Mutations improving production and secretion of extracellular lipase by Burkholderia glumae PG1.

Andreas Knapp,Rong Gao,Rolf Daniel,Michael Breuer,Sonja Voget,Rolf Müller,Nestor Zaburannyi,Karl-Erich Jaeger,Wolfgang R Streit,Bernhard Hauer,Dagmar Krysciak

doi:10.1007/s00253-015-7041-z

Abstract

Burkholderia glumae is a Gram-negative phytopathogenic bacterium known as the causative agent of rice panicle blight. Strain B. glumae PG1 is used for the production of a biotechnologically relevant lipase, which is secreted into the culture supernatant via a type II secretion pathway. We have comparatively analyzed the genome sequences of B. glumae PG1 wild type and a lipase overproducing strain obtained by classical strain mutagenesis. Among a total number of 72 single nucleotide polymorphisms (SNPs) identified in the genome of the production strain, two were localized in front of the lipAB operon and were analyzed in detail. Both mutations contribute to a 100-fold overproduction of extracellular lipase in B. glumae PG1 by affecting transcription of the lipAB operon and efficiency of lipase secretion. We analyzed each of the two SNPs separately and observed a stronger influence of the promoter mutation than of the signal peptide modification but also a cumulative effect of both mutations. Furthermore, fusion of the mutated LipA signal peptide resulted in a 2-fold increase in secretion of the heterologous reporter alkaline phosphatase from Escherichia coli.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-015-7041-z) contains supplementary material, which is available to authorized users.

Highlights

Burkholderia glumae belongs to the genus Burkholderia within the subphylum of the β-proteobacteria (Yabuuchi et al 1992)
The lipase production strain B. glumae LU8093 was constructed from B. glumae PG1 by repeated rounds of random mutagenesis and subsequent assays for increased extracellular lipase production R
We analyzed the increased lipase production of the industrial production strain B. glumae LU8093 (Fig. 2) which is a derivate of the wild type strain B. glumae PG1

Summary

Introduction

Burkholderia glumae (formerly known as Pseudomonas glumae) belongs to the genus Burkholderia within the subphylum of the β-proteobacteria (Yabuuchi et al 1992). B. glumae is a moderate rice pathogen (Ham et al 2011), which affects several other plants (Jeong et al 2003). All B. glumae strains studied so far infect rice panicles and produce a phytotoxin called toxoflavin Biotechnological applications of Burkholderia species mainly comprise their use as biofertilizers or bioremediation agents (Chiarini et al 2006; Paganin et al 2011; Suarez-Moreno et al 2012). The production of an extracellular lipase (Boekema et al 2007; Santambrogio et al 2013) and of rhamnolipid biosurfactants (Costa et al 2011) were described, but detailed studies regarding further biotechnological applications are missing

Methods

Results

Conclusion