The role of swnR gene on the biosynthesis pathway of the swainsonine in Metarhizium anisopliae

Swainsonine (SW) is the principal toxic ingredient of locoweeds, and is produced by fungi including Metarhizium anisopliae, Slafractonia leguminicola, and Alternaria oxytropis. Studies of the SW biosynthesis pathway in these fungi have demonstrated the requirement for a swnK gene and the presence of a variety of other SWN cluster genes, but have not determined a precise role for the swnR gene, which encodes a NADB Rossmann-fold reductase, nor if it is necessary for the biosynthesis of SW. In this study, we used homologous recombination (HR) to knock out the swnR gene of M. anisopliae to determine its effect on the SW biosynthesis pathway. The concentration of SW was measured in the fermentation broth of M. anisopliae at 1 d, 3 d, 5 d and 7 d using a Q Exactive Mass Spectrometer. The gene for swnR was detected by RT-qPCR. To determine the role of the swnR gene in the SW biosynthesis pathway of M. anisopliae, we used PEG-mediated homologous recombination (HR) to transform a wild-type strain (WT) with a Benomyl (ben)-resistant fragment to knock out the swnR gene producing a mutant-type strain (MT). A complemented-type (CT) strain was produced by adding a complementation vector that contains the glufosinate (herbicide) resistance (bar) gene as a marker. The content of SW decreased, but was not eliminated in the fermentation broth of the MT strain, and returned to the original level in the CT strain. These results indicate that the swnR gene plays a crucial role in the SW biosynthesis pathway of M. anisopliae, but suggests that another gene in the fungus may share the function of swnR.


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Metarhizium anisopliae is anentomopathogenic fungus that can infect a variety of 3 45 agricultural pest insects and produces swainsonine [1][2][3]. Swainsonine is an 46 indolizidine alkaloid that inhibits alpha-mannosidases. Swainsonine is produced by a  The precise roles for the other genes in the SWN cluster were not determined. 65 The aims of this study were to investigate the role of the swnR gene in the SW from M. anisopliae DNA using L1/R1 primers (Text S1).  andText S1).

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The primers L3 and R3 (Table S1) were used to amplify the ben resistance gene 100 from pBARGPE1-BenA (Wuhan Jingxiu Scientific Biotechnology Co., Ltd., China) 101 as a template. The primers L2/R2 and L4/R4 (Table S1) were used to amplify the 102 upstream target fragment (swnR-I) and the downstream target fragment (swnR-II), 103 respectively, of the swnR gene from the genomic DNA of M. anisopliae. The swnR-I, 104 ben, swnR-II and the double-cut pUC19 vector were ligated using In-Fusion cloning.

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Screening for optimal conditions for protoplast preparation 199 We explored the conditions affecting the preparation of protoplasts of M. anisopliaeto  (Fig.4C and4D). Subsequently, the L1/R1 primer set was used to identify the genomic DNA of the 216 transformant (MT) using electrophoresis and sequencing ( Fig.1 andText S2). To 217 verify the status of MT, a complement was produced by transforming the wild-type 218 swnR gene in pBARGPE1 into the MT. The complement transformant was grown on 219 SDA medium containing 2 mg/mL glufosinate and was identified as above. phenotypes, and growth rates did not change significantly (Fig.5B).  (Fig.S8A), 56.42 ± 10.82 μg/mg for CT (Fig.S8C) and 5.71± 2.23 232 μg/mg for MT (Fig.S8B). The content of SW in the fermentation broth of the MT was 233 significantly lower than that in the complemented strain and WT (Fig.6).

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[17] found that the swnK MT did not produce SW at all. However, in this study, after 246 the swnR gene was knocked out, the content of SW was reduced, rather than 247 completely absent. The cause of the low SW content may be due to the presence of a 248 catalytic enzyme gene having the same function as the swnR gene. Since swnN is also 249 a Rossman fold reductase, and is present in all SW-producing fungi, this could 250 provide a similar activity and thus explain why SW was decreased rather than 251 eliminated.

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To demonstrate that the decrease in the content of SW was caused by the knock  (Fig. 7). This study provides a preliminary             The protoplast release of M. anisopliae was the highest when digested with 1% snail 422 enzyme, 1% cellulase and 1% lysing enzymes for 3 h. Each data in the graph is the 423 mean ± SEM, n = 3, P<0.05. CT and WT. The content of SW in the fermentation broth of the WT was significantly 430 lower than that in CT and wild-type strain, and there was a significant difference.