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

Abstract

21Swainsonine (SW) is the principal toxic ingredient of locoweeds, and is produced by 22 fungi including Metarhizium anisopliae, Slafractonia leguminicola, and Alternaria 2 23 oxytropis. Studies of the SW biosynthesis pathway in these fungi have demonstrated 24 the requirement for a swnK gene and the presence of a variety of other SWN cluster 25 genes, but have not determined a precise role for the swnR gene, which encodes a 26 NADB Rossmann-fold reductase, nor if it is necessary for the biosynthesis of SW. In 27 this study, we used homologous recombination (HR) to knock out the swnR gene of 28 M. anisopliae to determine its effect on the SW biosynthesis pathway. The 29 concentration of SW was measured in the fermentation broth of M. anisopliae at 1 d, 30 3 d, 5 d and 7 d using a Q Exactive Mass Spectrometer. The gene for swnR was 31 detected by RT-qPCR. To determine the role of the swnR gene in the SW biosynthesis 32 pathway of M. anisopliae, we used PEG-mediated homologous recombination (HR) 33 to transform a wild-type strain (WT) with a Benomyl (ben)-resistant fragment to 34 knock out the swnR gene producing a mutant-type strain (MT). A complemented-type 35 (CT) strain was produced by adding a complementation vector that contains the 36 glufosinate (herbicide) resistance (bar) gene as a marker. The content of SW 37 decreased, but was not eliminated in the fermentation broth of the MT strain, and 38 returned to the original level in the CT strain. These results indicate that the swnR 39 gene plays a crucial role in the SW biosynthesis pathway of M. anisopliae, but 40 suggests that another gene in the fungus may share the function of swnR. 41 strain 43 Introduction 44 Metarhizium anisopliae is anentomopathogenic fungus that can infect a variety of 3 45 agricultural pest insects and produces swainsonine[1-3]. Swainsonine is an 46 indolizidine alkaloid that inhibits alpha-mannosidases. Swainsonine is produced by a 47 variety of fungi and is the major toxic component of locoweed plants, which are 48 widely found in western China and North America [4-9]. Swainsonine (SW) causes 49 locoism in grazing animals [10-12], when consumed for an extended time. SW also 50 has anti-tumor properties and may enhance the body's immunity [13-16]. 51 Recently, Cook et al. [17] sequenced the genome of two swainsonine-producing 52 fungi Slafractonia leguminicola and Alternaria oxytropis, and compared their 53 swainsonine producing genes to those in M. robertsiiand other swainsonine-producing 54 fungi. All the swainsonine-producing fungi contained a common gene cluster "SWN", 55 which included swnH 1 , swnH 2 , swnK, swnN, swnR, swnA, and swnT. Fungi that do not 56 produce swainsonine do not contain the SWN cluster. These genes encode catalytic 57 enzymes involved in the SW biosynthesis. swnN and swnR both encode NADB 58 Rossmann-fold reductases. After annotating the "SWN" gene cluster, Cook et al. [17] 59 successfully constructed a swnK knockout vector for the entomopathogen M. 60 robertsii, and obtained a swnK mutant strain by...