Dynamics of Amino Acid Metabolism, Gene Expression, and Circulomics in a Recombinant Chinese Hamster Ovary Cell Line Adapted to Moderate and High Levels of Extracellular Lactate

Chitwood, Dylan G.; Uy, Lisa; Fu, Wanfang; Klaubert, Stephanie R.; Harcum, Sarah W.; Saski, Christopher

doi:10.3390/genes14081576

Cited by 3 publications

(1 citation statement)

References 101 publications

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“…Analysis of eccDNA origins revealed eccDNA biogenesis on every chromosome with hotspots (high frequency of eccDNA formation) on chromosomes 1 and 7 (Figure 6). The identification of eccDNA originating from all over the genome is consistent with A. palmeri [1], as well as recent studies in mammalian cells [64,65]. These data provide further evidence that eccDNA biogenesis contributes to evolutionary innovation by contributing global genetic heterogeneity, plasticity, and a critical element to a plant's trajectory to rapid adaptation.…”

Section: Discussionsupporting

confidence: 82%

Sequence Characterization of Extra-Chromosomal Circular DNA Content in Multiple Blackgrass (Alopecurus myosuroides) Populations

Fu,

MacGregor,

Comont

et al. 2023

Genes

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Alopecurus myosuroides (blackgrass) is a problematic weed of Western European winter wheat, and its success is largely due to widespread multiple-herbicide resistance. Previous analysis of F2 seed families derived from two distinct blackgrass populations exhibiting equivalent non-target site resistance (NTSR) phenotypes shows resistance is polygenic and evolves from standing genetic variation. Using a CIDER-seq pipeline, we show that herbicide-resistant (HR) and herbicide-sensitive (HS) F3 plants from these F2 seed families as well as the parent populations they were derived from carry extra-chromosomal circular DNA (eccDNA). We identify the similarities and differences in the coding structures within and between resistant and sensitive populations. Although the numbers and size of detected eccDNAs varied between the populations, comparisons between the HR and HS blackgrass populations identified shared and unique coding content, predicted genes, and functional protein domains. These include genes related to herbicide detoxification such as Cytochrome P450s, ATP-binding cassette transporters, and glutathione transferases including AmGSTF1. eccDNA content was mapped to the A. myosuroides reference genome, revealing genomic regions at the distal end of chromosome 5 and the near center of chromosomes 1 and 7 as regions with a high number of mapped eccDNA gene density. Mapping to 15 known herbicide-resistant QTL regions showed that the eccDNA coding sequences matched twelve, with four QTL matching HS coding sequences; only one region contained HR coding sequences. These findings establish that, like other pernicious weeds, blackgrass has eccDNAs that contain homologs of chromosomal genes, and these may contribute genetic heterogeneity and evolutionary innovation to rapidly adapt to abiotic stresses, including herbicide treatment.

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Section: Discussionsupporting

confidence: 82%

Sequence Characterization of Extra-Chromosomal Circular DNA Content in Multiple Blackgrass (Alopecurus myosuroides) Populations

Fu,

MacGregor,

Comont

et al. 2023

Genes

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CHO stable pool fed‐batch process development of SARS‐CoV‐2 spike protein production: Impact of aeration conditions and feeding strategies

Reyes,

Pham,

Durocher

et al. 2024

Biotechnology Progress

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Technology scale‐up and transfer are a fundamental and critical part of process development in biomanufacturing. Important bioreactor hydrodynamic characteristics such as working volume, overhead gas flow rate, volumetric power input (P/V), impeller type, agitation regimen, sparging aeration strategy, sparger type, and kLa must be selected based on key performance indicators (KPI) to ensure a smooth and seamless process scale‐up and transfer. Finding suitable operational setpoints and developing an efficient feeding regimen to ensure process efficacy and consistency are instrumental. In this investigation, process development of a cumate inducible Chinese hamster ovary (CHO) stable pool expressing trimeric SARS‐CoV‐2 spike protein in 1.8 L benchtop stirred‐tank bioreactors is detailed. Various dissolved oxygen levels and aeration air caps were studied to determine their impact on cell growth and metabolism, culture longevity, and endpoint product titers. Once hydrodynamic conditions were tuned to an optimal zone, various feeding strategies were explored to increase culture performance. Dynamic feedings such as feeding based on current culture volume, viable cell density (VCD), oxygen uptake rate (OUR), and bio‐capacitance signals were tested and compared to standard bolus addition. Increases in integral of viable cell concentration (IVCC) (1.25‐fold) and protein yield (2.52‐fold), as well as greater culture longevity (extension of 5 days) were observed in dynamic feeding strategies when compared to periodic bolus feeding. Our study emphasizes the benefits of designing feeding strategies around metabolically relevant signals such as OUR and bio‐capacitance signals.

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Sequence characterization of eccDNA content in multiple blackgrass (Alopecurus myosuroides) populations

Fu,

MacGregor,

Comont

et al. 2023

Preprint

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Alopecurus myosuroides (blackgrass) is a problematic weed of Western European winter wheat and its success is largely due to widespread multiple-herbicide resistance. Previous analysis of F2 seed families derived from two distinct blackgrass populations exhibiting equivalent non-target site resistance (NTSR) phenotypes shows resistance is polygenic and evolves from standing genetic variation. Using a CIDER-seq pipeline we show that herbicide resistant (HR) and herbicide sensitive (HS) F3 plants from these F2 seed families as well as the parent populations they were derived from carry extra-chromosomal circular DNA (eccDNA). We identify the similarities and differences in the coding structures within and between resistant and sensitive populations. Although the numbers and size of detected eccDNAs varied between the populations, comparisons between HR and HS blackgrass populations identified shared and unique coding content, predicted genes, and functional protein domains. These include genes related to herbicide detoxification such as Cytochrome P450s, ATP-binding cassette transporters and glutathione transferases including AmGSTF1. eccDNA content was mapped to the Alopecurus myosuroides reference genome, revealing genomic regions at distal end of chromosome 5 and the near center of chromosome 1 and 7 as regions with high number of mapped eccDNA gene density. Mapping to 15 known herbicide-resistant QTL regions showed the eccDNA coding sequences match 12 with four QLT matching HS coding sequences one region only contained HR coding sequences. These findings establish that like other pernicious weeds, blackgrass has eccDNAs that contain homologs of chromosomal genes, and these may contribute genetic heterogeneity and evolutionary innovation to rapidly adapt to abiotic stresses including herbicide treatment.

show abstract

Dynamics of Amino Acid Metabolism, Gene Expression, and Circulomics in a Recombinant Chinese Hamster Ovary Cell Line Adapted to Moderate and High Levels of Extracellular Lactate

Cited by 3 publications

References 101 publications

Sequence Characterization of Extra-Chromosomal Circular DNA Content in Multiple Blackgrass (Alopecurus myosuroides) Populations

Sequence Characterization of Extra-Chromosomal Circular DNA Content in Multiple Blackgrass (Alopecurus myosuroides) Populations

CHO stable pool fed‐batch process development of SARS‐CoV‐2 spike protein production: Impact of aeration conditions and feeding strategies

Sequence characterization of eccDNA content in multiple blackgrass (Alopecurus myosuroides) populations

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