Self-Cycling Fermentation: Bioprocessing for the -omics era Self-Cycling Fermentation: Bioprocessing

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Text of Self-Cycling Fermentation: Bioprocessing for the -omics era Self-Cycling Fermentation: Bioprocessing

  • Self-Cycling Fermentation: Bioprocessing for the -omics era

    Department of Chemical Engineering and Materials Engineering

    University of Alberta

    Edmonton, Alberta, Canada

    Zachary J. Storms And

    Dominic Sauvageau

    BioPacific Rim Conference December 9, 2014

  • Microbial Cells as Factories

    Zack Storms

    Introduction

    212/9/2014

  • The -omics Era: Expanding our understanding of microbial cells

    Zack Storms

    Introduction

    312/9/2014

    Genomics:

    Gene function

    (DNA)

    Transcriptomics:

    Gene expression (RNA)

    Metabolomics:

    Biochemical Reactions

    How can we tailor large-scale bioprocessing to complement the knowledge we are gaining through –omics?

  • Biomass Production of E. coli during Self-Cycling Fermentation

    Self-Cycling Fermentation

    Zack Storms

    Features of SCF

    • Cyclical Semi-continuous Reactor

    • Contents halved upon depletion of limiting nutrient – Replenished with fresh media

    • Cycle period = cell doubling time

    • Maintains exponential growth – Reproducible, stable

    • Induces cell synchrony

    Storms et al. (2012) Self-Cycling Operation Increases Productivity of Recombinant Protein in Escherichia coli. Biotechnol Bioeng. 109: 2262-2270

    412/9/2014

  • Cell Synchrony

    Zack Storms

    Asynchronous cell growth Ideal synchronized cell growth

    Cell division occurs throughout entire cycle

    Cell division occurs at infinitely small time interval in cycle

    5

    Features of SCF

    12/9/2014

  • Growth of E. coli during a SCF Cycle

    Zack Storms

    • Cell division occurs in middle of cycle

    • Synchrony Index ≈ 0.6-0.7

    • Growth of culture behaves like individual cell

    • Cell metabolism slows down – Doubling time ~150 minutes

    Cell Density in synchronized cycle

    Sauvageau D, Storms ZJ, Cooper DG (2010) Synchronized Populations of Escherichia coli Using Simplified Self-Cycling Fermentation. J Biotechnol. 149: 67-73.

    6

    Note: Error bars represent the standard deviation

    Features of SCF

    How can we use –omics to take advantage of these properties in large scale fermentation?

    12/9/2014

  • Studies on synchronized cells with Bacteriophages

    Zack Storms

    • How does cell division cycle effect cell productivity?

    • Bacteriophage infections at different points in cell division cycle

    • Parameters measured – Burst size: phages/cell

    – Lysis time: phage incubation period

    – Intracellular RNA and DNA

    Storms ZJ, Brown T, Cooper DG, Sauvageau D, Leask RL (2014) Impact of the cell life-cycle on bacteriophage T4 infection FEMS Microbiol. Lett. 353: 63-68.

    7

    Increasing productivity using SCF

    Cell Growth and

    DNA Replication

    Cell Division

    12/9/2014

  • Studies on synchronized cells with Bacteriophages

    Zack Storms

    • How does cell division cycle effect cell productivity?

    • Burst size largest immediately preceding cell division

    • Lysis time shortest immediately preceding cell division

    Storms ZJ, Brown T, Cooper DG, Sauvageau D, Leask RL (2014) Impact of the cell life-cycle on bacteriophage T4 infection FEMS Microbiol. Lett. 353: 63-68.

    8

    Increasing productivity using SCF

    Results from cells infected by phage T4 at different points in their cell division cycle

    Cell Division

    12/9/2014

  • Studies on synchronized cells with Bacteriophages

    Zack Storms

    • How does cell division cycle effect cell productivity?

    • Burst size largest immediately preceding cell division

    • Lysis time shortest immediately preceding cell division

    • Productivity highest immediately preceding cell division

    9

    Increasing productivity using SCF

    Phage productivity of cells infected by phage T4 at different points in their cell division cycle

    Cell Division

    12/9/2014

    Storms ZJ, Brown T, Cooper DG, Sauvageau D, Leask RL (2014) Impact of the cell life-cycle on bacteriophage T4 infection FEMS Microbiol. Lett. 353: 63-68.

  • Studies on synchronized cells with Bacteriophages

    Zack Storms

    Storms ZJ, Brown T, Cooper DG, Sauvageau D, Leask RL (2014) Impact of the cell life-cycle on bacteriophage T4 infection FEMS Microbiol. Lett. 353: 63-68.

    10

    Increasing productivity using SCF

    12/9/2014

    • Why does cell productivity change with cell age?

    • Intracellular resources – RNA – Protein Synthesizing System – Transcriptomics

    • Burst size positively correlated to total cellular RNA

    • Lysis time negatively correlated to total cellular RNA

    • Productivity positively correlated with cellular RNA

  • Zack Storms

    • Synchronized host: – Maintains same level of

    phage production

    – Lower cell concentration

    – The number of phages per cell (burst size) is larger for a synchronized culture

    Sauvageau D and Cooper DG. (2010) Two-stage, self cycling process for the production of bacteriophages Microbial Cell Factories, 9:81

    11

    Increasing productivity using SCF

    Self-Cycling Fermentation

    Bacteriophage Production

    Implications for large scale production processes: bacteriophage production

    Infected E. coli cultures

    12/9/2014

  • Implications for large scale production processes: recombinant protein production

    • β-galactosidase production using recombinant bacteriophage – Induce production at different time

    points in SCF cycle

    Zack Storms 12

    Storms et al. (2012) Self-Cycling Operation Increases Productivity of Recombinant Protein in Escherichia coli. Biotechnol Bioeng. 109: 2262-2270

    Note: Error bars represent the standard deviation

    Increasing productivity using SCF

    12/9/2014

    0.0E+00

    1.0E+04

    2.0E+04

    3.0E+04

    4.0E+04

    5.0E+04

    6.0E+04

    7.0E+04

    0 50 100 150 200 250

    S p

    e c

    if ic

    I n

    te g

    ra te

    d P

    ro d

    u c

    ti v it

    y

    (U /L

    /h /O

    D )

    Induction Time (minutes) (Cell Age)

    Unsynchronized Culture

  • Implications for large scale production processes: recombinant protein production

    • β-galactosidase production using recombinant bacteriophage – Induce production at different time

    points in SCF cycle

    • Productivity of synchronized cultures

    – Maximum in productivity 50% larger than in non-synchronized culture

    – Maximum occurs 45 minutes earlier

    Zack Storms 13

    Storms et al. (2012) Self-Cycling Operation Increases Productivity of Recombinant Protein in Escherichia coli. Biotechnol Bioeng. 109: 2262-2270

    Note: Error bars represent the standard deviation

    Increasing productivity using SCF

    12/9/2014

    0.0E+00

    1.0E+04

    2.0E+04

    3.0E+04

    4.0E+04

    5.0E+04

    6.0E+04

    7.0E+04

    0 50 100 150 200 250

    S p

    e c

    if ic

    I n

    te g

    ra te

    d P

    ro d

    u c

    ti v it

    y

    (U /L

    /h /O

    D )

    Induction Time (minutes) (Cell Age)

    Unsynchronized Culture

    Synchronized Culture

  • Implications for large scale production processes: recombinant protein production

    • β-galactosidase production using recombinant bacteriophage – Induce production at different time

    points in SCF cycle

    • Productivity of synchronized cultures

    – Maximum in productivity 50% larger than in non-synchronized culture

    – Maximum occurs 45 minutes earlier

    – Two distinct maxima observed

    Zack Storms 14

    Storms et al. (2012) Self-Cycling Operation Increases Productivity of Recombinant Protein in Escherichia coli. Biotechnol Bioeng. 109: 2262-2270

    Note: Error bars represent the standard deviation

    Increasing productivity using SCF

    12/9/2014

    0.0E+00

    1.0E+04

    2.0E+04

    3.0E+04

    4.0E+04

    5.0E+04

    6.0E+04

    7.0E+04

    0 50 100 150 200 250

    S p

    e c

    if ic

    I n

    te g

    ra te

    d P

    ro d

    u c

    ti v it

    y

    (U /L

    /h /O

    D )

    Induction Time (minutes) (Cell Age)

    Synchronized Culture

  • Implications for large scale production processes: recombinant protein production

    • β-galactosidase production using recombinant bacteriophage

    • Productivity of synchronized cultures

    – Maximum in productivity 50% larger than in non-synchronized culture

    – Maximum occurs 45 minutes earlier

    – Two distinct maxima observed • Before and after cell division

    Zack Storms 15

    Storms et al. (2012) Self-Cycling Operation Increases Productivity of Recombinant Protein in Escherichia coli. Biotechnol Bioeng. 109: 2262-2270

    Note: Error bars represent the standard deviation

    Increasing productivity using SCF

    12/9/2014