Overproduction Mechanism And Nitrate Stimulating Effect On Lincomycin Biosynthesis

Lincomycin is a kind of highly effective broad-spectrum antibiotic with important clinical significance,which is widely used in the treatment of diseases caused by Gram-positive bacteria.The production of the existing industrial strains is 7 g/L,which is more than 200 times compared to the wild-type producing strain.Furthermore,with the supplementation of 0.8% sodium nitrate,the lincomycin yield of Streptomyces lincolnensis NRRL2936 was enhanced by 70%.In this work,the complete genome sequences of the lincomycin wild-type producer S.lincolnensis NRRL2936 and high-yield producer S.lincolnensis LC-G were determined,and the overproduction mechanism of lincomycin was illustrated by comparative functional genome and transcriptome analysis.In addition,the mechanism of nitrate stimulating effect on lincomycin biosynthesis was elaborated based on the well sequenced genome.The whole genome sequence of S.lincolnensis NRRL2936 was determined by 454 sequencing technology.Based on the draft genome,meta-pair technology was used to provide contig terminal information,and the gaps in the draft were closed according to the data analysis and molecular biology technology such as PCR.After the two telomere sequences of NRRL2936 genome were identified by a self-ligation method for PCR-sequencing,the complete genome sequence was finally acquired.NRRL2936 harbors a linear chromosome(10,319,054 bp)with 24-kb terminal inverted repeats.It was found that NRRL2936 has a relatively large core region(6.99 Mb)compared with other Streptomyces genomes,and the size of two non-core regions were 1.67 Mb and 1.66 Mb,respectively.The biosynthetic gene cluster of lincomycin is located in the non-core region of the chromosome,only 24 kb away from the left end of the chromosome.It was also found that there are at least other 31 potential secondary metabolic biosynthetic gene clusters in the NRRL2936 chromosome,including 5 polyketides(PKS),5 non-ribosomal peptides(NRPS),3 hybrid PKS-NRPSs,6 terpene,3 siderophore,2 butyrolactone,and 7 other types of gene clusters,most of them are located in the non-core regions of the chromosome.Under the condition of shaking flask fermentation,the lincomycin production of NRRL2936 and LC-G are 55.7 ± 12.8 mg/L and 970 ± 66.4 mg/L lincomycin,respectively.In order to explore the key factors responsible for lincomycin overproduction,the genome sequence of LC-G was also determined by 454 sequencing technology.Compared with NRRL2936,LC-G(9,513,637 bp)has two large deletion regions at its chromosomal right arm.These two regions are respectively 25.0 kb and 777.0 kb in length,together coding for 638 CDSs.Each region was deleted in the wild-type strain NRRL2936 to investigate its involvement in lincomycin production.The lincomycin yield was enhanced from 55.7 ± 12.8 mg/L in NRRL2936 to 77.7 ± 11.6 mg/L or 680.0 ± 53.9 mg/L in mutants with large region deletion.Especially,the higher productivity(680.0 ± 53.9 mg/L)was corresponding to 70% of the yield in LC-G,revealing the critical contribution of large region deletion on lincomycin overproduction.The transcription of lincomycin biosynthetic genes was not changed in DEL-I deletion mutant,while the cellular growth was significantly reduced,suggesting that the growth potential converts to antibiotics productivity.In addition,25 insertions/deletions(INEDL)and 129 single nucleotide variations(SNVs)were identified between these two genomes.It is interesting that there are multiple inversions and rearrangements on the linear chromosome of LC-G,which is rarely found in other actinomycetes.Furthermore,the transcriptome comparison of NRRL2936 and LC-G cultivated in industrial fermentation medium indicated that there are 821 differential expressed genes(DEGs),including genes related to lincomycin biosynthesis,carbon metabolism,and nitrogen metabolism,which resulted in the enhancement of lincomycin biosynthetic gene cluster and pentose phosphate pathway and attenuation of glycolytic pathway and TCA cycle at the same time.In the genome of S.lincolnensis NRRL 2936,two sets of ABC-type nitrate transporter genes,rarely found in sequenced actinobacterial genomes,were identified and proved to be involved in lincomycin biosynthesis by gene inactivation.When these two sets of ABC transporter genes were knocked out,the intracellular nitrate concentrations were reduced from 66.9 ± 6.8 ?g/mL to 56.3 ± 3.1 ?g/mL or 50.9 ± 1.5 ?g/mL,and a combined deletion resulted in the intracellular nitrate concentration decreased to 48.8±3.4 ?g/mL.The yields of lincomycin of these mutants were reduced by 21.0%,31.4%,and 35.9% of NRRL2936.With the supplementation of nitrate,the transcription of nitrogen assimilation genes,the two sets of ABC transporter genes,a lincomycin exporter gene lmrA was enhanced,and proved to be positively regulated by the nitrogen global regulator GlnR,whose expression was also improved.Therefore,it was speculatied the nitrate stimulating effect on lincomycin production was achieved by enhanced glnR expression,glutamate supply to form tyrosine,and exportation of lincomycin through LmrA.Gene nirC,a nitrite transporter gene from Amycolatopsis mediterranei was identified by comparative genomics,which has never been reported in actinomycetes.The introduction of ABC-type nitrate transporter genes or nitrite transporter gene nirC into Streptomyces coelicolor M145 resulted in increased actinorhodin production and GlnR expression.In addition,the introduction of a constructed nirC-ABC2 cassette further improved actinorhodin production.Integration of this cassette to the genomes caused increased titers of salinomycin,ansamitocin,lincomycin,and geldanamycin in S.albus BK3-25,Actinosynnema pretiosum ATCC31280,S.lincolnensis LC-G,and S.hygroscopicus XM201,respectively.Our work implicated the overproduction mechanism of lincomycin in LC-G through comparative functional genomics and transcriptome analysis;and elucidated the nitrate stimulating effect on the biosynthesis of lincomycin based on a well-sequenced genome of S.lincolnensis NRRL2936.Moreover,the nirC-ABC2 cassette could become a general tool for titer increase of antibiotics in actinomycetes with nitrate.