Marta Fedoryshyn

Marker removal from actinomycetes genome using Flp recombinase

We report here a system for the functional expression of the Flp recombinase in several actinomycetes: Streptomyces coelicolor, S. lividans, and Saccharotrix espanaensis. We have constructed a synthetic gene encoding the Flp recombinase with a GC content of 60.6% optimized for expression in high-GC bacteria. Using the synthetic flp(a) gene, we have removed an apramycin resistance gene flanked by FRT sites from the chromosome of actinomycetes with an efficiency of 40%. Sequencing the region of chromosome showed that excision of the apramycin cassette by Flp recombinase was specific.

Functional expression of the Cre recombinase in actinomycetes

Site-specific recombinases revolutionized “in vivo” genetic engineering because they can catalyze precise excisions, integrations, inversions, or translocations of DNA between their distinct recognition target sites. We have constructed a synthetic gene encoding Cre recombinase with the GC content 67.7% optimized for expression in high-GC bacteria and demonstrated this gene to be functional in Streptomyces lividans. Using the synthetic cre(a) gene, we have removed an apramycin resistance gene flanked by loxP sites from the chromosome of S. lividans with 100% efficiency. Sequencing of the chromosomal DNA part showed that excision of the apramycin cassette by Cre recombinase was specific.

Generation of novel landomycins M and O through targeted gene disruption.

Two genes from Streptomyces cyanogenous S136 that encode the reductase LanZ4 and the hydroxylase LanZ5, which are involved in landomycin A biosynthesis, were characterized by targeted gene inactivation. Analyses of the corresponding mutants as well as complementation experiments have allowed us to show that LanZ4 and LanZ5 are responsible for the unique C‐11‐hydroxylation that occurs during landomycin biosynthesis. Compounds accumulated by the lanZ4/Z5 mutants are the previously described landomycin F and the new landomycins M and O.

LanGT2 Catalyzes the First Glycosylation Step during landomycin A biosynthesis.

The glycosyltransferase LanGT2 is involved in the biosynthesis of the hexasaccharide side chain of the angucyclic antibiotic landomycin A. Its function was elucidated by targeted gene inactivation of lanGT2. The main metabolite of the obtained mutant was identified as tetrangulol (4), the progenitor of the landomycin aglycon (7). The lack of the sugar side chain indicates that LanGT2 catalyzes the priming glycosyl transfer in the hexasaccharide biosynthesis: the attachment of a D‐olivose to O‐8 of the polyketide backbone. Heterologous expression of urdGT2 from S. fradiae Tü2717 in this mutant resulted in the production of a novel C‐glycosylated angucycline (6).

IncP Plasmids are Most Effective in Mediating Conjugation between Escherichia coli and Streptomycetes

Mobilizable shuttle plasmids containing the origin of transfer (oriT) region of plasmid F (IncFI), ColIb-P9 (IncI1), and RP4/RP1 (IncPα) were constructed to test the ability of the cognate conjugation system to mediate gene transfer from Escherichia coli to Streptomyces. The conjugative system of the IncPα plasmids was shown to be most effective in conjugative transfer, giving peak values of (2.7 ± 0.2) × 10−2S. lividans TK24 exconjugants per recipient cell. To assess whether the mating-pair formation system or the DNA-processing apparatus of the IncPα plasmids is crucial in conjugative transfer, an assay with an IncQ-based mobilizable plasmid (RSF1010) specifying its own DNA-processing system was developed. Only the IncPα plasmid mobilized the construct to S. lividans indicating that the mating-pair formation system is primarly responsible for the promiscuous transfer of the plasmids between E. coli and Streptomyces. Dynamic of conjugative transfer from E. coli to S. lividans was investigated and exconjugants starting from the first hour of mating were obtained.

Engineering a Function into a Glycosyltransferase

As glycosyltransferases found in nature often show distinct substrate specificity, glycosyltransferase engineering is an important research field. In this work, we were able to introduce an activity into a glycosyltransferase involved in natural product (landomycin E) biosynthesis. This was achieved by recognizing hot spot amino acids in glycosyltransferases which are strongly involved in determining substrate specificity.

Function of lanGT3, a Glycosyltransferase Gene Involved in Landomycin A Biosynthesis

The glycosyltransferase gene lanGT3, involved in the biosynthesis of the angucyclic antibiotic landomycin A, has been characterised by targeted gene deletion. A lanGT3 mutant was shown to produce landomycin E, which consists of a trisaccharide side chain attached to the polyketide moiety. Expression of lanGT3 in the mutant restored landomycin A production. Our results indicate that LanGT3 is responsible for the transfer of the fourth sugar during landomycin A biosynthesis.