Enhancement of Carrimycin Production Via Traditional Mutagenesis with Metabolic Engineering in Streptomyces Spiramyceticus 54IA

Background: Carrimycin is a new approved class I antibiotic in China. The novel carrimycin producing strain, Streptomyces spiramyceticus 54IA, was constructed by CRISPR-Cas9 editing system without insertion of antibiotics resistant gene. The problem of low yield limits this strain in large scale fermentation. In this study, the carrimycin production was significantly improved by strain mutagenesis coupled metabolic engineering. Results: The sspD gene is responsible for degradation of triacylglycerol to provide precursors of the polyketide biosynthesis. The extra sspD gene controlled by the promoters of pks and bsm42 genes could moderately enhance carrimycin production. The Bsm42 was identified to play a pathway-specific positive regulator for carrimycin biosynthesis. Due to production of carrimycin significantly enhanced by bsm42 overexpression, the two different length promoters of bsm42 individually ligated with two reporter genes were used to monitor bsm42 expression for screening the higher carrimycin production mutants treated by plasma and ultraviolet. 47% of the 608 selected mutants had higher fermentation titer than the starting strain. The shorter promoter of bsm42 displayed more appropriate for selection of the carrimycin production improved mutants. The F2R-15 mutant had highest titer (1010±30 μg/mL), which was about 9 times higher than that of 54IA strain. Comparative analysis of transcriptome profiles of F2R-15 mutant and 54IA strains found 158 differential expression genes with more than 2 fold-changes. The up-regulated genes were associated with macrolide precursor biosynthesis, macrolide-inactivation, antibiotics transporter, oxidative phosphorylation; while the most down-regulated genes were referring to the primary metabolites synthetic genes and biosynthetic genes of other secondary metabolites. Conclusion: These results suggested that manipulation of the positive regulatory gene bsm42 and traditional mutagenesis coupled with reporter-guided mutant selection method facilitated selection of carrimycin high-yielding mutants.

Biosynthesis of fungal polyketides by collaborating and trans-acting enzymes

Investigations into fungal polyketide biosynthesis have revealed many examples of megasynthases and trans-acting accessory enzymes. This review collates the different classes of collaborating enzymes, demonstrating common themes and rarer examples.

Discovery of Mycothiogranaticins from Streptomyces vietnamensis GIMV4.0001 and the Regulatory Effect of Mycothiol on the Granaticin Biosynthesis

Granaticins are benzoisochromanequinone polyketides with remarkable antibacterial and anticancer activities. Three sulfur-containing granaticin congeners, mycothiogranaticins A (1), B (2) and granaticin MA (3) were discovered from a granaticin-producing strain of Streptomyces vietnamensis GIMV4.0001. Two of them were structurally determined with mycothiol or N-acetylcysteine moieties and found to be bio-actively reluctant. Disruption of the mshA gene (SVTN_RS20640) that encodes the D-inositol-3-phosphate glycosyltransferase crucial for mycothiol biosynthesis, fully abolished the production of mycothiogranaticins. The result substantiated that the newly discovered mycothiogranaticins are consequences of the combination of the granaticin and mycothiol biosynthetic pathways. The overall granaticin production of the ΔmshA mutant strain was unexpectedly decreased by at least more than 50%, while similar production level of granaticins to that of the wild type strain was observed in an mycothiol-S transferase gene (SVTN_RS22215) disruptant Δmst. These results indicated that the mycothiol deficiency was responsible for the decreased production of granaticins. Mycothiol may positively regulate the biosynthesis of granaticin possibly by maintaining the cellular redox balance. To the best of our knowledge, this is the first report that mycothiol can not only be a direct building block of polyketides but also play a regulatory role in the polyketide biosynthesis.

Conservation of griseofulvin genes in the gsf gene cluster among fungal genomes

The polyketide griseofulvin is a natural antifungal compound and research in griseofulvin has been key in establishing our current understanding of polyketide biosynthesis. Nevertheless, the griseofulvin gsf biosynthetic gene cluster (BGC) remains poorly understood in most fungal species, including Penicillium griseofulvum where griseofulvin was first isolated. To elucidate essential genes involved in griseofulvin biosynthesis, we performed third-generation sequencing to obtain the genome of Penicillium griseofulvum strain D-756. Furthermore, we gathered publicly available genome of 11 other fungal species in which gsf gene cluster was identified. In a comparative genome analysis, we annotated and compared the gsf BGC of all 12 fungal genomes. Our findings show no gene rearrangements at the gsf BGC. Furthermore, seven gsf genes are conserved by most genomes surveyed whereas the remaining six were poorly conserved. This study provides new insights into differences between gsf BGC and suggests that seven gsf genes are essential in griseofulvin production.

Initiating polyketide biosynthesis by on-line methyl esterification

Aurantinins (ARTs) are antibacterial polyketides featuring a unique 6/7/8/5-fused tetracyclic ring system and a triene side chain with a carboxyl terminus. Here we identify the art gene cluster and dissect ART’s C-methyl incorporation patterns to study its biosynthesis. During this process, an apparently redundant methyltransferase Art28 was characterized as a malonyl-acyl carrier protein O-methyltransferase, which represents an unusual on-line methyl esterification initiation strategy for polyketide biosynthesis. The methyl ester bond introduced by Art28 is kept until the last step of ART biosynthesis, in which it is hydrolyzed by Art9 to convert inactive ART 9B to active ART B. The cryptic reactions catalyzed by Art28 and Art9 represent a protecting group biosynthetic logic to render the ART carboxyl terminus inert to unwanted side reactions and to protect producing organisms from toxic ART intermediates. Further analyses revealed a wide distribution of this initiation strategy for polyketide biosynthesis in various bacteria.