Bjørn Madsen

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-β-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.

Characterization of a membrane-bound C-glucosyltransferase responsible for carminic acid biosynthesis in Dactylopius coccus Costa

Carminic acid, a glucosylated anthraquinone found in scale insects like Dactylopius coccus, has since ancient times been used as a red colorant in various applications. Here we show that a membrane-bound C-glucosyltransferase, isolated from D. coccus and designated DcUGT2, catalyzes the glucosylation of flavokermesic acid and kermesic acid into their respective C-glucosides dcII and carminic acid. DcUGT2 is predicted to be a type I integral endoplasmic reticulum (ER) membrane protein, containing a cleavable N-terminal signal peptide and a C-terminal transmembrane helix that anchors the protein to the ER, followed by a short cytoplasmic tail. DcUGT2 is found to be heavily glycosylated. Truncated DcUGT2 proteins synthesized in yeast indicate the presence of an internal ER-targeting signal. The cleavable N-terminal signal peptide is shown to be essential for the activity of DcUGT2, whereas the transmembrane helix/cytoplasmic domains, although important, are not crucial for its catalytic function.Carminic acid is a widely applied red colorant that is still harvested from insects because its biosynthesis is not fully understood. Here, the authors identify and characterize a membrane-bound C-glucosyltransferase catalyzing the final step during carminic acid biosynthesis.

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.

Patient education for alcohol cessation intervention at the time of acute fracture surgery: study protocol for a randomised clinical multi-centre trial on a gold standard programme (Scand-Ankle)

BackgroundPatients with hazardous alcohol intake are overrepresented in emergency departments and surgical wards. These patients have an increased risk of postoperative complications with prolonged hospital stays and admissions to intensive care unit after surgery. In elective surgery, preoperative alcohol cessation interventions can reduce postoperative complications, but no studies have investigated the effect of alcohol cessation intervention at the time of acute fracture surgery. This protocol describes a randomised clinical trial that aims to evaluate the effect of a new gold standard programme for alcohol cessation intervention in the perioperative period regarding postoperative complications, alcohol intake and cost-effectiveness.Methods/DesignPatients with hazardous alcohol intake undergoing ankle fracture surgery will be recruited into the trial from multiple orthopaedic wards at university hospitals in Denmark, Sweden and Norway. Included patients will be randomly allocated to either standard care or the gold standard programme aimed at complete alcohol abstinence before, during and 6 weeks after surgery. It includes a structured patient education programme and weekly interventions meetings at the orthopaedic outpatient clinic. Furthermore, patients are provided with thiamine and B-vitamins, alcohol withdrawal prophylaxis and treatment, and disulfiram to support abstinence. Alcohol intake is biochemically validated (blood, urine and breath tests) at the weekly intervention meetings and follow-up visits. Follow-up assessments will be conducted 6 weeks and 3, 6, 9 and 12 months after surgery for all patients. The effect of the gold standard programme will be assessed comparing the outcome measures between the intervention and control group at each follow-up point.DiscussionThe study will provide new knowledge about how to prevent alcohol-related postoperative complications at the time of acute fracture surgery. If effective, the results will be a benefit for the clinical course, patients and society alike.Trial registrationThe protocol is registered in ClinicalTrials.gov (Id: NCT00986791).