skip to main content

Title: Modeling of copy number variability in Pichia pastoris

Development of continuous biopharmaceutical manufacturing processes is an area of active research. This study considers the long‐term transgene copy number stability ofPichia pastorisin continuous bioreactors. We propose a model of copy number loss that quantifies population heterogeneity. An analytical solution is derived and compared with existing experimental data. The model is then used to provide guidance for stable operating timescales. The model is extended to consider copy number dependent growth such as in the case of Zeocin supplementation. The model is also extended to analyze a continuous seeding strategy. This study is a critical step towards understanding the impact of continuous processing on the stability ofPichia pastorisand the resultant products.

more » « less
Author(s) / Creator(s):
 ;  ;  ;  ;  ;  ;  
Publisher / Repository:
Wiley Blackwell (John Wiley & Sons)
Date Published:
Journal Name:
Biotechnology and Bioengineering
Page Range / eLocation ID:
p. 1832-1839
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. Abstract

    Hydrophobins are small highly surface‐active fungal proteins with potential as biosurfactants in a wide array of applications. However, practical implementation of hydrophobins at large scale has been hindered by low recombinant yields. In this study, the effects of increasing hydrophobin gene copy number and overexpressing endoplasmic reticulum resident chaperone proteins Kar2p, Pdi1p, and Ero1p were explored as a means to enhance recombinant yields of the class II hydrophobin HFBI in the eukaryotic expression hostPichia pastoris. One‐, 2‐, and 3‐copy‐HFBI strains were attained using an in vitro multimer ligation approach, with strains displaying copy number stability following subsequent transformations as measured by quantitative polymerase chain reaction. Increasing HFBI copy number alone had no effect on increasing HFBI secretion, but increasing copy number in concert with chaperone overexpression synergistically increased HFBI secretion. Overexpression ofPDI1orERO1caused insignificant changes in HFBI secretion in 1‐ and 2‐copy strains, but a statistically significant HFBI secretion increase in 3‐copy strain.KAR2overexpression consistently resulted in enhanced HFBI secretion in all copy number strains, with 3‐copy‐HFBI secreting 22±1.6 fold more than the 1‐copy‐HFBI/no chaperone strain. The highest increase was seen in 3‐copy‐HFBI/Ero1p overexpressing strain with 30±4.0 fold increase in HFBI secretion over 1‐copy‐HFBI/no chaperone strain. This corresponded to an expression level of approximately 330 mg/L HFBI in the 5 ml small‐scale format used in this study.

    more » « less
  2. Abstract

    Soybean growers widely use theResistance toHeteroderaglycines1 (Rhg1) locus to reduce yield losses caused by soybean cyst nematode (SCN).Rhg1is a tandemly repeated four gene block. Two classes of SCN resistance‐conferringRhg1haplotypes are recognized:rhg1‐a(“Peking‐type,” low‐copy number, three or fewerRhg1repeats) andrhg1‐b(“PI 88788‐type,” high‐copy number, four or moreRhg1repeats). Therhg1‐aandrhg1‐bhaplotypes encode α‐SNAP (alpha‐SolubleNSFAttachmentProtein) variants α‐SNAPRhg1LC and α‐SNAPRhg1HC, respectively, with differing atypical C‐terminal domains, that contribute to SCN resistance. Here we report thatrhg1‐asoybean accessions harbor a copia retrotransposon within theirRhg1 Glyma.18G022500(α‐SNAP‐encoding) gene. We termed this retrotransposon “RAC,” forRhg1alpha‐SNAPcopia. Soybean carries multipleRAC‐like retrotransposon sequences. TheRhg1 RACinsertion is in theGlyma.18G022500genes of all truerhg1‐ahaplotypes we tested and was not detected in any examinedrhg1‐borRhg1WT(single‐copy) soybeans.RACis an intact element residing within intron 1, anti‐sense to therhg1‐a α‐SNAPopen reading frame.RAChas intrinsic promoter activities, but overt impacts ofRACon transgenic α‐SNAPRhg1LC mRNA and protein abundance were not detected. From the nativerhg1‐a RAC+genomic context, elevated α‐SNAPRhg1LC protein abundance was observed in syncytium cells, as was previously observed for α‐SNAPRhg1HC (whoserhg1‐bdoes not carryRAC). Using a SoySNP50K SNP corresponding withRACpresence, just ~42% of USDA accessions bearing previously identifiedrhg1‐aSoySNP50K SNP signatures harbor theRACinsertion. Subsequent analysis of several of these putativerhg1‐aaccessions lackingRACrevealed that none encodedα‐SNAPRhg1LC, and thus, they are notrhg1‐a.rhg1‐ahaplotypes are of rising interest, withRhg4, for combating SCN populations that exhibit increased virulence against the widely usedrhg1‐bresistance. The present study reveals another unexpected structural feature of manyRhg1loci, and a selectable feature that is predictive ofrhg1‐ahaplotypes.

    more » « less
  3. Abstract

    A major challenge in the pursuit of higher‐energy‐density lithium batteries for carbon‐neutral‐mobility is electrolyte compatibility with a lithium metal electrode. This study demonstrates the robust and stable nature of acloso‐borate based gel polymer electrolyte (GPE), which enables outstanding electrochemical stability and capacity retention upon extensive cycling. The GPE developed herein has an ionic conductivity of 7.3 × 10−4 S cm−2at room temperature and stability over a wide temperature range from −35 to 80 °C with a high lithium transference number ( = 0.51). Multinuclear nuclear magnetic resonance and Fourier transform infrared are used to understand the solvation environment and interaction between the GPE components. Density functional theory calculations are leveraged to gain additional insight into the coordination environment and support spectroscopic interpretations. The GPE is also established to be a suitable electrolyte for extended cycling with four different active electrode materials when paired with a lithium metal electrode. The GPE can also be incorporated into a flexible battery that is capable of being cut and still functional. The incorporation of acloso‐borate into a gel polymer matrix represents a new direction for enhancing the electrochemical and physical properties of this class of materials.

    more » « less
  4. Abstract

    Chinese hamster ovary (CHO) cells are essential to biopharmaceutical manufacturing and production instability, the loss of productivity over time, is a long‐standing challenge in the industry. Accurate prediction of cell line stability could enable efficient screening to identify clones suitable for manufacturing saving significant time and costs. DNA repair genes may offer biomarkers to address this need. In this study, over 40 cell lines representing various host lineages from three companies/organizations were evaluated for expression of five DNA repair genes (Fam35a,Lig4,Palb2,Pari, andXrcc6). Expression measured in cells with less than 30 population doubling levels (PDLs) was correlated to stability profiles at 60+ PDL. Principal component analysis identified markers which separate stable and unstable CHO‐DG44 cell lines. Notably, two genes,Lig4andXrcc6, showed higher expression in unstable CHO‐DG44 cell lines with copy number loss identified as the mechanism of production instability. Expression levels across all cell ages showed lower DNA repair gene expression was associated with increased cell age. Collectively, DNA repair genes provide critical insight into long‐term behavior of CHO cells and their expression levels have potential to predict cell line stability in certain cases.

    more » « less
  5. Abstract

    Study of the late Quaternary geomagnetic field contributes significantly to understanding the origin of millennial‐scale paleomagnetic secular variations, the structure of geomagnetic excursions, and the long‐term shielding by the geomagnetic field. A compilation of paleomagnetic sediment records and archeomagnetic and lava flow data covering the past 100 ka enables reconstruction of the global geomagnetic field on such long‐term scales. We use regularized inversion to build the first global, time‐dependent, geomagnetic field model spanning the past 100 ka, namedGGF100k(GlobalGeomagneticField over the past100 ka). Spatial parametrization of the model is in spherical harmonics and time variations with cubic splines. The model is heavily constrained by more than 100 continuous sediment records covering extended periods of time, which strongly prevail over the limited number of discrete snapshots provided by archeomagnetic and volcanic data. Following an assessment of temporal resolution in each sediment's magnetic record, we have introduced smoothing kernels into the forward modeling when assessing data misfit. This accommodates the smoothing inherent in the remanence acquisition in individual sediment paleomagnetic records, facilitating a closer fit to both high‐ and low‐resolution records in regions where some sediments have variable temporal resolutions. The model has similar spatial resolution but less temporal complexity than current Holocene geomagnetic field models. Using the new reconstruction, we discuss dipole moment variations, the time‐averaged field, and paleomagnetic secular variation activity. The new GGF100k model fills the gap in the geomagnetic power spectrum in the frequency range 100–1,000 Ma−1.

    more » « less