Heterologous production of the widely used natural food colorant carminic acid in Aspergillus nidulans.

The natural red food colorants carmine (E120) and carminic acid are currently produced from scale insects. The access to raw material is limited and current production is sensitive to fluctuation in weather conditions. A cheaper and more stable supply is therefore desirable. Here we present the first proof-of-concept of heterologous microbial production of carminic acid in Aspergillus nidulans by developing a semi-natural biosynthetic pathway. Formation of the tricyclic core of carminic acid is achieved via a two-step process wherein a plant type III polyketide synthase (PKS) forms a non-reduced linear octaketide, which subsequently is folded into the desired flavokermesic acid anthrone (FKA) structure by a cyclase and a aromatase from a bacterial type II PKS system. The formed FKA is oxidized to flavokermesic acid and kermesic acid, catalyzed by endogenous A. nidulans monooxygenases, and further converted to dcII and carminic acid by the Dactylopius coccus C-glucosyltransferase DcUGT2. The establishment of a functional biosynthetic carminic acid pathway in A. nidulans serves as an important step towards industrial-scale production of carminic acid via liquid-state fermentation using a microbial cell factory.

On the biosynthetic origin of carminic acid.

The chemical composition of the scale insect Dactylopius coccus was analyzed with the aim to discover new possible intermediates in the biosynthesis of carminic acid. UPLC-DAD/HRMS analyses of fresh and dried insects resulted in the identification of three novel carminic acid analogues and the verification of several previously described intermediates. Structural elucidation revealed that the three novel compounds were desoxyerythrolaccin-O-glucosyl (DE-O-Glcp), 5,6-didehydroxyerythrolaccin 3-O-ß-D-glucopyranoside (DDE-3-O-Glcp), and flavokermesic acid anthrone (FKA). The finding of FKA in D. coccus provides solid evidence of a polyketide, rather than a shikimate, origin of coccid pigments. Based on the newly identified compounds, we present a detailed biosynthetic scheme that accounts for the formation of carminic acid (CA) in D. coccus and all described coccid pigments which share a flavokermesic acid (FK) core. Detection of coccid pigment intermediates in members of the Planococcus (mealybugs) and Pseudaulacaspis genera shows that the ability to form these pigments is taxonomically more widely spread than previously documented. The shared core-FK-biosynthetic pathway and wider taxonomic distribution suggests a common evolutionary origin for the trait in all coccid dye producing insect species.

Genetic transformation of Fusarium avenaceum by Agrobacterium tumefaciens mediated transformation and the development of a USER-Brick vector construction system.

BACKGROUND: The plant pathogenic and saprophytic fungus Fusarium avenaceum causes considerable in-field and post-field losses worldwide due to its infections of a wide range of different crops. Despite its significant impact on the profitability of agriculture production and a desire to characterize the infection process at the molecular biological level, no genetic transformation protocol has yet been established for F. avenaceum. In the current study, it is shown that F. avenaceum can be efficiently transformed by Agrobacterium tumefaciens mediated transformation. In addition, an efficient and versatile single step vector construction strategy relying on Uracil Specific Excision Reagent (USER) Fusion cloning, is developed. RESULTS: The new vector construction system, termed USER-Brick, is based on a limited number of PCR amplified vector fragments (core USER-Bricks) which are combined with PCR generated fragments from the gene of interest. The system was found to have an assembly efficiency of 97% with up to six DNA fragments, based on the construction of 55 vectors targeting different polyketide synthase (PKS) and PKS associated transcription factor encoding genes in F. avenaceum. Subsequently, the ΔFaPKS3 vector was used for optimizing A. tumefaciens mediated transformation (ATMT) of F. avenaceum with respect to six variables. Acetosyringone concentration, co-culturing time, co-culturing temperature and fungal inoculum were found to significantly impact the transformation frequency. Following optimization, an average of 140 transformants per 106 macroconidia was obtained in experiments aimed at introducing targeted genome modifications. Targeted deletion of FaPKS6 (FA08709.2) in F. avenaceum showed that this gene is essential for biosynthesis of the polyketide/nonribosomal compound fusaristatin A. CONCLUSION: The new USER-Brick system is highly versatile by allowing for the reuse of a common set of building blocks to accommodate seven different types of genome modifications. New USER-Bricks with additional functionality can easily be added to the system by future users. The optimized protocol for ATMT of F. avenaceum represents the first reported targeted genome modification by double homologous recombination of this plant pathogen and will allow for future characterization of this fungus. Functional linkage of FaPKS6 to the production of the mycotoxin fusaristatin A serves as a first testimony to this.