Entomopathogenic fungi utilize specific secondary metabolites to defend against insect immunity, thereby enabling colonization of their specific hosts. We are particularly interested in the polyketide synthesis gene pks15, which is involved in metabolite production, and its role in fungal virulence. targeted disruption of pks15 followed by genetic complementation with a functional copy of the gene would allow for functional characterization of this secondary metabolite biosynthesis gene. Using a Beauveria bassiana ∆pks15 mutant previously disrupted by a bialophos-resistance (bar) cassette, we report here an in-cis complementation at bar cassette using CRISPR/Cas9 gene editing. A barspecific short guide RNA was used to target and cause a double-strand break in bar, and a donor DnA carrying a wild-type copy of pks15 was co-transformed with the guide RNA. Isolate G6 of ∆pks15 complemented with pks15 was obtained and verified by PCR, Southern analyses and DNA sequencing. Compared to ∆pks15 which showed a marked reduction in sporulation and insect virulence, the complementation in G6 restored with insect virulence, sporulation and conidial germination to wildtype levels. Atomic force and scanning electron microscopy revealed that G6 and wild-type conidial wall surfaces possessed the characteristic rodlet bundles and rough surface while ∆pks15 walls lacked the bundles and were relatively smoother. Conidia of ∆pks15 were larger and more elongated than that of G6 and the wild type, indicating changes in their cell wall organization. Our data indicate that PKS15 and its metabolite are likely not only important for fungal virulence and asexual reproduction, but also cell wall formation. Beauveria bassiana, an entomopathogenic fungus, has a broad host spectrum and is considered to have high potential for insect biocontrol in agriculture. While B. bassiana can cause mycosis in several insect species 1,2 , insect killing is fairly slow due to several limiting factors, particularly in the field. The fungus is also vulnerable to environmental stress factors such as UV radiation, high temperature and drought 3. A better understanding of the biological and physiological characteristics of this entomopathogen should allow us to improve its virulence and stress tolerance. Secondary metabolites are abundant in entomopathogenic fungi and include polyketides, nonribosomal peptides, terpenes and alkaloids that play important roles in various aspects of the fungal life cycle. B. bassiana BCC2660, a widely used biocontrol fungus in Thailand, has 12 polyketide synthase (PKS) genes in its genome 4. Two PKS genes, pks15 and pks14, have crucial roles in virulence against insects, as previously demonstrated by targeted gene deletion 4,5. The pks15 mutant exhibits loss in phagocytic survival ability, a phenotype likely associated with changes in the cell wall, the outermost layer of fungal conidia. Unfortunately, little is known regarding the relationship between polyketides and the fungal wall. In a few reports, melanin, the metabolite of a n...

An enzymatic method for specific determination of stevioside content was established. Recombinant β-glucosidase BT_3567 (rBT_3567) from Bacteroides thetaiotaomicron HB-13 exhibited selective hydrolysis of stevioside at β-1,2-glycosidic bond to yield rubusoside and glucose. Coupling of this enzyme with glucose oxidase and peroxidase allowed for quantitation of stevioside content in Stevia samples by using a colorimetric-based approach. The series of reactions for stevioside determination can be completed within 1 h at 37 °C. Stevioside determination using the enzymatic assay strongly correlated with results obtained from HPLC quantitation (r = 0.9629, n = 16). The percentages of coefficient variation (CV) of within day (n = 12) and between days (n = 12) assays were lower than 5%, and accuracy ranges were 95-105%. This analysis demonstrates that the enzymatic method developed in this study is specific, easy to perform, accurate, and yields reproducible results.

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