Characterization Of Type Ⅲ Polyketide Synthase From Streptomyces Sp.CB03234-S And Its Effect On The Yield Of Enediyne Tiancimycin

Background and aim: Type Ⅲ polyketide synthases(PKS)are a class of polyketide synthases with the simplest structure widely existing in microorganisms,and their products have a variety of structures and biological activities.Streptomyces sp.CB03234-S is a high-yielding strain of ten-membered anthraquinone enediyne(tiancimycins,TNMs).Whole-genome resequencing found that there are two adjacent type Ⅲ PKS(No.2.and No.3).Based on the guidance of genomic and transcriptomic data,this topic will systematically study the effect of type Ⅲ PKS in CB03234-S on the production of TNMs,and heterologously express and metabolize the unknown type Ⅲ PKS in the type strain Streptomyces albus.The specific research contents are as follows:Methods and Results: 1.By comparing the biological information with the known bacterial type Ⅲ PKS gene,it is found that the similarity between type Ⅲ PKS No.3 and alkylresorcinolase is 100%,while the similarity between type Ⅲ PKS No.2 and Rpp A(producing 1,3,6,8-tetrahydroxynaphthalene(THN))was only 72.38%.Transcriptome analysis showed that the expression of these two type Ⅲ PKS genes was significantly increased in CB03234-S compared with wild-type CB03234.Therefore,we knocked out two type Ⅲ PKS genes in CB03234-S respectively.The fermentation results showed that knockout of type Ⅲ PKS No.In Δ2PKS/S,the yield of TNMs reached 19.1 ± 0.9 mg/L,which was 56.32% higher than CB03234-S.To explore whether the increase in TNMs production is related to the competition of precursor malonyl-Co A,we further knocked out three other core genes of type I PKS except TNMs biosynthesis gene cluster(tnm)in CB03234-S,but the yield of TNMs did not change significantly in these mutants.2.Through antismash analysis,we found that there are P450 oxidase and cupin monooxygenase encoding genes adjacent to and downstream of the No.2 type Ⅲ PKS gene,but no related products were found in the fermentation metabolites of CB03234-S.Therefore,we combined this group of genes(pks,p450 and cupin)for heterologous expression in Streptomyces albus J1074,respectively.The spores and fermentation broth of the two constructed mutant strains,P/white(pks+p450)and PC/white(pks+p450+cupin),were obviously red,and the results of LC/MS and high-resolution mass spectrometry showed that the red secondary The main metabolites are flaviolin and its dimer.Combined with the known biosynthesis of flaviolin,type Ⅲ PKS gene No.2encodes a homologous Rpp A enzyme to synthesize THN,and then generates flaviolin and its dimer under the combined action of P450 enzyme and cupin monooxygenase.In addition,we also overexpressed these two groups of genes in CB03234-S,respectively,and observed the production of red flaviolin in the obtained two mutant strains P/S and PC/S,but no dimer was found,and the production of TNMs all decreased,up to 67.51%.Conclusion: In summary,through genetic verification and heterologous expression,we determined that type Ⅲ PKS type 2 in CB03234-S is the Rpp A enzyme that synthesizes THN,which is a potential factor affecting the production of TNMs as a precursor competition pathway.After removal,a high-yielding strain Δ2-PKS/S with TNMs production increased by more than 50% was obtained.This study revealed that Rpp A type Ⅲ PKS is likely to have a general competitive effect on polyketide natural products using malonyl-Co A as a substrate,providing a new idea and research for the production of polyketide natural products direction.