BioChem - DOS

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

Microbial cell factories

Hamed, Mohamed Belal; Karamanou, Spyridoula; Ó lafsdottir, Solveig; Basí lio, Joana Sofia Martins; Simoens, Kenneth; Tsolis, Kostantinos C.; Van Mellaert, Lieve; Guðmundsdó ttir, Eik Elí sabet; Hreggvidsson, Gudmundur Oli; Anné , Jozef; Bernaerts, Kristel; Fridjonsson, Olafur H.; Economou, Anastassios

<P><B>Background</B></P><P>The gene encoding a thermostable cellulase of family 12 was previously isolated from a <I>Rhodothermus marinus</I> 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 <I>Escherichia coli</I> strains and not at all in others. Here we present an alternative approach for its heterologous production as a secreted polypeptide in <I>Streptomyces</I>.</P><P><B>Results</B></P><P>CelA was successfully over-expressed as a secreted polypeptide in <I>Streptomyces lividans</I> 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&#x2013;507, 2006) from <I>Streptomyces venezuelae</I> 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&#x2013;90&nbsp;mg/L or 100&#x2013;120&nbsp;mg/g of dry cell weight) are secreted in the spent growth media after 7&nbsp;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 <I>S. lividans</I>.</P><P><B>Conclusion</B></P><P>This study further validates and extends the use of the <I>S. lividans</I> 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 <I>S. lividans</I>.</P><P><B>Electronic supplementary material</B></P><P>The online version of this article (10.1186/s12934-017-0847-x) contains supplementary material, which is available to authorized users.</P>

2017

BioMed Central

cc-by

1475-2859

16

pp.232

10.1186/s12934-017-0847-x

http://click.ndsl.kr/servlet/OpenAPIDetailView?keyValue=05787966&target=NART&cn=NART90266550

https://microbialcellfactories.biomedcentral.com/track/pdf/10.1186/s12934-017-0847-x

Cellulase; Protein translocase; Signal peptide; Secretion; Streptomyces lividans; Protein secretion biotechnology