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

Rasmus J N Frandsen,Birger Lindberg Møller,Majse Nafisi,Bjørn Madsen,Ulf Thrane,Kenneth T Kongstad,Kim Binderup,Finn T Okkels,Rubini M Kannangara,Uffe H Mortensen,Paiman Khorsand-Jamal,Dan Staerk

doi:10.1038/s41598-018-30816-9

Abstract

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.

Highlights

Towards construction of a microbial cell factory for carminic acid production, we have previously shown that the first intermediate in the carminic acid synthesis is the polyketide flavokermesic acid[7] and that the glucose moiety of carminic acid is added by an endoplasmatic reticulum (ER) membrane-bound C-glucosyltransferase, UGT28
Chiang et al have previously demonstrated that deletion of the gene clusters that are responsible for formation of the major endogenous polyketide synthase (PKS) products in A. nidulans[11], improved the potential of A. nidulans as a cell factory for heterologous production of polyketides
As a first step towards a fungal cell factory for carminic acid production, we eliminated the biosynthetic pathways for the dominating aromatic polyketides that could potentially interfere with carminic acid production and complicate the analysis and downstream purification

Summary

Introduction

Towards construction of a microbial cell factory for carminic acid production, we have previously shown that the first intermediate in the carminic acid synthesis is the polyketide flavokermesic acid[7] and that the glucose moiety of carminic acid is added by an endoplasmatic reticulum (ER) membrane-bound C-glucosyltransferase, UGT28. In the absence of the natural PKS, we have adopted an alternative strategy for constructing a microbial cell factory for carminic acid production. We explored the possibility of establishing a semi-natural carminic acid pathway in the filamentous fungus Aspergillus nidulans, a well-characterized producer of a wide range of aromatic polyketides, using a synthetic biology approach. Using this strategy we have established proof-of-concept of stable microbial production of carminic acid

Methods

Results

Conclusion