Cloning of separate meilingmycin biosynthesis gene clusters by use of acyltransferase-ketoreductase didomain PCR amplification

Abstract

Five meilingmycins, A to E, with A as the major component, were isolated from Streptomyces nanchangensis NS3226. Through nuclear magnetic resonance (NMR) characterization, meilingmycins A to E proved to be identical to reported milbemycins alpha11, alpha13, alpha14, beta1, and beta9, respectively. Sequencing of a previously cloned 103-kb region identified three modular type I polyketide synthase genes putatively encoding the last 11 elongation steps, three modification proteins, and one transcriptional regulatory protein for meilingmycin biosynthesis. However, the expected loading module and the first two elongation modules were missing. In meilingmycin, the presence of a methyl group at C-24 and a hydroxyl group at C-25 suggests that the elongation module 1 contains a methylmalonyl-coenzyme A (CoA)-specific acyltransferase (ATp) domain and a ketoreductase (KR) domain. Based on the conserved motifs of the ATp and KR domains, a pair of primers was designed for PCR amplification, and a 1.40-kb expected fragment was amplified, whose sequence shows significant homology with the elongation module 1 of the aveA1-encoded enzyme AVES1. A polyketide synthase (PKS) gene encoding one loading and two elongation modules, with a downstream C-5-O-methyltransferase gene, meiD, was subsequently localized 55 kb apart from the previously sequenced region, and its deletion abolishes meilingmycin production. A series of deletions within the 55-kb intercluster region rules out its involvement in meilingmycin biosynthesis. Furthermore, gene deletion of meiD eliminates meilingmycins D and E, with methyls at C-5. Our work provides a more specific strategy for the cloning of modular type I PKS gene clusters. The cloning of the meilingmycin gene clusters paves the way for its pathway engineering.

FIG. 1.

HPLC profile of meilingmycins and structure comparison between meilingmycins and avermectin. (A) HPLC profile of meilingmycins. (B) Structures of meilingmycins and avermectin A1a. The carbons of meilingmycins are numbered. The five meilingmycins are different at R1 to R5, and the differences are shown in the table. Meilingmycins A, B, and C contain a furan ring between R4 and R5 and a side chain at R3. No furan ring and side chain are identified in meilingmycins D and E, which are methylated at C-5. mAU, milli-absorbance units.

FIG. 2.

Physical map and gene organization of meilingmycin biosynthetic gene clusters. (A) Physical map of meilingmycin biosynthetic gene clusters. The vertical dashed lines indicate the positions of primers for chromosome walking. The horizontal lines indicate individual cosmid/fosmids, and those shown in pink are sequenced regions, including the 17-kb ClaI fragment from 2G6 and the 8.5-kb SacI fragment from 26H12. (B) Gene organization of meilingmycin biosynthetic gene clusters. ave, avermectin biosynthetic gene cluster (14); mei, meilingmycin biosynthetic gene cluster; red, PKS genes; blue, genes for post-PKS modifications; green, regulatory genes; blank arrows, genes probably not related to meilingmycin biosynthesis. The ORFs boxed with dashed lines are from the previously sequenced 103-kb region.

FIG. 3.

PCR amplification of the ATp-KR region. (A) Hypothetical domain organization of MeiA1. (B) Sequence alignment of the mei AT domains. The top six ATs are malonyl-CoA-specific AT domains (ATa). The rest are methylmalonyl-CoA-specific AT (ATp) domains. The conserved motif of ATp is underlined, and the primer ATp1 was designed according to this motif. (C) Sequence alignment of the mei KR domains. The conserved motif of KR for NADP(H) binding is underlined, and the degenerate primer KRp was designed according to this motif. (D) PCR amplification of the ATp-KR didomain. The expected 1.40-kb fragment was amplified with primers ATp1 and KRp.

FIG. 4.

Gene deletion of meiA1 and upstream genes. (A) Schematic description of gene deletion. The 18.6-kb region, including most of meiA1, and 10 upstream ORFs were replaced by aac(3)IV through double crossover. Using a portion of the 1.60-kb SacI fragment at the left end of the deletion region as a probe for Southern hybridization, the SacI-digested genomic DNA from wild-type NS3226 will give a 1.60-kb signal, whereas the deletion mutant HYL23 will yield a 1.90-kb signal. (B) Confirmation through Southern hybridization. The 562-bp probe was amplified using primers 10D8-C1 and 10D8-C2 and cosmid 10D8 as a template. The Southern hybridization revealed that there is a 1.90-kb signal in mutant HYL23 and a 1.60-kb signal in wild-type NS3226. (C) Comparison between wild-type NS3226 and deletion mutants through LC-MS. HYL23, deletion (Δ) of orf60-meiA1 containing the amplified ATp-KR fragment; HYL25, deletion of orf24-orf32 within the 55-kb region; HYL22, deletion of orf31-orf55 within the 55-kb region; HYL26, deletion of orf56-orf59 within the 55-kb region; HYL27, deletion of orf60-orf69 within the 55-kb region; HYL24, deletion of orf15-orf21 to determine the left boundary of the mei gene clusters; HYL29, deletion of orf70-orf79 to determine the right boundary of the mei gene clusters.

FIG. 5.

Gene replacement of meiD. (A) Schematic representation of the replacement of a 615-bp internal fragment of meiD by the 1.40-kb oriT-acc(3)IV cassette. Wild-type NS3226 should yield a 1.20-kb PCR-amplified product, and mutant HYL28 should yield a 1.90-kb product by using primers meiD-C1 and meiD-C2. (B) Confirmation of the meiD-deleted mutant via PCR amplification. (C) HPLC analysis of wild-type NS3226 and meiD mutant HYL28. The peaks of meilingmycins D and E disappeared in mutant HYL28, and meilingmycins A, B, and C are still produced by the mutant.

FIG. 6.

Proposed biosynthetic pathway to meilingmycins. R, H or CH₃; green circles, loading module or starter units; orange circles, methylmalonyl-CoA-specific modules or acetate-derived extender units; blue circles, malonyl-CoA-specific modules or propionate-derived extender units; pink circle, the thioesterase (TE) domain; KR*, nonfunctional ketoreductase domain deduced from meilingmycin structure; KR^I, inactive ketoreductase domain; AT, acyltransferase; ATa, malonyl-CoA-specific AT; ATp, methylmalonyl-CoA-specific AT; ACP, acyl carrier protein; DH, dehydratase; ER, enoyl reductase; KR, ketoreductase; KS, ketosynthase.