Amino acid uptake profiling of wild type and recombinant Streptomyces lividans TK24 batch fermentations.
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.
D'Huys
Pieter-Jan
PJ
Chemical and Biochemical Process Technology and Control Section (BioTeC), Department of Chemical Engineering, Katholieke Universiteit Leuven, Willem de Croylaan 46, B-3001 Leuven, Belgium.
Lule
Ivan
I
Van Hove
Sven
S
Vercammen
Dominique
D
Wouters
Christine
C
Bernaerts
Kristel
K
Anné
Jozef
J
Van Impe
Jan F M
JF
D000596:Amino Acids / Q000493:pharmacokinetics*; D000818:Animals; D001224:Aspartic Acid / Q000378:metabolism; D018533:Biomass; D001709:Biotechnology / Q000379:methods*; D005285:Fermentation / Q000502:physiology*; D018698:Glutamic Acid / Q000378:metabolism; D055442:Metabolome / Q000235:genetics; D051379:Mice; D014176:Protein Biosynthesis / Q000502:physiology*; D013045:Species Specificity; D048170:Streptomyces lividans / Q000378:metabolism* / Q000502:physiology; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
D000596:Amino Acids; D014409:Tumor Necrosis Factor-alpha; C017721:casamino acids; D001224:Aspartic Acid; D018698:Glutamic Acid