The 2015 Nobel Prize in Physiology or Medicine has been awarded to avermectins and artemisinin,respectively.Avermectins produced by Streptomyces avermitilis are excellent anthelmintic and potential antibiotic agents.Because wild-type strains only produce low levels of avermectins,much research effort has focused on improvements in avermectin production to meet the ever increasing demand for such compounds.This review describes the strategies that have been widely employed and the future prospects of synthetic biology applications in avermectin yield improvement.With the help of genome sequencing of S.avermitilis and an understanding of the avermectin biosynthetic/regulatory pathways,synthetic and systems biotechnology approaches have been applied for precision engineering.We focus on the design and synthesis of biological chassis,parts,devices,and modules from diverse microbes to reconstruct and optimize their dynamic processes,as well as predict favorable effective overproduction of avermectins by a 4Ms strategy(Mine,Model,Manipulation,and Measurement).
Erythromycin A is a widely used antibiotic produced by Saccharopolyspora erythraea;however,its biosynthetic cluster lacks a regulatory gene,limiting the yield enhancement via regulation engineering of S.erythraea.Herein,six TetR family transcriptional regulators(TFRs)belonging to three genomic context types were individually inactivated in S.erythraea A226,and one of them,SACE_3446,was proved to play a negative role in regulating erythromycin biosynthesis.EMSA and qRT-PCR analysis revealed that SACE_3446 covering intact N-terminal DNA binding domain specifically bound to the promoter regions of erythromycin biosynthetic gene eryAI,the resistant gene ermE and the adjacent gene SACE_3447(encoding a longchain fatty-acid CoA ligase),and repressed their transcription.Furthermore,we explored the interaction relationships of SACE_3446 and previously identified TFRs(SACE_3986 and SACE_7301)associated with erythromycin production.Given demonstrated relatively independent regulation mode of SACE_3446 and SACE_3986 in erythromycin biosynthesis,we individually and concomitantly inactivated them in an industrial S.erythraea WB.Compared with WB,the WBΔ3446 and WBΔ3446Δ3986 mutants respectively displayed 36%and 65%yield enhancement of erythromycin A,following significantly elevated transcription of eryAI and ermE.When cultured in a 5 L fermentor,erythromycin A ofWBΔ3446 and WBΔ3446Δ3986 successively reached 4095 mg/L and 4670 mg/L with 23%and 41%production improvement relative to WB.The strategy reported here will be useful to improve antibiotics production in other industrial actinomycete.
Beauvericin, a cyclohexadepsipeptide-possessing natural product with synergistic antifungal, insecticidal, and cytotoxic activities. We isolated and characterized the fpBeas gene cluster, devoted to beauvericin biosynthesis, from the filamentous fungus Fusarium proliferatum LF061. Targeted inactivation of the F. proliferatum genomic copy of fpBeas abolished the production of beauvericin. Comparative sequence analysis of the FpBEAS showed 74% similarity with the BbBEAS that synthesizes the cyclic trimeric ester beauvericin in Beauveria bassiana, which assembles N-methyl-dipeptidol monomer intermediates by the programmed iterative use of the nonribosomal peptide synthetase modules. Differences between the organization of the beauvericin loci in F. proliferaturm and B. bassiana revealed the mechanism for high production of beauvericin in F. proliferatum. Our work provides new insights into beauvericin biosynthesis, and may lead to beauvericin overproduction and creation of new analogs via synthetic biology approaches.
The complete genome of methanol-utilizing Amycolatopsis methanolica strain 239T was generated,revealing a single 7,237,391 nucleotide circular chromosome with 7074 annotated protein-coding sequences(CDSs).Comparative analyses against the complete genome sequences of Amycolatopsis japonica strain MG417-CF17T,Amycolatopsis mediterranei strain U32 and Amycolatopsis orientalis strain HCCB10007 revealed a broad spectrum of genomic structures,including various genome sizes,core/quasi-core/non-core configurations and different kinds of episomes.Although polyketide synthase gene clusters were absent from the A.methanolica genome,12 gene clusters related to the biosynthesis of other specialized(secondary)metabolites were identified.Complete pathways attributable to the facultative methylotrophic physiology of A.methanolica strain 239T,including both the mdo/mscR encoded methanol oxidation and the hps/hpi encoded formaldehyde assimilation via the ribulose monophosphate cycle,were identified together with evidence that the latter might be the result of horizontal gene transfer.Phylogenetic analyses based on 16S rDNA or orthologues of AMETH_3452,a novel actinobacterial class-specific conserved gene against 62 or 18 Amycolatopsis type strains,respectively,revealed three major phyletic lineages,namely the mesophilic or moderately thermophilic A.orientalis subclade(AOS),the mesophilic Amycolatopsis taiwanensis subclade(ATS)and the thermophilic A.methanolica subclade(AMS).The distinct growth temperatures of members of the subclades correlated with corresponding genetic variations in their encoded compatible solutes.This study shows the value of integrating conventional taxonomic with whole genome sequence data.
Two potent anti-MRSA tanshinone glycosides(1 and 2)were discovered by targeted microbial biotransformation,along with rapid identification via MS/MS networking.Serial reactions including dehydrogenation,demethylations,reduction,glycosylation and methylation have been observed after incubation of tanshinone IIA and fungus Mucor rouxianus AS 3.3447.In addition,tanshinosides B(2)showed potent activities against serial clinical isolates of oxacillin-resistant Staphylococcus aureus with MIC values of 0.78 mg/mL.This is the first study that shows a significant increase in the level and activities of tanshinone glycosides relative to the substrate tanshinone IIA.
