| Daptomycin, a lipopeptide antibiotic, has been approved by FDA for the treatment of complicated skin and skin-structure infections caused by gram-positive bacteria, and bacteremia and endocarditis caused by Staphylococcus aureus. Streptomyces sp. HCCB10043 can produce A21978C, which is the leading compound of daptomycin. To learn the capability of HCCB 10043 to produce secondary metabolites and genes related to A21978C biosynthesis, the genome of this strain was sequenced. Based on the genome information, arylomycin biosynthesis gene cluster was found and analyzed. Furthermore, the genes involved in fatty acid side-chains biosynthesis and impacting A21978C production were also investigated. The main results are as follows:1. Genome sequencing, annotation and mining for potential secondary metabolites biosynthesis gene clustersThe genome of HCCB 10043 was sequenced utilizing Solexa technology. The resulting reads were assembled into 449 scaffolds spanning 8.65 Mb. The draft genome contains 6145 genes encoding proteins. Among these proteins,5584 proteins show high homology to proteins from S. roseosporus, and 3987 proteins can be assigned to COG families.In addition to A21978C biosynthesis gene cluster,12 gene clusters for secondary metabolites biosynthesis were discovered in HCCB 10043, including four NRPS gene clusters (N1-N4), three PKS gene clusters (P1-P3), three hybrid NRPS-PKS gene clusters and two other type gene clusters (O1-O2). Among these clusters, N2, P3, NP3, O1 and 02 are respectively responsible for napsamycins, alkylresorcinols, collismycins, desferrioxamine E and FR900098 biosynthesis, while the function of other gene clusters are still unknown.RT-PCR analysis of related genes in the above clusters revealed that nearly all the selected genes were transcripted at high levels in media F1 and F2 at 48 h, which confirmed that these gene were active and with potential to produce secondary metabolites.2. Identification of metabolites corresponding to the function-unknown gene clustersFor those function-unknown gene clusters, N1, N4, NP2 and P2 were chosen for further study due to their high transcript levels in Fl and F2. Firstly, genes n1-1, n4-1, np2-1 and p2-1 were deleted in HCCB10043 via homologous recombination. UPLC-MS analysis showed that mutants except Δn4-1 had a similar metabolite profile to HCCB 10043. Compared with HCCB 10043, four compounds were lost in the fermentation broth of An4-1 and were identified as arylomycin A2, A4, A5 and A6.PCR amplifications were performed to obtain complete N4 cluster sequence with genome sequences of HCCB 10043 and S. roseosporus as references. N4 cluster contains 10 genes. aryA, aryB and aryD encode NRPSs and are responsible for hexapeptide of arylomycin biosynthesis. aryF, aryG and aryH encode hydroxymandelate synthase, hydroxymandelate oxidase and hydroxyphenylglycine aminotransferase, which share high sequence similarities with the enzymes involved in the biosynthesis of HPG residue in chloroeremomycin. Gene deletion of aryF and HPG-fed fermentation of the AaryF mutant proved they were indeed responsible for HPG biosynthesis.In the arylomycin biosynthesis gene cluster, aryC encodes a P450 protein, which shows similarity with other P450 enzymes involved in biaryl oxidative coupling reactions, such as HmtS, P-450mel and OxyC. Inactivation of aryC resulted in non-production of arylomycins and generation of four novel linear lipopentapeptides, which lost antibacterial activity against Staphylococcus epidermidis. When a single copy of aryC was introduced into AaryC mutant, the resulting mutant restored arylomycin production, which confirmed that aryC is essential for arylomycin production and involved in the formation of biaryl bridge between HPG residue and Tyr residue.Arylomycins can act as type Ⅰ signal peptidase (SPase I) inhibitors. Besides mutation in Spase I, there were some still unknown factors contributed to arylomycins resistance in some bacteria. To confirm whether the ABC transporter encoding genes aryl and aryJ are related to arylomycin resistance, Aaryl and AaryJ mutants derived from HCCB 10043 and ΔaryF were constructed respectively. The results showed that when either aryl or aryJ was deleted in HCCB 10043, the resulting mutant produced significantly fewer spores after cultured on MS plate for 4 days, while the phenomena would not occur since the production of arylomycin was arrested simultaneously. Based on these facts, aryl and aryJ were proposed to improve arylomycin resistance in HCCB10043 besides Pro mutations existing in Spase I.3. Function of fabH and bkd during biosynthesis of fatty acid side-chains in A21978C and arylomycinA21978C and arylomycin are both lipopeptides, while no fatty acid (FA) biosynthetic gene was found in their biosynthesis gene clusters. The main components of A21978C and arylomycin contain branched chain fatty acid (BCFA) side-chains, while daptomycin was the n-decanoyl analog of A21978C, containing a straight chain fatty acid (SCFA) side-chain. To identified the genes involved in FAs biosynthesis and genetically modify these genes subsequently to improve the ratios of the components containing SCFAs (daptomycin) in the fermentation broth, the roles of fabH which is essential for FAs biosynthesis, and bkd which is involved in branched amino acids metabolism, during A21978C and arylomycin biosynthesis were investigated.There are seven fabH genes in HCCB 10043 genome, and one of them is clustered with fabD, fabB and acpP, which has been proved involved in an essential primary metabolic process. To confirm which fabH gene is responsible for FA side-chains biosynthesis, the fabH genes beyond the fab cluster were deleted singly and simultaneously. HPLC analysis showed that the resulting mutants produced A21978C and arylomycin at similar levels to that of HCCB 10043, which revealed that these genes, are not responsible for biosynthesis of FA side-chains in A21978C and arylomycin. Next, we attempted to replace the fabH gene in the fab cluster by the fabH from Escherichia coli (ecfabH). Firstly, pWMH-ecfabHl-4 which can express ecFabH were constructed and introduced in HCCB 10043, then fabH in the fab cluster was tried to be deleted via homologous recombination, but it was failed, which may because the fabH is essential for HCCB 10043 and adverse influence would be generated when it was replaced.In HCCB10043, two bkd gene cluster:bkdA1B1C1 and bkdA2B2C2, were found. Deletion of bkdA2B2C2 had no effect on production of A21978C and arylomycin, while AbkdA1B1C1 mutant produced components containing SCFAs rather than those containing BCFAs. When isobutyrate and 2-methylbutyrate, catabolic products of Val and Ile, were supplied, generation of the components containing BCFAs (such as C1-3, A2 and A4) by AbkdA1B1C1 mutant could be restored, which suggested that the short BCFA precursors used for BCFA side-chains biosynthesis in A21978C and arylomycin were derived from catabolic products of Val and Ile catalyzed by BkdA1B1C1. We also tried to feed ΔbkdA1B1C1 mutant n-decanoic acid, but the autolysis of mycelia occurred prematurely and no daptomycin was accumulated, which may because too many SCFAs were incorporated into cell membranes and led to less rigid membrane to against the toxicity of decanoic acid.4. Transcriptome analysis of A21978C high-and low-producing mutants, and verification of related differential expression genesTo identify the factors impacting the production of A21978C and can be used for further strain improvement, the transcriptomes of A21978C high-producing mutant A9 and low-producing mutant C4 after cultured for 48 h were analyzed by RNA-seq. There are 66 differential expression genes (BDEG1-66) both detected in C4 and A9. Among these genes,10 genes were up-regulated and 56 genes were down-regulated in A9. Moreover, there are 494 genes only detected in A9 and 579 genes only detected in C4. The genes in A21978C biosynthesis gene cluster were analyzed by RT-PCR. The results demonstrated that the A21978C skeleton biosynthetic genes and resistance genes showed similar transcript levels in C4 and A9, which are consistent with RNA-seq results and suggested that the different production of A21978C was not resulted from the distinct expression levels of these biosynthetic genes.Differential expression genes BDEG4, DEG14, BDEG22, BDEG28, BDEG41 and BDEG61 were further verified via RT-PCR, and they showed changes in C4 and A9 similar to the results of RNA-seq. Then BDEG14 (glpK) and BDEG28 (ssgG) were selected for further research. Deletion of glpK in C4 had no effect on A21978C production, while the production of A21978C was improved about 10-30% when ssgG was deleted in C4, which revealed that the low expression of ssgG contributes to the high production of A21978C in A9.In addition, there are 425 SNV sites existing in A9 compared with C4, and one of them was observed at nt 129 in rpsL encoding ribosomal S12 protein. The appearance of this SNV caused great improvement of streptomycin and kanamycin resistance in A9, and it has been reported in A21978 high producing strains. so, the SNV is also one of the important reasons for A21978C overproduction in A9. |
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