Abstract
BackgroundWhilst undergoing differentiation, Streptomyces produce a large quantity of hydrolytic enzymes and secondary metabolites, and it is this very ability that has focussed increasing interest on the use of these bacteria as hosts for the production of various heterologous proteins. However, within this genus, the exploration and understanding of the metabolic burden associated with such bio-products has only just begun. In this study our overall aim was to apply metabolomics approaches as tools to get a glimpse of the metabolic alterations within S.lividans TK24 when this industrially relevant microbe is producing recombinant murine tumour necrosis factor alpha (mTNFα), in comparison to wild type and empty (non-recombinant protein containing) plasmid-carrying strains as controls.ResultsWhilst growth profiles of all strains demonstrated comparable trends, principal component-discriminant function analysis of Fourier transform infrared (FT-IR) spectral data, showed clear separation of wild type from empty plasmid and mTNFα-producing strains, throughout the time course of incubation. Analysis of intra- and extra-cellular metabolic profiles using gas chromatography–mass spectrometry (GC–MS) displayed similar trends to the FT-IR data. Although the strain carrying the empty plasmid demonstrated metabolic changes due to the maintenance of the plasmid, the metabolic behaviour of the recombinant mTNFα-producing strain appeared to be the most significantly affected. GC–MS results also demonstrated a significant overflow of several organic acids (pyruvate, 2-ketoglutarate and propanoate) and sugars (xylitol, mannose and fructose) in the mTNFα-producing strain.ConclusionThe results obtained in this study have clearly demonstrated the metabolic impacts of producing mTNFα in S. lividans TK24, while displaying profound metabolic effects of harbouring the empty PIJ486 plasmid. In addition, the level of mTNFα produced in this study, further highlights the key role of media composition towards the efficiency of a bioprocess and metabolic behaviour of the host cells, which directly influences the yield of the recombinant product.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0350-1) contains supplementary material, which is available to authorized users.
Highlights
Whilst undergoing differentiation, Streptomyces produce a large quantity of hydrolytic enzymes and secondary metabolites, and it is this very ability that has focussed increasing interest on the use of these bacteria as hosts for the production of various heterologous proteins
The current study is aimed at investigating the impact of heterologous murine tumour necrosis factor α (mTNFα) production on the metabolite pools and growth behaviour of S. lividans TK24 grown in a defined minimal medium using gas chromatography–mass spectrometry (GC–MS) as metabolic profiling and footprinting approaches, whilst employing Fourier transform infrared (FT-IR) as a rapid metabolic fingerprinting strategy
Growth profile and mTNFα production The growth behaviour of all three S. lividans strains in minimal medium (NMMP) was monitored by measuring the dry cell weight (DCW) of each strain at different time points during the incubation period
Summary
Streptomyces produce a large quantity of hydrolytic enzymes and secondary metabolites, and it is this very ability that has focussed increasing interest on the use of these bacteria as hosts for the production of various heterologous proteins. Whilst undergoing differentiation, leading to sporulation, a large quantity of hydrolytic enzymes and secondary metabolites are known to be secreted [5], and it is perhaps this very ability that has focused increasing interest on the use of these bacteria as hosts for the production of various heterologous proteins. These include the production of antifungal compounds and antibacterial agents, in addition to anti-parasitic [6] and anticancer drugs [7, 8]. For an overview of recombinant protein production and some recent examples of heterologous (mammalian and microbial) proteins produced using S. lividans as the host organism, as well as how systems biology (including metabolomics) approaches can be developed to improve protein production, the reader is directed to the following excellent reviews [17, 18]
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