Volume 4, Issue 5, Pages 530-542.e6 (May 2017) Mammalian Systems Biotechnology Reveals Global Cellular Adaptations in a Recombinant CHO Cell Line  Faraaz Noor Khan Yusufi, Meiyappan Lakshmanan, Ying Swan Ho, Bernard Liat Wen Loo, Pramila Ariyaratne, Yuansheng Yang, Say Kong Ng, Tessa Rui Min Tan, Hock Chuan Yeo, Hsueh Lee Lim, Sze Wai Ng, Ai Ping Hiu, Chung Ping Chow, Corrine Wan, Shuwen Chen, Gavin Teo, Gao Song, Ju Xin Chin, Xiaoan Ruan, Ken Wing Kin Sung, Wei-Shou Hu, Miranda Gek Sim Yap, Muriel Bardor, Niranjan Nagarajan, Dong-Yup Lee  Cell Systems  Volume 4, Issue 5, Pages 530-542.e6 (May 2017) DOI: 10.1016/j.cels.2017.04.009 Copyright © 2017 The Author(s) Terms and Conditions

Cell Systems 2017 4, 530-542.e6DOI: (10.1016/j.cels.2017.04.009) Copyright © 2017 The Author(s) Terms and Conditions

Figure 1 Schematic Illustration of the Proposed Mammalian Systems Biotechnology Framework Combining the Multi-omics Data and Genome-Scale Model To unravel the global cellular adaptations between the wild-type CHO-K1 and antibody-producer SH-87 cell lines, multi-omics data were generated and then systematically analyzed in conjunction with GEM, thereby discovering the underlying mechanisms. Cell Systems 2017 4, 530-542.e6DOI: (10.1016/j.cels.2017.04.009) Copyright © 2017 The Author(s) Terms and Conditions

Figure 2 Genomic and Transcriptional Changes between CHO-K1 and SH-87 (A) Amplifications and rearrangements unique to SH-87 compared with the CHO-K1 genome. Note that only five of the six transgene integration sites are shown. The sixth integration site occurred on a scaffold, which was too small and excluded from the plot. (B) Amplifications and rearrangements in the vector sequence. (C) Impact of genomic rearrangements on vector-encoded transcripts. Cell Systems 2017 4, 530-542.e6DOI: (10.1016/j.cels.2017.04.009) Copyright © 2017 The Author(s) Terms and Conditions

Figure 3 The Effect of Genomic Variations on Gene Expression (A) Pie charts depicting the proportion of genes significantly up- or downregulated among genes with varying copy number. (B) Differentially expressed genes and their respective pathway. Summary of cellular components/functions enriched according to transcriptomics data. Cell Systems 2017 4, 530-542.e6DOI: (10.1016/j.cels.2017.04.009) Copyright © 2017 The Author(s) Terms and Conditions

Figure 4 Differential Expression of Individual Reactions Visualized Using the CHO Genome-Scale Metabolic Network For visualization purposes, the negative or positive log10 of the p value is presented in the network diagram. The color intensity in the network diagram indicates the significance of up- or downregulation and not fold change. Cell Systems 2017 4, 530-542.e6DOI: (10.1016/j.cels.2017.04.009) Copyright © 2017 The Author(s) Terms and Conditions

Figure 5 Culture Profiling and Metabolic Modeling of CHO-K1 and SH-87 Cell Lines (A–D) (A) Viable cell density and % viability in batch cultures, (B) metabolic profiles of glucose, lactate and IgG titer, (C) metabolic profiles of NH4, glutamine and glutamate and (D) the sampling results of a few key reactions of central metabolism whose feasible flux distributions are significant are shown. The error bars in (A), (B), and (C) denote the confidence intervals (mean ± SD). Highlighted regions in (A), (B), and (C) correspond to the exponential growth phase. The blue and red lines in (A), (B), and (C) denote CHO-K1 and SH-87, respectively. Diamonds and circles in (A) denote viability and VCD concentration, respectively. Diamonds, circles, and squares in (B) denote lactate, glucose, and IgG concentrations, respectively. Diamonds, circles, and squares in (B) denote NH4, glutamate, and glutamine concentrations, respectively. Blue and red lines in the insert graphs in (D) indicate the flux distributions of CHO-K1 and SH-87, respectively. Metabolite abbreviations are as follows: 13DPG, 1,3-diphosphoglycerate; 2PG, 2-phosphoglycerate; 3PG, 3-phosphoglycerate; ACCOA, acetyl-CoA; AKG, α-ketoglutaric acid; CIT, citrate; DHAP, dihydroxy acetone phosphate; E4P, erythrose-4-phosphate; F6P, fructose 6-phosphate; FDP, fructose diphosphate; FUM, fumarate; G3P, glyceraldehyde 3-phosphate; G6P, glucose 6-phosphate; GLC, glucose; LAC, lactate; MAL, malate; OAA, oxaloacetic acid; PEP, phosphoenolpyruvate; PYR, pyruvate; Q, oxidized ubiquinone; QH2, reduced ubiquinone; S7P, sedoheptulose 7-phosphate; SUCC, succinate; X5P, xylose 5-phosphate; XU5P, xylulose 5-phosphate. Reaction/gene abbreviations are as follows: ACON, aconitase; ALD, aldolase; ATPS, ATP synthase; ENO, enolase; CS, citrate synthase; CYB, cytochrome b reductase; FH, fumarase; G6PI, glucose-6-phoshate isomerase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HXK, hexokinase; IDH, isocitrate dehydrogenase; LDH, lactate dehydrogenase; MDH, malate dehydrogenase; NDH, NADH dehydrogenase; OGDH, α-ketoglutaric acid dehydrogenase; PDH, pyruvate dehydrogenase; PFK, phosphofructokinase; PGK, phosphoglycerate kinase; PGM, phosphoglycerate mutase; PYK, pyruvate kinase; SDH, succinate dehydrogenase; RPE, ribose epimerase; RPI, ribose isomerase; TAL, transaldolase; TKT, transketolase; TPI, triose phosphate isomerase. Cell Systems 2017 4, 530-542.e6DOI: (10.1016/j.cels.2017.04.009) Copyright © 2017 The Author(s) Terms and Conditions

Figure 6 Summary of Primary Metabolic Changes between CHO-K1 and SH-87 Cell Lines Composition of metabolite classes most distinctly different between CHO-K1 and SH-87 cell lines and illustration of the energy metabolism, phospholipid metabolism, and ceramide metabolism detailing the main changes in gene expression and metabolite level between the CHO-K1 and SH-87 cell lines. The nodes in blue indicate the reporter metabolites, whereas the nodes circled with red or blue are the metabolites measured by liquid chromatography-mass spectrometry with increased and decreased levels in SH-87, respectively. The red and green edges show up- and downregulated genes, respectively. Metabolite abbreviations are as follows: ACCOA, acetyl-CoA; AKG, α-ketoglutaric acid; CDPCHO, CDP-choline; CER, ceramide; CHO, choline; CIT, citrate; DHCER, dihydroceramide; FUM, fumarate; G3P, glycerol 3-phosphate; GALCER, galactosyl ceramide; GLCER, glucosyl ceramide; GLU, glutamate; GLY, glycine; GPC, glycerophosphocholine; GSH, reduced glutathione; GSSG, oxidized glutathione; LAC, lactate; LPC, lysophosphocholine; MAL, malate; OAA, oxaloacetic acid; PC, phosphatidyl choline; PCHO, phosphocholine; PEP, phosphoenolpyruvate; PYR, pyruvate; SM, sphingomyelin; SPH, sphinganine; SUCC, succinate. Reaction/gene abbreviations are as follows: CCT, choline phosphate cytididyltransferase; CK, choline kinase; CPT, choline phosphotransferase; CS, citrate synthase; FH, fumarase; GPP, glycerophosphodiester phosphodiesterase; IDH, isocitrate dehydrogenase; LDH, lactate dehydrogenase; LPL, lysophospholipase; MDH, malate dehydrogenase; OGDH, α-ketoglutaric acid dehydrogenase; PLA, phospholipase; PDH, pyruvate dehydrogenase; PYK, pyruvate kinase; SDH, succinate dehydrogenase. Cell Systems 2017 4, 530-542.e6DOI: (10.1016/j.cels.2017.04.009) Copyright © 2017 The Author(s) Terms and Conditions