yield undecylprodiginine (Stanley et al., 2006). To confirm the role of RedH, genetic complementation of the redH mutant was performed, resulting in restoration of prodiginine production. Heterologous expres- sion of redH in Streptomyces venezuelae and feeding with synthetic 2-UP and MBC resulted in efficient undecylprodiginine production, unambiguously demonstrating the role of RedH in the biosynthetic pathway (Haynes et al., 2008).