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Amino acid uptake profiling of wild type and recombinant Streptomyces lividans TK24 batch fermentations

Paper ID Volume ID Publish Year Pages File Format Full-Text
24038 43491 2011 12 PDF Available
Title
Amino acid uptake profiling of wild type and recombinant Streptomyces lividans TK24 batch fermentations
Abstract

Streptomyces lividans is considered an interesting host for the secretory production of heterologous proteins. To obtain a good secretion yield of heterologous proteins, the availability of suitable nitrogen sources in the medium is required. Often, undefined mixtures of amino acids are used to improve protein yields. However, the understanding of amino acid utilization as well as their contribution to the heterologous protein synthesis is poor.In this paper, amino acid utilization by wild type and recombinant S. lividans TK24 growing on a minimal medium supplemented with casamino acids is profiled by intensive analysis of the exometabolome (metabolic footprint) as a function of time. Dynamics of biomass, substrates, by-products and heterologous protein are characterized, analyzed and compared. As an exemplary protein mouse Tumor Necrosis Factor Alpha (mTNF-α) is considered.Results unveil preferential glutamate and aspartate assimilation, together with glucose and ammonium, but the associated high biomass growth rate is unfavorable for protein production. Excretion of organic acids as well as alanine is observed. Pyruvate and alanine overflow point at an imbalance between carbon and nitrogen catabolism and biosynthetic fluxes. Lactate secretion is probably related to clump formation. Heterologous protein production induces a slowdown in growth, denser clump formation and a shift in metabolism, as reflected in the altered substrate requirements and overflow pattern. Besides glutamate and aspartate, most amino acids are catabolized, however, their exact contribution in heterologous protein production could not be seized from macroscopic quantities.The metabolic footprints presented in this paper provide a first insight into the impact and relevance of amino acids on biomass growth and protein production. Type and availability of substrates together with biomass growth rate and morphology affect the protein secretion efficiency and should be optimally controlled, e.g., by appropriate medium formulation and substrate dosing. Overflow metabolism as well as high biomass growth rates must be avoided because they reduce protein yields. Further investigation of the intracellular metabolic fluxes should be conducted to fully unravel and identify ways to relieve the metabolic burden of plasmid maintenance and heterologous protein production and to prevent overflow.

Keywords
mTNF-α, mouse Tumor Necrosis Factor alpha; WT, S. lividans strain TK24; PL, S. lividans strain TK24 harboring empty pIJ486 plasmid; REC, S. lividans strain TK24 harboring mTNF-α gene on pIJ486 plasmid; GP, growth phase; GABA, gamma-aminobutyric acidStrept
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Amino acid uptake profiling of wild type and recombinant Streptomyces lividans TK24 batch fermentations
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 152, Issue 4, 10 April 2011, Pages 132–143
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us