Design of an Aspergillus nidulans cell factory for the production of the natural food pigment carmine based on an artificial type II/III PKS system

Rasmus J.N. Frandsen 1 Paiman Khorsand-Jamal 1 Kenneth T. Kongstad 3 Majse Nafisi 2 Rubini M. Kannangara 2 Dan Staerk 3 Finn Okkels 2 Kim Binderup 2 Bjørn Madsen 2 Birger Lindberg Møller 4 Ulf Thrane 1 Uffe H. Mortensen 1
1Bioengineering, The Technical University of Denmark, Kgs. Lyngby, Denmark
2Natural Colors, Chr. Hansen A/S, Hoersholm, Denmark
3Drug Design and Pharmacology, University of Copenhagen, Copenhagen, Denmark
4Plant and Environmental Sciences, University of Copenhagen, Frederiksberg, Denmark

The natural red food pigments carmine (E120/cochineal) and carminic acid have been utilized by humans since 3000 BC. Industrial production of the pigments are based on extraction from the scale insect Dactylopius coccus. The market value of carmine is ~200 million USD/year, however, the market price experiences large year-to-year fluctuations due to changing production levels. Carminic acid is a glucosylated non-reduced aromatic polyketide that includes an octaketide with a C2-C15/C5-C14/C7-C12 backbone fold. The natural biosynthetic pathway remains enigmatic, and only a single enzyme, a C-glucosyltransferase (DcUGT2) responsible for the last step in the pathway, has been identified. We can here present the first fermentable microbial cell factory for the production of carminic acid, the dye component of carmine. The major challenge for construction of the cell factory was the lack of known natural PKS systems for forming the polyketide core of carminic acid. However, de novo biosynthesis was achieved by constructing an artificial PKS system, by combining a plant type III PKS (OKS) with an aromatase (ZhuI) and a cyclase (ZhuJ) from a bacterial type II PKS system. Co-expression of the three genes in Aspergillus nidulans resulted in production of the desired aromatic structure (flavokermesic acid anthrone). Further analysis of the strain revealed the production of flavokermesic acid and kermesic acid, both known downstream intermediates in the natural D. coccus pathway, showing that endogenous A. nidulans monooxygenases can catalyze several essential steps of the pathway. Final conversion to carminic acid was achieved by introducing DcUGT2. The artificial PKS system represents the first successful hyphenation of type II and III PKS systems. This synthetic biological solution can be generalized to form the first programmable PKS platform which allows for the production of a wide variety of different aromatic polyketides, by offering control over polyketide chain length and folding pattern.