The Cellular Mechanisms that Ensure an Efficient Secretion in Streptomyces

Gram-positive soil bacteria included in the genus Streptomyces produce a large variety of secondary metabolites in addition to extracellular hydrolytic enzymes. From the industrial and commercial viewpoints, the S. lividans strain has generated greater interest as a host bacterium for the overproduction of homologous and heterologous hydrolytic enzymes as an industrial application, which has considerably increased scientific interest in the characterization of secretion routes in this bacterium. This review will focus on the secretion machinery in S. lividans.This work was funded by the Spanish Ministry of Economy, Industry, and Competitivity/ European Regional Development Fund grant BIO2015-71504-R (AEI/FEDER, UE

Clase de Máster de Biotecnología

Clase de Máster en Biotecnología MicrobianaOrganizado por la Universidad Autónoma de MadridPeer reviewe

Aislamiento y caracterización de genes implicados en la biosíntesis del antitumoral estefimicina en Streptomyces steffisburgensis

Estefimicina pertenece a un grupo de policétidos llamados antraciclinas al que pertenecen también doxorrubicina y daunorrubicina usadas en el tratamiento de diferentes tipos de cáncer. Estefimicina no posee una actividad biológica destacada, sin embargo esta molécula posee una serie de características estructurales que no están presentes en otras antraciclinas y que hacen atractivo el estudio de los genes implicados en la biosíntesis de estefimicina para contribuir a la generación de nuevos derivados del tipo antraciclina, con una potencial actividad antitumoral, a través de la biosíntesis combinatoria. En este trabajo se describe la clonación y secuenciación del agrupamiento génico implicado en la biosíntesis de estefimicina en S. steffisburgensis. Tanto la expresión heteróloga de algunos de los genes implicados en la biosíntesis de estefimicina así como la inactivación génica de algunos de ellos, ha permitido identificar tres intermediarios en la ruta de biosíntesis de estefimicina así como establecer la secuencia de reacciones biosintéticas que tienen lugar durante la biosíntesis de estefimicina. El objetivo fundamental para el futuro es la utilización de la información obtenida a partir de los genes de biosíntesis de estefimicina para contribuir a la generación de nuevos derivados a través de biosíntesis combinatoria

Four thiol-oxidoreductases involved in the formation of disulphide bonds in the Streptomyces lividans TK21 secretory proteins

[Background] Bacterial secretory proteins often require the formation of disulphide bonds outside the cell to acquire an active conformation. Thiol-disulphide oxidoreductases are enzymes that catalyse the formation of disulphide bonds. The bacterium Streptomyces lividans is a well-known host for the efficient secretion of overproduced homologous and heterologous secretory proteins of industrial application. Therefore, the correct conformation of these extracellular proteins is of great importance when engineering that overproduction.[Results] We have identified four acting thiol-disulphide oxidoreductases (TDORs) in S. lividans TK21, mutants in all TDOR candidates affect the secretion and activity of the Sec-dependent alpha-amylase, which contains several disulphide bonds, but the effect was more drastic in the case of the Sli-DsbA deficient strain. Thus, the four TDOR are required to obtain active alpha-amylase. Additionally, only mutations in Sli-DsbA and Sli-DsbB affect the secretion and activity of the Tat-dependent agarase, which does not form a disulphide bond, when it is overproduced. This suggests a possible role of the oxidised Sli-DsbA as a chaperone in the production of active agarase.[Conclusions] Enzymes involved in the production of the extracellular mature active proteins are not fully characterised yet in Streptomyces lividans. Our results suggest that the role of thiol-disulphide oxidoreductases must be considered when engineering Streptomyces strains for the overproduction of homologous or heterologous secretory proteins of industrial application, irrespective of their secretion route, in order to obtain active, correctly folded proteins.This work was funded by Spanish Ministry of Science, Innovation and Universities (MCIU)/State Research Agency (AEI)/European Regional Development Fund (FEDER) Grant BIO2015-71504-R (MCIU/AEI/FEDER, UE) to RPM.Peer reviewe

Modelling the metabolism of protein secretion through the Tat route in Streptomyces lividans

[Background] Streptomyces lividans has demonstrated its value as an efficient host for protein production due to its ability to secrete functional proteins directly to the media. Secretory proteins that use the major Sec route need to be properly folded outside the cell, whereas secretory proteins using the Tat route appear outside the cell correctly folded. This feature makes the Tat system very attractive for the production of natural or engineered Tat secretory proteins. S. lividans cells are known to respond differently to overproduction and secretion of Tat versus Sec proteins. Increased understanding of the impact of protein secretion through the Tat route can be obtained by a deeper analysis of the metabolic impact associated with protein production, and its dependence on protein origin, composition, secretion mechanisms, growth phases and nutrients. Flux Balance Analysis of Genome-Scale Metabolic Network models provides a theoretical framework to investigate cell metabolism under different constraints. Conclusions This work provides a detailed look to metabolic changes associated to Tat-dependent protein secretion reproducing experimental observations and identifying changes that are specific to each secretory route, presenting a novel, improved, more accurate and strain-independent model of S. lividans, thus opening the way for enhanced metabolic engineering of protein overproduction in S. lividans.[Results] We have built new models for various S. lividans strains to better understand the mechanisms associated with overproduction of proteins secreted through the Tat route. We compare models of an S. lividans Tat-dependent agarase overproducing strain with those of the S. lividans wild-type, an S. lividans strain carrying the multi-copy plasmid vector and an α-amylase Sec-dependent overproducing strain. Using updated genomic, transcriptomic and experimental data we could extend existing S. lividans models and produce a new model which produces improved results largely extending the coverage of S. lividans strains, the number of genes and reactions being considered, the predictive behaviour and the dependence on specification of exchange constraints. Comparison of the optimized solutions obtained highlights numerous changes between Tat- and Sec-dependent protein secreting strains affecting the metabolism of carbon, amino acids, nucleotides, lipids and cofactors, and variability analysis predicts a large potential for protein overproduction.[Conclusions] This work provides a detailed look to metabolic changes associated to Tat-dependent protein secretion reproducing experimental observations and identifying changes that are specific to each secretory route, presenting a novel, improved, more accurate and strain-independent model of S. lividans, thus opening the way for enhanced metabolic engineering of protein overproduction in S. lividans.This work was funded by Spanish Ministry of Economy, Industry and Competitivity / European Regional Development Fund grant BIO2015–71504-R (AEI/FEDER, UE). The funding bodies had no role in the design of the study, in data collection, analysis or interpretation, or in writing this manuscript.Peer reviewe

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

© 2019 The Authors.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.This work was supported by the Spanish Ministry of Science, Innovation and Universities (MCIU)/State Research Agency (AEI)/ European Regional Development Fund (FEDER) grant BIO2015- 71504-R (MCIU/AEI/FEDER, UE) to R.P.M. Acknowledgements. The proteomic analysis was performed in the proteomics facility of Centro Nacional de Biotecnología-CSIC, member of ProteoRed (PRB3-ISCIII), funded by ISCIII and FEDER (grant PT13/0001)