Insights into the Role of Ketoreductases in the Biosynthesis of Partially Reduced Bacterial Aromatic Polyketides*

Abstract Partially reduced aromatic polyketides are bioactive secondary metabolites or intermediates in the biosynthesis of deoxygenated aromatics. For the antibiotic GTRI‐02 (mensalone) in different Streptomyces spp., biosynthesis involving the reduction of a fully aromatized acetyltrihydroxynaphthalene by a naphthol reductase has been proposed and shown in vitro with a fungal enzyme. However, more recently, GTRI‐02 has been identified as a product of the ActIII biosynthetic gene cluster from Streptomyces coelicolor A3(2), for which the reduction of a linear polyketide precursor by ActIII ketoreductase, prior to cyclization and aromatization, has been suggested. We have examined three different ketoreductases from bacterial producer strains of GTRI‐02 for their ability to reduce mono‐, bi‐, and tricyclic aromatic substrates. The enzymes reduced 1‐ and 2‐tetralone but not other aromatic substrates. This strongly suggests a reduction of a cyclized but not yet aromatic polyketide intermediate in the biosynthesis of GTRI‐02. Implications of the results for the biosynthesis of other secondary polyketidic metabolites are discussed.


I. General remarks
All chemical reagents and solvents were obtained from Sigma Aldrich and HiMedia. NMR spectra were recorded at 24 °C on a DRX 400 spectrometer (Bruker) operating at 400 and 100 MHz for 1 H and 13 C acquisitions, respectively. Chemical shifts (δ) of the 1    Streptomyces coelicolor A3(2) was cloned using genomic DNA.

Cloning and sequence analysis
For the cloning of the ketoreductase genes into pET19b vector the In-Fusion HD Cloning Kit from Clontech Laboratories was used. First, the vector was linearized by digestion with XhoI.
Then, the respective genes were cloned from genomic DNA using primers, which had a 5'-end complementary for the respective ends of the linearized vector, and a 3'-end complementary to the start or end of the gene of interest. Furthermore, the originally included XhoI cut site was preserved. After In-Fusion cloning, the resulting circular plasmids were transformed by heat shock at 42 °C for 45 s into DH5α cells, which were consequently grown under selective pressure (ampicillin). Correct sequences and orientation of the amplified genes in the vector were confirmed by sequence analysis through GATC Biotech.

Primers for cloning of KR1 of Streptomyces sp. GW4184
C220_ORF9_for CAAGCATATGCTCGAGATGGCGCAGGACAAGC

Primers for cloning of KR2 of Streptomyces sp. GW4184
C313_ORF14_for CAAGCATATGCTCGAGATGTCACAGGCAGTCAAGCC

Cultivation and expression
The overnight cultures were diluted to 500 mL of medium each (ampicillin 100 µg·mL -1 ).
IPTG (0.1 mM) was added after the mid-log phase (OD600nm = 0.6) was reached. The cultures were incubated for 20 h at 25 °C and 160 rpm.

Workup and storage
The harvested E. coli cells were resuspended in lysis buffer (50 mM Tris HCl, 5
The solution was filtered through a sintered glass filter with a silica bed, and washed with EtOAc (10 mL). The aqueous layer was extracted with EtOAc (2 ×10 mL) and the combined S10 organic layer was washed with brine (10 mL). The organic layer was dried over Na2SO4 and filtered, and the solvent was removed under reduced pressure to afford the residue. Product formation was analyzed through TLC and 1 H NMR spectra recorded in acetone-d6. U/mL) was added and the mixture was stirred at room temperature. After 24 h, the solution was acidified with 10% of H2SO4 (0.25 mL) and EtOAc (10 mL) was added and stirred vigorously, precipitating the enzyme. The solution was filtered with sintered glass with silica bed and washed with EtOAc (10 mL). The aqueous layer was extracted (x2) with EtOAc and the combined organic layer was washed with brine (10 mL). The organic layer was dried over Na2SO4, filtered and the solvent was removed under reduced pressure to afford a dark brown residue 16, which was purified by using column chromatography.  mg, 0.013 mmol, 0.1 equiv., 10%) and MAE (500 µL, 40 U/mL) were added and the mixture was stirred slowly under argon at room temperature. After 1h, the substrate, 1-tetralone (15) (20.0 mg, 0.136 mmol, 1 equiv.) in 2-propanol (0.5 mL, 5% v/v) was added slowly, while stirring at 100 rpm. At last, the enzyme (KR1_his, KR2_his and ActIII KR_his) (2 mL, 3.3 U/mL) was added and the mixture was stirred at room temperature. After 24 h, the solution S12 was acidified with 10% of H2SO4 (0.25 mL) and EtOAc (10 mL) was added and stirred vigorously, precipitating the enzyme. The solution was filtered with sintered glass with silica bed and washed with EtOAc (10 mL). The aqueous layer was extracted (x2) with EtOAc and the combined organic layer was washed with brine (10 mL). The organic layer was dried over Na2SO4, filtered and the solvent was removed under reduced pressure to afford a dark brown residue 17, which was purified by using column chromatography.