A <i>Streptomyces lividans</i> SipY deficient strain as a host for protein production: standardization of operational alternatives for model proteins

Gabarró, Marcel·la Vidal; Gullón, Sonia; Vicente, Rebeca L.; Caminal, Glòria; Mellado, Rafael P.; López-Santı́n, Josep

doi:10.1002/jctb.4933

Cited by 12 publications

(10 citation statements)

References 30 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Using these less-constrained models, we could monitor the uptake and excretion rates of numerous metabolites whose exchange could not be considered in previous simulations (see Additional file 5 , MTF and FVA results for iJV1220). The results agree with observations from growth in minimal medium: besides numerous ions not considered in previous models, the model identified amino acids as the preferred nutrients, in agreement with experimental observations (when grown with casamino acids, amino acids are the preferred carbon sources, and as they start to diminish, the cells start using other carbon sources [ 19 , 22 , 43 ]). Overall, the computed MTF fluxes and their respective FVA limits were remarkably similar irrespective of whether they were computed with extensive experimental constraints, with relaxed or even with no constrains at all (other than biomass and minimal secreted protein production) (See Additional file 5 , MTF and FVA results for iJV1220) and agreed with experimentally observed exchange rates, which were within the predicted FVA limits.…”

Section: Resultssupporting

confidence: 89%

“…The fluxes computed with the more complete model, iJV1220 show statistically significant changes related to cell envelope, cofactor and nucleotide biosynthesis similarly to iJV710, with differences that can be ascribed to the inclusion of previously unconsidered relevant routes. Being more comprehensive, iJV1220 provides additional details and reproduces better the known experimental behaviour [ 11 , 19 , 22 , 26 , 43 ]. The iJV1220 model displayed a better predictive behaviour, reproducing microarray expression data [ 19 ] and experimental observations of metabolite exchange rates when most constraints on exchange flux limits were relaxed or removed.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

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

2018

Self Cite

View full text Add to dashboard Cite

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.Electronic supplementary materialThe online version of this article (10.1186/s12866-018-1199-3) contains supplementary material, which is available to authorized users.

show abstract

Section: Resultssupporting

confidence: 89%

Section: Discussionmentioning

confidence: 99%

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

2018

Self Cite

View full text Add to dashboard Cite

show abstract

“…Use of L-alanine resulted in a corresponding reduction in NH 4 + needs, which automatically started being excreted. The observed shoulder in growth rate matched a less-defined region (identified by a larger dispersion) in published growth curves [27, 28].…”

Section: Resultssupporting

confidence: 62%

“…We now proceed to show the use of Adaptive DFBA to explore the metabolism of systems with limited knowledge: first, we analyse S. lividans TK21 grown in a complex medium (NMMP complemented with mannitol and casamino acids), over-expressing secretory proteins cloned in the multi-copy plasmid pIJ486 and secreting them through the minor Tat or the major Sec secretion routes, using model proteins that have been shown to display secretion patterns clearly dissociated from growth [28, 29]. Previous studies have shown that the strains of S. lividans TK21 behave similar to those of S. lividans TK24, when grown on similar media [28, 29], and that the metabolic model should be (to the extent known) transferable between them [6].…”

Section: Resultsmentioning

confidence: 99%

Dynamic metabolic modelling of overproduced protein secretion in Streptomyces lividans using adaptive DFBA

et al. 2019

Self Cite

View full text Add to dashboard Cite

BackgroundStreptomyces lividans is an appealing host for the production of proteins of biotechnological interest due to its relaxed exogenous DNA restriction system and its ability to secrete proteins directly to the medium through the major Sec or the minor Tat routes. Often, protein secretion displays non-uniform time-dependent patterns. Understanding the associated metabolic changes is a crucial step to engineer protein production. Dynamic Flux Balance Analysis (DFBA) allows the study of the interactions between a modelled organism and its environment over time. Existing methods allow the specification of initial model and environment conditions, but do not allow introducing arbitrary modifications in the course of the simulation. Living organisms, however, display unexpected adaptive metabolic behaviours in response to unpredictable changes in their environment. Engineering the secretion of products of biotechnological interest has systematically proven especially difficult to model using DFBA. Accurate time-dependent modelling of complex and/or arbitrary, adaptive metabolic processes demands an extended approach to DFBA.ResultsIn this work, we introduce Adaptive DFBA, a novel, versatile simulation approach that permits inclusion of changes in the organism or the environment at any time in the simulation, either arbitrary or interactively responsive to environmental changes. This approach extends traditional DFBA to allow steering arbitrarily complex simulations of metabolic dynamics. When applied to Sec- or Tat-dependent secretion of overproduced proteins in S. lividans, Adaptive DFBA can overcome the limitations of traditional DFBA to reproduce experimental data on plasmid-free, plasmid bearing and secretory protein overproducing S. lividans TK24, and can yield useful insights on the behaviour of systems with limited experimental knowledge such as agarase or amylase overproduction in S. lividans TK21.ConclusionsAdaptive DFBA has allowed us to overcome DFBA limitations and to generate more accurate models of the metabolism during the overproduction of secretory proteins in S. lividans, improving our understanding of the underlying processes. Adaptive DFBA is versatile enough to permit dynamical metabolic simulations of arbitrarily complex biotechnological processes.

show abstract

“…Heterologous protein yields obtained in S. lividans are often low or inconsistent, driving the research for uncovering production bottlenecks and applying improvement strategies [1, 4]. Screening for alternative promoters and signal peptides [5], codon optimization [6], and optimization of operational conditions [7] have been applied as strategies—with varying success—for improving protein production in S. lividans . Additional increases in protein production might be obtained by finding genetic targets based on a thorough understanding of the metabolic burden caused by recombinant protein production.…”

Section: Introductionmentioning

confidence: 99%

Transcriptomic and fluxomic changes in Streptomyces lividans producing heterologous protein

et al. 2018

View full text Add to dashboard Cite

BackgroundThe Gram-positive Streptomyces lividans TK24 is an attractive host for heterologous protein production because of its high capability to secrete proteins—which favors correct folding and facilitates downstream processing—as well as its acceptance of methylated DNA and its low endogeneous protease activity. However, current inconsistencies in protein yields urge for a deeper understanding of the burden of heterologous protein production on the cell. In the current study, transcriptomics and -based fluxomics were exploited to uncover gene expression and metabolic flux changes associated with heterologous protein production. The Rhodothermus marinus thermostable cellulase A (CelA)—previously shown to be successfully overexpressed in S. lividans—was taken as an example protein.ResultsRNA-seq and -based metabolic flux analysis were performed on a CelA-producing and an empty-plasmid strain under the same conditions. Differential gene expression, followed by cluster analysis based on co-expression and co-localization, identified transcriptomic responses related to secretion-induced stress and DNA damage. Furthermore, the OsdR regulon (previously associated with hypoxia, oxidative stress, intercellular signaling, and morphological development) was consistently upregulated in the CelA-producing strain and exhibited co-expression with isoenzymes from the pentose phosphate pathway linked to secondary metabolism. Increased expression of these isoenzymes matches to increased fluxes in the pentose phosphate pathway. Additionally, flux maps of the central carbon metabolism show increased flux through the tricarboxylic acid cycle in the CelA-producing strain. Redirection of fluxes in the CelA-producing strain leads to higher production of NADPH, which can only partly be attributed to increased secretion.ConclusionsTranscriptomic and fluxomic changes uncover potential new leads for targeted strain improvement strategies which may ease the secretion stress and metabolic burden associated with heterologous protein synthesis and secretion, and may help create a more consistently performing S. lividans strain. Yet, links to secondary metabolism and redox balancing should be further investigated to fully understand the S. lividans metabolome under heterologous protein production.Electronic supplementary materialThe online version of this article (10.1186/s12934-018-1040-6) contains supplementary material, which is available to authorized users.

show abstract

A Streptomyces lividans SipY deficient strain as a host for protein production: standardization of operational alternatives for model proteins

Cited by 12 publications

References 30 publications

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

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

Dynamic metabolic modelling of overproduced protein secretion in Streptomyces lividans using adaptive DFBA

Transcriptomic and fluxomic changes in Streptomyces lividans producing heterologous protein

Contact Info

Product

Resources

About