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Protein glycosylation is critically important in vivo; current estimates are that more than half of the proteins in the SWISS‐PROT database are glycoproteins. Glycosylation plays a substantial role wide a range of physiological and pathological processes including development, immunology, cancer, and infectious disease. Protein glycosylation is also vitally important in the development of therapeutic bioproducts. Currently, more than 165 recombinant protein pharmaceuticals are approved for human use, with another 500 in preclinical and clinical trials. Of these, approximately 70% are glycosylated proteins. Glycosylation affects the structure, activity, immunogenicity, protease sensitivity, stability, and biological clearance of glycoproteins. Hence, an understanding of the mechanisms by which proteins are glycosylated, and strategies for analyzing and controlling glycoforms has become increasingly important in the development of biopharmaceuticals. Advances in chromatography and mass spectrometry have permitted more detailed identification of glycans, while cellular and protein engineering strategies have allowed manipulation of the glycoforms. In this chapter, we review the biology of protein glycosylation, methods for identifying and characterizing glycans and glycoproteins, and the effects of host cell line, culture conditions, and cellular engineering on the glycoforms of recombinant glycoproteins, providing a comprehensive overview of glycosylation of recombinant protein therapeutics.

Fungal secondary metabolites are a rich source of valuable natural prod-5 ucts. Genome sequencing have revealed an enormous potential from predicted biosyn-6 thetic gene clusters. It is however currently a time consuming task and an unfeasible 7 task to characterize all biosynthetic gene cluster and to identify possible uses of the 8 compounds. A rational approach is needed to identify promising gene clusters respon-9 sible for producing valuable compounds. Several valuable bioactive clusters have been 10 shown to include a resistance gene which is a paralog of the target gene inhibited by 11 the compound. This mechanism can be used to design a rational approach selecting 12 those clusters. 13 We have developed a pipeline FRIGG (Fungal ResIstance Gene-directed Genome 14 mining) identifying putative resistance genes found in biosynthetic gene clusters based 15 on homology patterns of the cluster genes. The FRIGG pipeline has been run using 51 16 Aspergillus and Penicillium genomes, identifying 72 unique protein families with putative 17 resistance genes using various settings in the pipeline. The pipeline was also able to 18 identify the characterized resistance gene inpE from the Fellutamide B cluster thereby 19 validating the approach. 20 We have successfully developed an approach identifying putative valuable bio-21 active clusters based on a specific resistance mechanism. This approach will be highly 22 ms Submission Template mSystems Submission Template mSystems Submission Template mSystems Submission Template mSystems Submission Template mSystems Submission Tem 1 Kjaerbølling et al. number of secondary metabolite gene clusters than the number of characterized 41 secondary metabolites thus revealing a much larger potential (2, 3, 4, 1). The number 42 of sequenced genomes is ever increasing mainly due to large sequencing efforts such as 43 the 1000 Fungal Genomes Project of the Department of Energy Joint Genomes Initiative 44 (http://1000.fungalgenomes.org/home/) and the 300 Aspergillus genome project (5, 6) 45 and therefore the number of predicted secondary metabolite gene clusters is steadily 46 increasing. 47 Despite progress in molecular tools and methods for characterization of secondary 48 metabolite gene clusters, it is still a time-consuming task, making it unfeasible to 49 investigate all predicted secondary metabolite gene clusters. Therefore only a small 50 fraction of the predicted clusters are characterized and investigated experimentally. 51 With the plethora of predicted secondary metabolite gene clusters (clusters) and the 52 aim of discovering novel bio-active compounds useful as drugs, the question emerges: 53 How do we select the most interesting predicted clusters producing potential valuable 54 drugs such as anti-fungicides, anti-cancer drugs and anti-microbial compounds? To 55 meet this need we have created a pipeline FRIGG (Fungal ResIstance Gene-directed 56 Genome mining) identifying clusters producing likely bio-active compounds based on 57 resistance genes. Many bio-active...

Glutathione (GSH) plays a central role in the redox balance maintenance in mammalian cells. The study of industrial CHO cell lines have demonstrated a close link between GSH metabolism and clone productivity. However, a deep investigation is still required to understand this correlation and highlights new potential targets for cell engineering. In this study, we have modulated the GSH intracellular content of an industrial cell line under bioprocess conditions in order to further elucidate the role of the GSH synthesis pathway. Two strategies were used : the variation of cystine supply and the direct inhibition of the GSH synthesis using buthionine sulfoximine (BSO). Cysteine supply modulation have revealed a correlation between intracellular GSH and product titer over time. Analysis of metabolites uptake/secretion rates and proteome comparison between BSO-treated cells and non-treated cells has highlighted a slow down of the TCA cycle leading to a secretion of lactate and alanine in the extracellular environment. Moreover, an adaptation of the glutathione related proteome has been observed with a up-regulation of the regulatory subunit of glutamate cysteine ligase and a down-regulation of a specific glutathione transferase subgroup, the Mu family. Surprisingly, the main impact of BSO treatment was observed on a global down-regulation of the cholesterol synthesis pathways. As cholesterol is required for protein secretion, it can be the missing part of the jigsaw to finally elucidate the link between GSH synthesis and productivity.

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CHO cell lines are a workhorse for the production of pharmaceutical proteins, but show some limitations in the variability and stability of N-glycosylation profiles. One promising approach to addressing this at the required systems-level is miRNA, which can regulate a large number of genes and have predictable targets. Herein, we first identified de novo 656 potential miRNAs in the CHO genome based on a combination of literature, database searching, and miRNA sequencing. We further sequenced mRNA from the same cultures, and used a combination of mRNA-miRNA correlation analysis, target prediction and literature searches to find miRNAs potentially targeting N-glycosylation. Our ten best miRNA candidates were subjected to miRNA overexpression, knockdown, or knock-out in CHO cell lines. Out of the ten candidates, four (miR-128, miR-34c, miR-30b, and miR-449a) showed positive effects on N-glycosylation and could be applied directly for CHO cell engineering. The fact that 40% of the screened targets had a desired effect, and the prediction of 656 miRNAs illustrates the massive potential of miRNA engineering in CHO.

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