Partial secretome analysis of Caldariomyces fumago reveals extracellular production of the CPO co-substrate H2O2 and provides a coproduction concept for CPO and glucose oxidase.

The culture supernatant of Caldariomyces fumago strains grown in a minimal medium with fructose contains mainly the biotechnologically relevant enzyme chloroperoxidase (CPO) and only minor amounts of other proteins. Our approach to identify the nature of these proteins via peptide mass fingerprinting and transcriptome analysis demonstrated the presence of putative glycosyl hydrolase and glucose oxidase (GOx) enzymes. These activities had been described earlier as parts of the fungus´ halogenation machinery, as they provide CPO with the co-substrate H2O2. The GOx activity was found to have a pH optimum of 5. Compared to the wild type values, GOx activity and glucose-driven MCD chlorination activity in the culture of a white mutant were found to be strongly increased to values of 1-2 U mL-1. As most CPO-catalyzed peroxidation reactions also show pH optima at around 5, the C. fumago culture supernatant can provide a highly convenient CPO/GOx source for many reactions with in situ H2O2 production.

Caldariomyces fumago DSM1256 Contains Two Chloroperoxidase Genes, Both Encoding Secreted and Active Enzymes.

Inspection of transcriptome data from the chloroperoxidase (CPO)-producing fungus Caldariomyces fumago DSM1256 led to the discovery of two distinct CPO mRNA sequences. This strain could be shown to contain the newly identified isogene as well as produce and secrete both isoenzymes. The CPO2 enzyme bears high sequence similarity to the well-characterized CPO (87% identity for the mature proteins). It shows two insertions in the signal peptide and in the C-terminal propeptide, and one deletion in the mature polypeptide close to the C-terminus. Furthermore, it lacks one of the serine residues known to be O-glycosylated in the CPO sequence. The demonstration of a CPO isogene which is expressed as a secreted and active CPO clarifies the nature of this isoenzyme already identified in earlier reports. A structure model comparison shows a high conservation of the active site and the substrate channel, suggesting very similar catalytic properties.

Identification of a Caldariomyces fumago mutant secreting an inactive form of chloroperoxidase lacking the heme group and N-glycans.

By mutant colony screening of Caldariomyces fumago a mutant was isolated which was slightly greenish on fructose minimal medium and grew slower in comparison to the wild type. The supernatant samples lacked the Soret band typical for the heme group of the CPO and nearly no CPO activity was detected. SDS-PAGE analysis of mutant culture supernatant samples showed production of a 38-40 kDa protein while wild type samples contain the 42 kDa CPO protein. Protein identification using nanoLC-ESI-MS/MS was performed and based on three peptides the protein in the mutant culture was identified as CPO. No differences in the CPO gene sequences of wild type and mutant were found indicating a post-translational defect in protein maturation. Deglycosylation experiments using CPO from wild type and mutant were carried out. After removing N-linked oligosaccharides from wild type CPO a protein band at 38-40 kDa was detected. Our results reveal that the mutant protein lacks the heme group as well as the N-glycans.

Enzymatic production and in situ separation of natural ß-ionone from ß-carotene.

A biotechnological process concept for generation and in situ separation of natural ß-ionone from ß-carotene is presented. The process employs carotenoid cleavage dioxygenases (CCDs), a plant-derived iron-containing nonheme enzyme family requiring only dissolved oxygen as cosubstrate and no additional cofactors. Organophilic pervaporation was found to be very well suited for continuous in situ separation of ß-ionone. Its application led to a highly pure product despite the complexity of the reaction solution containing cell homogenates. Among three different pervaporation membrane types tested, a polyoctylmethylsiloxane active layer on a porous polyetherimide support led to the best results. A laboratory-scale demonstration plant was set up, and a highly pure aqueous-ethanolic solution of ß-ionone was produced from ß-carotene. The described process permits generation of high-value flavor and fragrance compounds bearing the desired label "natural" according to US and European food and safety regulations and demonstrates the potential of CCD enzymes for selective oxidative cleavage of carotenoids.

White mutants of chloroperoxidase-secreting Caldariomyces fumago as superior production strains, revealing an interaction between pigmentation and enzyme secretion.

By mutant-colony screening of Caldariomyces fumago, several white mutants were isolated that are superior strains for the production of the valuable enzyme chloroperoxidase (CPO). Their culture supernatant lacks the contaminating dark pigment, which simplifies downstream processing. Furthermore, the CPO content increased significantly faster than the wild-type rate, which uncovers possible interactions between pigmentation and enzyme secretion.

Over-expression of chloroperoxidase in Caldariomyces fumago.

The filamentous fungus Caldariomyces fumago secretes a chloroperoxidase (CPO). To increase its production, we integrated a CPO-expression cassette into the non-transcribed spacer regions of the rDNA in C. fumago. One strain was obtained that had twice the CPO activity when grown in shake-flask and bioreactor compared to the wild-type. The highest CPO activity from the bioreactor cultivation was 3,236 U ml(-1). This is the highest value reported so far.