Large-scale production of a thermostable Rhodothermus marinus cellulase by heterologous secretion from Streptomyces lividans

Mohamed Belal Hamed,Jozef Anné,Olafur H Fridjonsson,Lieve Van Mellaert,Joana Sofia Martins Basílio,Spyridoula Karamanou,Eik Elísabet Guðmundsdóttir,Kostantinos C Tsolis,Kristel Bernaerts,Anastassios Economou,Kenneth Simoens,Solveig Ólafsdottir,Gudmundur Oli Hreggvidsson

doi:10.1186/s12934-017-0847-x

Abstract

BackgroundThe gene encoding a thermostable cellulase of family 12 was previously isolated from a Rhodothermus marinus through functional screening. CelA is a protein of 260 aminoacyl residues with a 28-residue amino-terminal signal peptide. Mature CelA was poorly synthesized in some Escherichia coli strains and not at all in others. Here we present an alternative approach for its heterologous production as a secreted polypeptide in Streptomyces.ResultsCelA was successfully over-expressed as a secreted polypeptide in Streptomyces lividans TK24. To this end, CelA was fused C-terminally to the secretory signal peptide of the subtilisin inhibitor protein (Sianidis et al. in J Biotechnol. 121: 498–507, 2006) from Streptomyces venezuelae and a new cloning strategy developed. Optimal growth media and conditions that stall biomass production promote excessive CelA secretion. Under optimal growth conditions in nutrient broth medium, significant amounts of mature CelA (50–90 mg/L or 100–120 mg/g of dry cell weight) are secreted in the spent growth media after 7 days. A protocol to rapidly purify CelA to homogeneity from culture supernatants was developed and specific anti-sera raised against it. Biophysical, biochemical and immmuno-detection analyses indicate that the enzyme is intact, stable and fully functional. CelA is the most thermostable heterologous polypeptide shown to be secreted from S. lividans.ConclusionThis study further validates and extends the use of the S. lividans platform for production of heterologous enzymes of industrial importance and extends it to active thermostable enzymes. This study contributes to developing a platform for poly-omics analysis of protein secretion in S. lividans.

Highlights

The gene encoding a thermostable cellulase of family 12 was previously isolated from a Rhodothermus marinus through functional screening
We have extended the use of the S. lividans secretion system for the production of thermostable enzymes of industrial interest, using as an example a cellulase of glycosyl hydrolase family 12 (CelA) from Rhodothermus marinus [13]
Production of recombinant RmCelA in S. lividans High-level production of CelA was originally achieved by cloning the celA gene in E. coli in a pET23bAH vector with a C-terminal hexahistidinyl tag [13]

Summary

Introduction

The gene encoding a thermostable cellulase of family 12 was previously isolated from a Rhodothermus marinus through functional screening. We present an alternative approach for its heterologous production as a secreted polypeptide in Streptomyces. Streptomyces lividans has been used for the heterologous secretion of several polypeptides of bacterial and eukaryotic origin (for examples see [1,2,3,4,5]). Hamed et al Microb Cell Fact (2017) 16:232 avoidance of inclusion body formation, render S. lividans secretion an attractive biotechnology platform. In many instances, it can provide alternative solutions when established workhorses, like Escherichia coli, fail or are more costly in bio-processing terms [8]. One successful approach has been the use of the transcription elements and the signal peptide of the Streptomyces venezuelae CBS762.70 subtilisin inhibitor gene [12] (hereafter: vsi)

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Results

Conclusion