Functional identification of a Streptomyces lividans FKBP-like protein involved in the folding of overproduced secreted proteins.

R L Vicente,S Gullón,R P Mellado,J R Valverde,C Palomino,S Marín

doi:10.1098/rsob.190201

Abstract

Some bacterial peptidyl-prolyl cis/trans isomerases (PPIases) are involved in secretory protein folding after the translocation step. Streptomyces lividans has been used as a host for engineering extracellular overproduction of homologous and heterologous proteins in industrial applications. Although the mechanisms governing the major secretory pathway (Sec route) and the minor secretory pathway (Tat route) are reasonably well described, the function of proteins responsible for the extracellular secretory protein folding is not characterized as yet. We have characterized a Tat-dependent S. lividans FK506-binding protein-like lipoprotein (FKBP) that has PPIase activity. A mutant in the sli-fkbp gene induces a secretion stress response and affects secretion and activity of the Sec-dependent protein α-amylase. Additionally, propagation in high copy number of the sli-fkbp gene has a positive effect on the activity of both the overproduced α-amylase and the overproduced Tat-dependent agarase, both containing proline cis isomers. Targeted proteomic analyses showed that a relevant group of secreted proteins in S. lividans TK21 are affected by Sli-FKBP, revealing a wide substrate range. The results obtained indicate that, regardless of the secretory route used by proteins in S. lividans, adjusting the expression of sli-fkbp may facilitate folding of dependent proteins when engineering Streptomyces strains for the overproduction of homologous or heterologous secretory proteins.

Highlights

Streptomycetes are Gram-positive soil bacteria that secrete an array of hydrolytic enzymes [1,2], among other metabolites, to ensure their survival in this harsh environment
The results obtained indicate that, regardless of the secretory route used by proteins in S. lividans, adjusting the expression of sli-fkbp may facilitate folding of dependent proteins when engineering Streptomyces strains for the overproduction of homologous or heterologous secretory proteins
The 1222 bp long amplified DNA fragment was sequenced and its sequence was aligned with the equivalent ones from the S. coelicolor and the S. lividans TK24 genomes and found to coincide in 99% and 100%, respectively, with that of the genes encoding their respective FK506-binding protein-like lipoprotein (FKBP)-like proteins

Summary

Introduction

Streptomycetes are Gram-positive soil bacteria that secrete an array of hydrolytic enzymes [1,2], among other metabolites, to ensure their survival in this harsh environment. In S. lividans, the Tat system has three components (TatA, TatB and TatC), and the Tat signal peptide, containing the highly conserved twin-arginine motif SRRXFLK, at its amino end, is thought to be recognized by the TatA–TatB heterocomplex [11]. This complex theoretically interacts with TatC in the membrane, and, oligomerization of TatA forms the membrane pore to support secretion of the Tat proteins [11]. In Escherichia coli the signal peptide recognition is mediated by the TatB–TatC complex [12]

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Discussion

Conclusion