Abstract
BackgroundStreptomyces 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.ResultsWe 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.ConclusionsThis 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.
Highlights
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
To better understand the aspects of protein secretion in S. lividans TK21 that are specific of the Tat route, we modelled production of α-amylase and its secretion through the Sec route using data obtained in S. lividans TK21 for comparison
In this work we describe the utilization of metabolic models to describe the experimental growth and secretion rates of Tat-secreted agarase and Sec-secreted α-amylase overproduced in S. lividans TK21
Summary
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. Streptomyces are non-pathogenic gram-positive soil bacteria, members of the Actinobacteria phylum, displaying mycelial growth, and involved in the breakdown of soil material. They are well known for their ability to synthesize antibiotics and other compounds of biotechnological interest, as well as to produce large quantities of extracellular proteins. Hybridizations of the genomes of various strains of S. lividans (66, TK21, TK24) among themselves and with those of S. coelicolor A3(2) and M145, have shown a considerable genome plasticity, accommodating large deletions and extensive amplifications, often linked to conjugative elements such as SLP1 or SLP3 [6,7,8]
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