The highly potent antitumor agent ansamitocin P3 is a macrolactam isolated from Actinosynnema pretiosum ATCC 31565. A 120-kb DNA fragment was previously identified as the ansamitocin biosynthetic gene cluster, and contains genes for polyketide assembly, precursor synthesis, post-polyketide synthesis modification, and regulation. Within the biosynthetic gene cluster, asm8 encodes an 1117-amino-acid protein with a high degree of similarity to the large ATP-binding LuxR family-type regulators. In the current study, we determined that inactivation of asm8 by gene replacement in ATCC 31565 resulted in the complete loss of ansamitocin production, and that complementation with a cloned asm8 gene restored ansamitocin biosynthesis. Interestingly, the disruption of asm8 decreased the transcription of genes responsible for 3-amino-5-hydroxybenzoate (AHBA) formation, the starter unit required for ansamitocin biosynthesis. Subsequently, feeding of exogenous AHBA to the asm8 mutant restored ansamitocin biosynthesis, which showed that Asm8 is a specific positive regulator in AHBA biosynthesis. In addition, investigation of asm8 homologs identified two new ansamitocin producers, and inactivation of the asm8 homolog in A. pretiosum ATCC 31280 abolished ansamitocin production in this strain. Characterization of the positive regulator Asm8 and discovery of the two new ansamitocin producers paves the way for further improving production of this important antitumor agent.
PAN WenQinKANG QianJinWANG LeiBAI LinQuanDENG ZiXin
An ideal surrogate host for heterologous production of various natural products is expected to have efficient nutrient utilization,fast growth,abundant precursors and energy supply,and a pronounced gene expression.Streptomyces albus BK3-25 is a high-yield industrial strain producing type-Ⅰ polyketide sahnomycin,with a unique ability of bean oil utilization.Its potential of being a surrogate host for heterologous production of PKS was engineered and evaluated herein.Firstly,introduction of a three-gene cassette for the biosynthesis of ethylmalonyl-CoA resulted in accumulation of ethylmalonyl-CoA precursor and sahnomycin,and subsequent deletion of the sahnomycin biosynthetic gene cluster resulted in a host with rich supplies of common polyketide precursors,including malonyl-CoA,methylmalonyl-CoA,and ethylmalonyl-CoA.Secondly,the energy and reducing force were measured,and the improved accumulation of ATP and NADPH was observed in the mutant.Furthermore,the strength of a series of selected endogenous promoters based on microarray data was assessed at different growth phases,and a strong constitutive promoter was identified,providing a useful tool for further engineered gene expression.Finally,the potential of the BK3-25 derived host ZXJ-6 was evaluated with the introduction of the actinorhodin biosynthetic gene cluster from Streptomyces coelicolor,and the heterologous production of actinorhodin was obtained.This work clearly indicated the potential of the high-yield sahnomycin producer as a surrogate host for heterologous production of polyketides,although more genetic manipulation should be conducted to streamline its performance.
