• Journal of Microbiology and Biotechnology
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• v.31 no.1
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• pp.8-15
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• 2021

More and more available fungal genome sequence data reveal a large amount of secondary metabolite (SM) biosynthetic 'dark matter' to be discovered. Heterogeneous expression is one of the most effective approaches to exploit these novel natural products, but it is limited by having to clone entire biosynthetic gene clusters (BGCs) without errors. So far, few effective technologies have been developed to manipulate the specific large DNA fragments in filamentous fungi. Here, we developed a fungal BGC-capturing system based on CRISPR/Cas9 cleavage in vitro. In our system, Cas9 protein was purified and CRISPR guide sequences in combination with in vivo yeast assembly were rationally designed. Using targeted cleavages of plasmid DNAs with linear (8.5 kb) or circular (8.5 kb and 28 kb) states, we were able to cleave the plasmids precisely, demonstrating the high efficiency of this system. Furthermore, we successfully captured the entire Nrc gene cluster from the genomic DNA of Neosartorya fischeri. Our results provide an easy and efficient approach to manipulate fungal genomic DNA based on the in vitro application of Cas9 endonuclease. Our methodology will lay a foundation for capturing entire groups of BGCs in filamentous fungi and accelerate fungal SMs mining.

• Biotechnology and Bioprocess Engineering:BBE
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• v.10 no.2
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• pp.162-165
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• 2005

A series of fluorine and hydroxyl containing jasmonate derivatives, which were chemically synthesized in our institute, were investigated for their effects on the biosynthesis and heterogeneity of ginsenosides in suspension cultures of Panax notoginseng cells. Com-pared to the control (without addition of elicitors), $100{\mu}M$ of each of the jasmonate was added on day 4 to the suspension cultures of P. notoginseng cells. It was observed that, jasmonates greatly enhanced the ginsenoside content and the ratio of Rb group to Rg group (i.e. $(Rb_1\;+\;Rd)/(Rg_1\;+\;Re)$ in the P. notoginseng cells. Some of the synthetic jasmonates, such as pentafluoropropyl jasmonate (PFPJA), 2-hydroxyethyl jasmonate (HEJA) and 2-hydroxye-thoxyethyl jasmonate (HEEJA), could promote the ginsenoside content to $2.55\;\pm\;0.11,\;3.65\;\pm\;0.13\;and\;2.94\;\pm\;0.06$mg/100 mg DW, respectively, compared to that of $0.64\;\pm\;0.06$mg/100 mg DW for the control and $2.17\;\pm\;0.04$ mg/100 mg DW by the commercially available methyl jasmonate (MJA); and they could change the respective Rb:Rg ratio to $1.60\;\pm\;0.04,\;1.87\;\pm\;0.01\;and\;1.56\;\pm\;0.05$, compared to that of $0.47\;\pm\;0.01$ for the control and $1.42\;\pm\;0.06$ by MJA. The results suggest that suitable esterification of MJA with fluorine or hydroxyl group could in-crease the elicitation activity to induce plant secondary metabolism. The information obtained from this study is useful for hyper-production of heterogeneous products by plant cell cultures.