Study On The Regulation Of Oxytetracycline Biosynthesis In Streptomyces Rimosus By Two-component Systems RimA1A2 And AfrQ1Q2

Oxytetracycline(OTC)is one of aromatic polyketides synthesized by bacterial type ?polyketide synthases(PKSs)in S.rimosus M4018.Nowadays,S.rimosus has become one of the main OTC commercial producing strains.The regulation genes related to cell growth and oxytetracycline production need to be mined from the genomic information,which could improve the regulation network of oxytetracycline biosynthesis.As a global regulator,two-component systems(TCSs)play a very complex role in the cell growth,sporulation of bacteria and the biosynthesis of secondary metabolites.At present,most studies about TCS from Streptomyces are based on S.coelicolor,and there are still few studies on the TCS in S.rimosus.Meanwhile,the regulatory network of oxytetracycline is not comprehensive due to the lack of genomic information of S.rimosus.In view of the above shortcomings,this study has explored the TCSs in S.rimosus based on the genomic information to investigate the relationship between the OTC biosynthesis and TCSs,thus further broadening the regulatory network of oxytetracycline.Due to characteristics of high oxygen consumption in OTC fermentation process,studies on the expression of Vitreoscilla hemoglobin gene(vgb)in S.rimosus were carried out to investigate its effects on the cell growth of S.rimosus and OTC production.The results are listed as follows:1.S.rimosus genomic DNA sequencing,classification and identification of TCSsTotal size of S.rimosus M4018 genome is estimated to 9.2 Mb based on S.rimosus genomic DNA sequencing results.The draft genome of S.rimosus M4018 is estimated to have a total of 8584 protein-coding genes.Putative open reading frames(ORFs)and the corresponding proteins in the genome were annotated based on the PFAM database.77 putative response regulators(RR)and 171 putative histidine kinase(HK)homologs were identified in the genome.50 pairs of typical TCS comprising a histidine kinase and a cognate response regulator protein were screened,considering that HK and RR of a typical TCS are adjacent to each other on the chromosome,and they are generally transcribed in the same orientation.Two putative TCSs with high homology to the known TCSs in S.coelicolor were found in S.rimosus M4018 genome sequence,which were designated as RimA1A2 and AfrQ1Q2,respectively.RimA1A2 and AfrQ1Q2 in S.rimosus M4018 were proved to be typical TCSs by amino acid sequence alignment.RT-PCR confirmed that HK and RR coding genes of these two newly identified TCSs were co-transcribed.2.RimA1A2 and AfrQ1Q2 regulated the OTC production negatively in a medium-dependent mannerRimA1-disrupted strain S.rimosus M4018 ?rimA1 and afrQ1-disrupted strain S.rimosus M4018 ?afrQl were constructed.Compared to the parental strain S.rimosus M4018,M4018 vrimA1 and M4018 ?afrQ1 showed no visible change in the culture pigmentation,OTC biosynthesis,morphology or any other phenotypic properties,when cultivated on rich media.When cultivated on minimal medium(MM)with different nitrogen source as the sole nitrogen source,significant increase in pigmentation intensity was observed in M4018 ?rimAl and M4018 ?afrQ1 compared with M4018.The most significant difference in pigment production was observed when MM was supplemented with glycine among all the nitrogen sources tested,thus indicating that RimA1A2 and AfrQ1Q2 may negatively regulate OTC biosynthesis in a medium-dependent manner.3.TCS RimA1A2 had a different role in OTC biosynthesis under different environmental stress conditionsThe complementary mutant M4018 ?rimAl(pIB-KA-rim)and its corresponding control M4018 ?rimA1(pIB-KA),the overexpression strain M4018(pIB-KA-rim)and its corresponding control M4018(pIB-KA)were constructed.No significant difference in OTC production was observed when comparing M4018(pIB-KA)and M4018,or when comparing M4018 ?rim and M4018 ?rim(pIB-KA),indicating that the introduction of the empty plasmid pIB-KA had no effect on the OTC production in M4018 and M4018 ?rim in MM supplemented with glycine.Under the oxidative stress environment caused by the addition of H2O2 into MM+glycine,the rimA1-disrupted mutant M4018 ?rim(pIB-KA)exhibited the highest oxidative stress tolerance in relation to the OTC production.In contrast to the osmotic stress caused by KCl addition,where the overexpression of RimA1A2 showed the highest osmotic stress tolerance in relation to the OTC production.4.TCS AfrQ1Q2 had a different role in OTC biosynthesis under different environmental stresses conditionsThe complementary mutant M4018 ?afrQl(pIB139-KA-afr)and the overexpression strain M4018(pIB139-KA-afr)were constructed.Under the oxidative stress environment caused by the addition of H2O2 into MM+glycine,the inactivation of AfrQ1Q2 was beneficial to resist oxidative stress and reduced the decline of OTC production in M4018-?afrQ1.When the initial concentration of glucose in MM+Gly was increased from 1.0%to 8.0%,it was found that the inactivation of AfrQ1Q2 could help M4018-?afrQ1 to reduce the decline of OTC production under the stress condition caused by high glucose concentration.5.Role of RimA1A2 and AfrQlQ2 in regulation of OTC biosynthesisTo investigate the possible reasons that enhance OTC production in the disruption mutant,one OTC biosynthesis(oxyB)and four regulation-related genes(otrB,otcG,otcR and otrC)which were reported to be positively correlated with OTC production,were selected to quantify their transcriptional levels.When RR gene rimA1 or afrQ1 was disrupted,the transcriptional level of five genes was all increased with different degree compared to M4018,which was in agreement with the OTC production level.It can be inferred that RimA1A2 or AfrQ1Q2 had an effect on OTC biosynthesis and regulation-related genes as a global regulator to realize the regulation of OTC production.6.Effect of Vitreoscilla hemoglobin gene(vgb)expression on growth and oxytetracycline biosynthesis by S.rimosusA pSET152-derived integrative plasmid was constructed with vgb gene expression under the control of a constitutive promoter PermE*,and it was introduced into S.rimosus M4018 chromosome through conjugation.The expression of vgb in recombinant S.rimosus enhanced the oxygen affinity of the strain,promoted OTC biosynthesis under high and low dissolved oxygen tensions,and significantly improved the growth of S.rimosus under low dissolved oxygen tensions.