ABSTRACT
The culture of Chinese Hamster Ovary (CHO) cells for modern industrial applications, such as expression of recombinant proteins, requires media that support growth and production. Such media must support high viable cell densities while also stimulating the synthesis and extracellular transport of biologic products. Early media development efforts in this area yielded basic formulations to sustain growth, viability, and cellular function, albeit comprising animal sourced components, and complex constituents used in batch culture mode. Subsequent improvements included the development of serum-free and chemically defined (CD) media, the identification of critical nutrients, growth factors, and potentially inhibitory or toxic cellular metabolites, and the use of fed-batch and perfusion culture techniques to optimize nutrient delivery while minimizing accumulation of unwanted waste products. This review is comprised of sections covering milestones in the evolution of mammalian cell culture media, nutrient composition and formulation requirements, optimization strategies, consistency and scalability of powder and liquid media preparation for industrial applications, and key recent advances driving progress in CHO cell culture medium design and development. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1407-1426, 2018.
Subject(s)
Recombinant Proteins/metabolism , Animals , CHO Cells , Cell Culture Techniques , Cricetinae , Cricetulus , Culture Media , Recombinant Proteins/geneticsABSTRACT
Streptomyces strain LL-P018 produces the phaeochromycins, novel anti-inflammatory polyketides. This organism was identified as a strain of Streptomyces phaeochromogenes by physiological and genetic taxonomic analysis. In order to gain greater taxonomic perspective, LL-P018 was compared to related strains from major culture collections by 16S rRNA gene sequence, ribotype, HPLC-MS metabolite profile, and rpoB sequence. Using BioNumerics software, genetic and chemical fingerprint data were integrated via multivariate cluster analysis into a single, robust comparison. Based upon this analysis, strain LL-P018 is very closely related to the type strains of both S. phaeochromogenes and Streptomyces ederensis, indicating that these two types may in fact represent a single species. This novel comparative multi-cluster analysis is most useful for clarifying relationships between closely related species.
Subject(s)
Acetogenins/metabolism , Phenotype , Streptomyces/classification , Streptomyces/physiology , Acetogenins/chemistry , Bacterial Proteins/genetics , Base Sequence , Carbohydrate Metabolism , Chromatography, High Pressure Liquid , Cluster Analysis , Culture Media , Molecular Sequence Data , RNA, Ribosomal, 16S/genetics , Ribotyping , Sequence Alignment , Species Specificity , Streptomyces/genetics , Streptomyces/metabolismABSTRACT
Five new polyketide metabolites, phaeochromycins A-E (1-5), were isolated from an actinomycete designated Streptomyces phaeochromogenes LL-P018, cultured from a soil sample collected from a riverbank in Westevenger, Germany. Phaeochromycins A and C were found to be weak inhibitors of MAPKAP kinase-2 (IC50 = 39 and 130 microM, respectively). The structures of the compounds were determined by spectroscopic analysis, primarily two-dimensional NMR, and revealed that phaeochromycins A, B, C, and E were octaketides, elaborated from a C4 starter unit, related to shunt products of the actinorhodin pathway, namely, mutactin, dehydromutactin, SEK34b, and BSM1. Phaeochromycin D (4) is an unusual partially cyclized degraded octaketide intermediate.
Subject(s)
Anti-Inflammatory Agents/isolation & purification , Enzyme Inhibitors/isolation & purification , Protein Serine-Threonine Kinases/antagonists & inhibitors , Pyrones/isolation & purification , Streptomyces/chemistry , Anti-Inflammatory Agents/chemistry , Anti-Inflammatory Agents/pharmacology , Arthritis, Rheumatoid/drug therapy , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Germany , Humans , Inhibitory Concentration 50 , Intracellular Signaling Peptides and Proteins , Molecular Structure , Nuclear Magnetic Resonance, Biomolecular , Pyrones/chemistry , Pyrones/pharmacology , Soil MicrobiologyABSTRACT
The natural product rapamycin, produced during fermentation by Streptomyces hygroscopicus, is known for its potent antifungal, immunosuppressive, and anticancer activities. During rapamycin biosynthesis, the amino acid l-pipecolate is incorporated into the rapamycin molecule. We investigated the use of precursor-directed biosynthesis to create new rapamycin analogs by substitution of unusual l-pipecolate analogs in place of the normal amino acid. Our results suggest that the l-pipecolate analog (+/-)-nipecotic acid inhibits the biosynthesis of l-pipecolate, thereby limiting the availability of this molecule for rapamycin biosynthesis. We used (+/-)-nipecotic acid in our precursor-directed biosynthesis studies to reduce l-pipecolate availability and thereby enhance the incorporation of other pipecolate analogs into the rapamycin molecule. We describe here the use of this method for production of two new sulfur-containing rapamycin analogs, 20-thiarapamycin and 15-deoxo-19-sulfoxylrapamycin, and report measurement of their binding to FKBP12.
Subject(s)
Gene Expression Regulation, Bacterial , Protein Precursors/metabolism , Sirolimus/analogs & derivatives , Sirolimus/metabolism , Streptomyces/metabolism , Biotechnology/methods , Nipecotic Acids/metabolism , Pipecolic Acids/metabolism , Streptomyces/genetics , Streptomyces/growth & development , Tacrolimus Binding Protein 1A/metabolismABSTRACT
[reaction: see text] Two novel sulfur-containing analogs of the immunosuppressive natural product rapamycin (1) were obtained by feeding cultures of Streptomyces hygroscopicus with l-nipecotic acid (4) and either (S)-1,3-thiazane-4-carboxylic acid (5) or (S)-1,4-thiazane-3-carboxylic acid (6). The structures of the two new compounds, 20-thiarapamycin (2) and 15-deoxo-19-sulfoxylrapamycin (3), were determined by spectroscopic methods.
