High-throughput assay for effect screening of amphotericin B and bioactive components on filamentous Candida albicans.

AIMS: The aim of this study was to develop a high-throughput robotic microtiter plate-based screening assay for Candida albicans, optimizing growth conditions to replicate the filamentous biofilm growth found in vivo, and subsequently, to demonstrate the assay by evaluating the effect of nutritional drinks alone and in combination with the antifungal amphotericin B (AmB). METHODS AND RESULTS: Candida albicans cultured in a defined growth medium showed filamentous growth in microcolonies, mimicking the morphology of oral mucosal disease (oral candidiasis). Addition of nutrient drinks containing fruit juices, fish oil and whey protein to the medium resulted in changed morphology and promoted growth as free yeast cells and with weak biofilm structures. Minimum inhibitory concentration of AmB on the biofilms was 0.25 µg ml-1 , and this was eightfold reduced (0.0038 µg ml-1 ) in the presence of the nutritional drinks. CONCLUSIONS: The established assay demonstrated applicability for screening of antifungal and anti-biofilm effects of bioactive substances on C. albicans biofilm with clinically relevant morphology. SIGNIFICANCE AND IMPACT OF THE STUDY: Candida albicans is the causative agent of the majority of fungal infections globally. The filamentous morphology of C. albicans and the ability to form biofilm are traits known to increase virulence and resistance towards antifungals. This study describes the development of a plate-based in vitro screening method mimicking the filamentous morphology of C. albicans found in vivo. The assay established can thus facilitate efficient antifungal drug discovery and development.

Engineering chitinolytic activity into a cellulose-active lytic polysaccharide monooxygenase provides insights into substrate specificity.

Lytic polysaccharide monooxygenases (LPMOs) catalyze oxidative cleavage of recalcitrant polysaccharides such as cellulose and chitin and play an important role in the enzymatic degradation of biomass. Although it is clear that these monocopper enzymes have extended substrate-binding surfaces for interacting with their fibrous substrates, the structural determinants of LPMO substrate specificity remain largely unknown. To gain additional insight into substrate specificity in LPMOs, here we generated a mutant library of a cellulose-active family AA10 LPMO from Streptomyces coelicolor A3(2) (ScLPMO10C, also known as CelS2) having multiple substitutions at five positions on the substrate-binding surface that we identified by sequence comparisons. Screening of this library using a newly-developed MS-based high-throughput assay helped identify multiple enzyme variants that contained four substitutions and exhibited significant chitinolytic activity and a concomitant decrease in cellulolytic activity. The chitin-active variants became more rapidly inactivated during catalysis than a natural chitin-active AA10 LPMO, an observation likely indicative of suboptimal substrate binding leading to autocatalytic oxidative damage of these variants. These results reveal several structural determinants of LPMO substrate specificity and underpin the notion that productive substrate binding by these enzymes is complex, depending on a multitude of amino acids located on the substrate-binding surface.

Engineering lytic polysaccharide monooxygenases (LPMOs).

Lytic polysaccharide monooxygenases (LPMOs) are mono-copper enzymes that catalyze the hydroxylation of glycosidic bonds found in the most abundant and recalcitrant polysaccharides on Earth. Since their discovery in 2010, these enzymes have received extensive attention in both fundamental and applied research due to their remarkable oxidative power and synergistic interplay with hydrolytic enzymes. The harsh and unnatural conditions used in industrial enzymatic saccharification processes and the sensitivity of LPMOs for damage induced by reactive oxygen species call for enzyme engineering to develop LPMOs to become robust industrial biocatalysts. Other engineering targets include improved catalytic activity, adjusted substrate specificity and the introduction of completely new activities. Reaching these targets not only requires appropriate methods for measuring enzyme activity, but also requires in-depth knowledge of the active site and the reaction mechanism, which is yet to be achieved in the LPMO field. Here we describe what has been done in the LPMO engineering field so far. Furthermore, we address the difficulties involved in properly assessing LPMO functionality, which are due to common side reactions taking place in LPMO reactions and which complicate screening methods.