Developing enzyme cocktails for cellulosic biomass hydrolysis complementary to current cellulase systems is a critical step needed for economically viable biofuels production. Recent genomic analysis indicates that some plant pathogenic fungi are likely a largely untapped resource in which to prospect for novel hydrolytic enzymes for biomass conversion. In order to develop high throughput screening assays for enzyme bioprospecting, a standardized microplate assay was developed for rapid analysis of polysaccharide hydrolysis by fungal extracts, incorporating biomass substrates. Fungi were grown for 10 days on cellulose-or switchgrass-containing media to produce enzyme extracts for analysis. Reducing sugar released from filter paper, Avicel, corn stalk, switchgrass, carboxymethylcellulose, and arabinoxylan was quantified using a miniaturized colorimetric assay based on 3,5-dinitrosalicylic acid. Significant interactions were identified among fungal species, growth media composition, assay substrate, and temperature. Within a small sampling of plant pathogenic fungi, some extracts had crude activities comparable to or greater than T. reesei, particularly when assayed at lower temperatures and on biomass substrates. This microplate assay system should prove useful for high-throughput bioprospecting for new sources of novel enzymes for biofuel production.

The yeast Kluyveromyces marxianus grows at high temperatures and on a wide range of carbon sources, making it a promising host for industrial biotechnology to produce renewable chemicals from plant biomass feedstocks. However, major genetic engineering limitations have kept this yeast from replacing the commonly used yeast Saccharomyces cerevisiae in industrial applications. Here, we describe genetic tools for genome editing and breeding K. marxianus strains, which we use to create a new thermotolerant strain with promising fatty acid production. These results open the door to using K. marxianus as a versatile synthetic biology platform organism for industrial applications.

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Advances in enzyme stabilization and immobilization make the use of enzymes for industrial applications increasingly feasible. The lactoperoxidase (LPO) system is a naturally occurring enzyme system with known antimicrobial activity. Stabilized LPO and glucose oxidase (GOx) enzymes were combined with glucose, potassium iodide, and ammonium thiocyanate to create an anti-fungal formulation, which inhibited in-vitro growth of the plant pathogenic oomycete Pythium ultimum, and the plant pathogenic fungi Fusarium graminearum and Rhizoctonia solani. Pythium ultimum was more sensitive than F. graminearum and R. solani, and was killed at LPO and GOx concentrations of 20 nM and 26 nM, respectively. Rhizoctonia solani and F. graminearum were 70% to 80% inhibited by LPO and GOx concentrations of 242 nM and 315 nM, respectively. The enzyme system was tested for compatibility with five commercial fungicides as co-treatments. The majority of enzyme + fungicide co-treatments resulted in additive activity. Synergism ranging from 7% to 36% above the expected additive activity was observed when P. ultimum was exposed to the enzyme system combined with Daconil ® (active ingredient (AI): chlorothalonil 29.6%, GardenTech, Lexington, KY, USA), tea tree oil, and mancozeb at select fungicide concentrations. Antagonism was observed when the enzyme system was combined with Tilt ® (AI: propiconazole 41.8%, Syngenta, Basel, Switzerland) at one fungicide concentration, resulting in activity 24% below the expected additive activity at that concentration.