ABSTRACT
Copper concentration can impact lactate metabolism in Chinese Hamster ovary (CHO) cells. In our previous study, a 20-fold increase in initial copper concentration enabled CHO cultures to shift from net lactate production to net lactate consumption, and achieve higher cell growth and productivity. In this follow-up study, we used transcriptomics to investigate the mechanism of action (MOA) of copper that mediates this beneficial metabolism shift. From microarray profiling (days 0-7), the number of differentially expressed genes increased considerably after the lactate shift (>day 3). To uncouple the effects of copper at early time points (days 0-3) from that of lactate per se (>day 3), and to validate microarray hits, we analyzed samples before the lactate shift by RNA-Seq. Out of 6,398 overlapping genes analyzed by both transcriptomic methods, only the early growth response 1 gene-coding for a transcription factor that activates signaling pathways in response to environmental stimuli-satisfied the differential expression criteria (fold change ≥ 1.5; P < 0.05). Gene expression correlation and biological pathway analyses further confirmed that copper differences exerted minimal transcriptional impact on the CHO cultures before the lactate shift. By contrast, genes associated with hypoxia network and oxidative stress response were upregulated after the lactate shift. These upregulations should boost cell proliferation and survival, but do not account for the preceding shift in lactate metabolism. The findings here indicate that the primary MOA of copper that enabled the shift in lactate metabolism is not at the transcriptional level.
Subject(s)
Copper/toxicity , Gene Expression/drug effects , Transcriptome/drug effects , Animals , CHO Cells , Cell Line , Cell Survival/drug effects , Cluster Analysis , Cricetinae , Cricetulus , Early Growth Response Transcription Factors/analysis , Early Growth Response Transcription Factors/genetics , Early Growth Response Transcription Factors/metabolism , Gene Expression Profiling , Humans , Mice , Oligonucleotide Array Sequence Analysis , Prostaglandin-Endoperoxide Synthases/analysis , Prostaglandin-Endoperoxide Synthases/genetics , Prostaglandin-Endoperoxide Synthases/metabolismABSTRACT
The most important approach to the development of platform organisms for recombinant protein production relies on random mutagenesis and phenotypic selection. Complex phenotypes, including those associated with significantly elevated expression and secretion of heterologous proteins, are the result of multiple genomic mutations. Using next generation sequencing, a parent and derivative hypersecreter strain (B41) of Escherichia coli were sequenced with an average coverage of 52.8X and 55X, respectively. A new base-pair calling program, revealed a single nucleotide polymorphism in the B41 genome at position 1,074,787, resulting in translation termination near the N-terminus of a transcriptional regulator protein, RutR, coded by the ycdC gene. We verified the hypersecretion phenotype in a ycdC::Tn5 mutant and observed a 3.4-fold increase in active hemolysin secretion, consistent with the increase observed in B41 strain. mRNA expression profiling showed decreased expression of tRNA-synthetases and some amino acid transporters in the ycdC::Tn5 mutant. This study demonstrates the power of next generation sequencing to characterize mutants leading to successful metabolic engineering strategies for strain improvement.
Subject(s)
Amino Acyl-tRNA Synthetases/genetics , Escherichia coli/genetics , Genes, Bacterial , Polymorphism, Single Nucleotide , Base Sequence , Blotting, Western , DNA Primers , DNA, Bacterial/genetics , Molecular Sequence Data , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Nucleic AcidABSTRACT
MicroRNAs (miRNAs) are small, non-coding RNAs that regulate multiple aspects of cell physiology. The differential expression of conserved miRNAs in two Chinese hamster ovary (CHO) cell lines producing recombinant proteins was examined relative to the CHO-K1 cell line. A total of 190 conserved CHO miRNAs were identified through homology with known human and rodent miRNAs. More than 80% of these miRNAs showed differential expression in recombinant CHO cell lines. The small RNA sequencing data were analyzed in context of the CHO-K1 genome to examine miRNA organization and develop sequence-specific miRNA resources for CHO cells. The identification and characterization of CHO miRNAs will facilitate the use of miRNA tools in cell line engineering efforts to improve product yield and quality.
Subject(s)
Cricetulus/genetics , Gene Expression Regulation , MicroRNAs/biosynthesis , Animals , CHO Cells , Cricetinae , MicroRNAs/genetics , Recombination, Genetic , Sequence Analysis, DNA/methodsABSTRACT
BACKGROUND: Chinese hamster ovary (CHO) cells are among the most widely used hosts for therapeutic protein production. Yet few genomic resources are available to aid in engineering high-producing cell lines. RESULTS: High-throughput Illumina sequencing was used to generate a 1x genomic coverage of an engineered CHO cell line expressing secreted alkaline phosphatase (SEAP). Reference-guided alignment and assembly produced 3.57 million contigs and CHO-specific sequence information for ~ 18,000 mouse and ~ 19,000 rat orthologous genes. The majority of these genes are involved in metabolic processes, cellular signaling, and transport and represent attractive targets for cell line engineering. CONCLUSIONS: This demonstrates the applicability of next-generation sequencing technology and comparative genomic analysis in the development of CHO genomic resources.
