Abstract
Disorazol, a macrocyclic polykitide produced by the myxobacterium Sorangium cellulosum So ce12 and it is reported to have potential cytotoxic activity towards several cancer cell lines, including multi-drug resistant cells. The disorazol biosynthetic gene cluster (dis) from Sorangium cellulosum (So ce12) was identified by transposon mutagenesis and cloned in a bacterial artificial chromosome (BAC) library. The 58-kb dis core gene cluster was reconstituted from BACs via Red/ET recombineering and expressed in Myxococcus xanthus DK1622. For the first time ever, a myxobacterial trans-AT polyketide synthase has been expressed heterologously in this study. Expression in M. xanthus allowed us to optimize the yield of several biosynthetic products using promoter engineering. The insertion of an artificial synthetic promoter upstream of the disD gene encoding a discrete acyl transferase (AT), together with an oxidoreductase (Or), resulted in 7-fold increase in disorazol production. The successful reconstitution and expression of the genetic sequences encoding for these promising cytotoxic compounds will allow combinatorial biosynthesis to generate novel disorazol derivatives for further bioactivity evaluation.
Highlights
Large clusters of genes that encode the enzymes for natural product biosynthesis have been difficult to engineer using conventional technology
Several complete biosynthetic pathways from fastidious bacteria have been heterologously expressed via Red/ET recombineering, e.g. myxochromide S and myxothiazol in Pseudomonas putida and M. xanthus[16,17,18,19], epothilones in M. xanthus[19], human alpha-defensin 5 mature peptide in Pichia pastoris[20], nikkomycin in Streptomyces ansochromogenes[21], pretubulysin in P. putida and M. xanthus[22], luminmycin and glidobactin in E. coli Nissle191723,24, salinomycin in Streptomyces coelicolor[25] and even a minimal set of genes for magnetosome biosynthesis from the magnetotactic bacterium in Rhodospirillum rubrum[26]
The backbone of pBeloBAC11-dis was replaced by a cassette containing the p15A replication origin (p15A ori), the origin of transfer for conjugation purposes, two inverted repeats (IRs), a MycoMar transposase gene (Tps) for transposition, an inducible promoter tetR-Ptet for driving the dis gene cluster upstream of disA and a kanamycin resistance gene for selection in M. xanthus DK1622
Summary
Reconstitution of the disorazol A biosynthetic gene cluster. The disorazol A biosynthetic gene cluster has been cloned, sequenced and identified previously from a BAC library of So ce1237. We have found small amounts of various disorazol compounds (including disrazols A1, A2, A3, A4, B2, B4 and F2) in both extracts of M. xanthus:: p15A-dis and M. xanthus:: p15A-dis-est (Figs 3 and S3, Table S2), upon comparing the secondary metabolite profiles from M. xanthus wild type strain and mutants. As expected, these results indicate that the chosen set of genes is sufficient to produce the polyketide-nonribosomal peptide skeleton of the disorazols. To discover the actual function of the orf[9] gene in disorazol biosynthesis, we inactivated it on the expression construct p15A-dis and performed heterologous production in M. xanthus. We have established a pioneer protocol to overexpress an independent AT resulted in increased yield of the final product, which can be used for the production optimzation of trans-AT directed natural products in the native or heterologous hosts
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