Roxana Disela, Tim Neijenhuis, Olivier Le Bussy, Geoffroy Geldhof, Marieke Klijn, Martin Pabst, Marcel Ottens
Purification of recombinantly produced biopharmaceuticals involves removal of host cell material, such as host cell proteins (HCPs). For lysates of the common expression host Escherichia coli (E. coli) over 1500 unique proteins can be identified. Currently, understanding the behavior of individual HCPs for purification operations, such as preparative chromatography, is limited. Therefore, we aim to elucidate the elution behavior of individual HCPs from E. coli strain BLR(DE3) during chromatography. Understanding this complex mixture and knowing the chromatographic behavior of each individual HCP improves the ability for rational purification process design. Specifically, linear gradient experiments were performed using ion exchange (IEX) and hydrophobic interaction chromatography, coupled with mass spectrometry-based proteomics to map the retention of individual HCPs. We combined knowledge of protein location, function, and interaction available in literature to identify trends in elution behavior. Additionally, quantitative structure–property relationship models were trained relating the protein 3D structure to elution behavior during IEX. For the complete data set a model with a cross-validated R2 of 0.55 was constructed, that could be improved to a R2 of 0.70 by considering only monomeric proteins. Ultimately this study is a significant step toward greater process understanding.
{"title":"Experimental characterization and prediction of Escherichia coli host cell proteome retention during preparative chromatography","authors":"Roxana Disela, Tim Neijenhuis, Olivier Le Bussy, Geoffroy Geldhof, Marieke Klijn, Martin Pabst, Marcel Ottens","doi":"10.1002/bit.28840","DOIUrl":"10.1002/bit.28840","url":null,"abstract":"<p>Purification of recombinantly produced biopharmaceuticals involves removal of host cell material, such as host cell proteins (HCPs). For lysates of the common expression host <i>Escherichia coli</i> (<i>E. coli</i>) over 1500 unique proteins can be identified. Currently, understanding the behavior of individual HCPs for purification operations, such as preparative chromatography, is limited. Therefore, we aim to elucidate the elution behavior of individual HCPs from <i>E. coli</i> strain BLR(DE3) during chromatography. Understanding this complex mixture and knowing the chromatographic behavior of each individual HCP improves the ability for rational purification process design. Specifically, linear gradient experiments were performed using ion exchange (IEX) and hydrophobic interaction chromatography, coupled with mass spectrometry-based proteomics to map the retention of individual HCPs. We combined knowledge of protein location, function, and interaction available in literature to identify trends in elution behavior. Additionally, quantitative structure–property relationship models were trained relating the protein 3D structure to elution behavior during IEX. For the complete data set a model with a cross-validated <i>R</i><sup>2</sup> of 0.55 was constructed, that could be improved to a <i>R</i><sup>2</sup> of 0.70 by considering only monomeric proteins. Ultimately this study is a significant step toward greater process understanding.</p>","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"121 12","pages":"3848-3859"},"PeriodicalIF":3.5,"publicationDate":"2024-09-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/bit.28840","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142231604","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Sucrose is a commonly utilized nutritive sweetener in food and beverages due to its abundance in nature and low production costs. However, excessive intake of sucrose increases the risk of metabolic disorders, including diabetes and obesity. Therefore, there is a growing demand for the development of nonnutritive sweeteners with almost no calories. d-Allulose is an ultra-low-calorie, rare six-carbon monosaccharide with high sweetness, making it an ideal alternative to sucrose. In this study, we developed a cell factory for d-allulose production from sucrose using Escherichia coli JM109 (DE3) as a chassis host. The genes cscA, cscB, cscK, alsE, and a6PP were co-expressed for the construction of the synthesis pathway. Then, the introduction of ptsG-F and knockout of ptsG, fruA, ptsI, and ptsH to reprogram sugar transport pathways resulted in an improvement in substrate utilization. Next, the carbon fluxes of the Embden-Meyerhof-Parnas and the pentose phosphate pathways were regulated by the inactivation of pfkA and zwf, achieving an increase in d-allulose titer and yield of 154.2% and 161.1%, respectively. Finally, scaled-up fermentation was performed in a 5 L fermenter. The titer of d-allulose reached 11.15 g/L, with a yield of 0.208 g/g on sucrose.
{"title":"Biosynthesis of nonnutritive monosaccharide d-allulose by metabolically engineered Escherichia coli from nutritive disaccharide sucrose","authors":"Ling-Jie Zheng, Wei-Xiang Chen, Shang-He Zheng, Irfan Ullah, Hui-Dong Zheng, Li-Hai Fan, Qiang Guo","doi":"10.1002/bit.28842","DOIUrl":"10.1002/bit.28842","url":null,"abstract":"<p>Sucrose is a commonly utilized nutritive sweetener in food and beverages due to its abundance in nature and low production costs. However, excessive intake of sucrose increases the risk of metabolic disorders, including diabetes and obesity. Therefore, there is a growing demand for the development of nonnutritive sweeteners with almost no calories. <span>d</span>-Allulose is an ultra-low-calorie, rare six-carbon monosaccharide with high sweetness, making it an ideal alternative to sucrose. In this study, we developed a cell factory for <span>d</span>-allulose production from sucrose using <i>Escherichia coli</i> JM109 (DE3) as a chassis host. The genes <i>cscA</i>, <i>cscB</i>, <i>cscK</i>, <i>alsE</i>, and <i>a6PP</i> were co-expressed for the construction of the synthesis pathway. Then, the introduction of <i>ptsG-F</i> and knockout of <i>ptsG</i>, <i>fruA</i>, <i>ptsI</i>, and <i>ptsH</i> to reprogram sugar transport pathways resulted in an improvement in substrate utilization. Next, the carbon fluxes of the Embden-Meyerhof-Parnas and the pentose phosphate pathways were regulated by the inactivation of <i>pfkA</i> and <i>zwf</i>, achieving an increase in <span>d</span>-allulose titer and yield of 154.2% and 161.1%, respectively. Finally, scaled-up fermentation was performed in a 5 L fermenter. The titer of <span>d</span>-allulose reached 11.15 g/L, with a yield of 0.208 g/g on sucrose.</p>","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"121 12","pages":"3684-3693"},"PeriodicalIF":3.5,"publicationDate":"2024-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142166363","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Melanie Maier, Stefan Schneider, Linus Weiss, Simon Fischer, Daniel Lakatos, Joey Studts, Matthias Franzreb
The cover image is based on the Article Tailoring polishing steps for effective removal of polysorbate-degrading host cell proteins in antibody purification by Melanie Maier et al., https://doi.org/10.1002/bit.28767.