MaSp2 Spider Silk Production Modeling - Utah State University Silk Team Meeting... · MaSp2 Spider Silk Production Modeling. Utah State University August 26, 2016 Spider Silk Team

August 26, 2016Utah State University

H. Scott HintonSpider Silk Team Meeting - MaSp2 Modeling -1-

MaSp2 Spider Silk Production Modeling



MaSp2 Modeling• Modeling Limitations• Bioproduct Identification• Select Host Strain• Bioproduct Pathway

Defining pathway Adding pathway to the model Adding the plasmid to the model

• Strain Design Bioproduct Maximum Production Carbon Sources Nutrient & Amino Acid Limitations

• Stress Response Stringent Response

Future Opportunities



Assumptions & Limitations• Model cell steady-state phenotypes during exponential

growth phase. Can model the different phenotypes that can exist

during the exponential growth phase. Can understand the capabilities of each phenotype Can identify and modify cellular pathways to favor

specific bioproduct producing phenotypes Constraint-based models do not model transitions

between phenotypes Most genome-scale models do not include the genes

required for the stationary phase (proteases, etc.) Most genome-scale models do not include the

complete transcription and translation pathways• The biomass function represents the average metabolic

load required during exponential cell growth. The biomass function represents the average

percentages of the component parts (amino acids, nucleotides, energy, etc.) that are included in 1 gm of cell biomass.

Decline PhaseDeath Phase

Log (Exponential) Phase

Lag Phase

Cell

Den

sity

(OD

)Time (hours)

Stationary Phase

Acceleration Phase

Purpose: Identify phenotypes that can exist in exponential growth phase










Spider Silk

Sarah Allred, “Metabolic Modeling of Spider Silk Production in E. coli,” MS Thesis, USU, 2014

• Wound dressings

• Tissue and bone scaffolds

• Sutures

• Artificial nerves

• Vessels for drug delivery

• Coatings for biomedical implants

• Ligament and tendon replacements

• Parachute cords

• Composite materials in aircrafts

• Construction materials

Potential Applications

Dragline Silk• Composed of two spider silk proteins;

MaSp1 (80%) and MaSp2 (20%)



MaSp2 Amino Acid Repeated Sequence

KLHMTGPGQQGPGGYGPGQQGPSGPGSAAAAAAAAGPGQQGPGGYGPGQQGPGGYGPGQQGPSGPGSAAAAAAAAGPGGYGPGQQGPGGYGPGQQGPGGYGPGQQGPSGPGSAAAAAAAAGPGQQGPGGYGPGQQGPGGYGPGQQGPSGPGSAAAAAAAAGPGQQGPGGYGPGQQGPSGPGSAAAAAAAAGPGQQGPGGYGPGQQGPGGYGPGQQGPSGPGSAAAAAAAAGPGGYGPGQQGPGGYGPGQQGPGGYGPGQQGPSGPGSAAAAAAAAGPGQQGPGGYGPGQQGPGGYGPGQQGPSGPGSAAAAAAAAGPGQQGPGGYGPGQQGPSGPGSAAAAAAAASGGD

• MaSp2 spider silk amino acid sequence (349 Amino acids with MW of 29.5 Daltons)

A: 72 Ala AlanineR: 0 Arg ArginineN: 0 Asn AsparagineD: 1 Asp Aspartic acid (Aspartate)C: 0 Cys CysteineQ: 48 Gln GlutamineE: 0 Glu Glutamic acid (Glutamate)G: 128 Gly GlycineH: 1 His HistidineI: 0 Ile IsoleucineL: 1 Leu LeucineK: 1 Lys LysineM: 1 Met MethionineF: 0 Phe PhenylalanineP: 59 Pro ProlineS: 19 Ser SerineT: 1 Thr ThreonineW: 0 Trp TryptophanY: 17 Tyr TyrosineV: 0 Val Valine

Number of each amino acid in sequence using Matlab

Bioinformatics function “aacount”

• Most used Amino acids

Glycine - 128 Alanine – 72 Proline – 59 Glutamine – 48










Host Selection• Select host that naturally produces the target bioproduct

• Select heterologous host that can be engineered Commonly used strains of E.coli

http://openwetware.org/wiki/E._coli_genotypes

• Commercial competent cell strains Invitrogen – Top10 -

http://www.thermofisher.com/order/catalog/product/K456001

Promega -https://www.promega.com/products/vectors/bacterial-expression-vectors/

Qiagen - https://www.qiagen.com/us/shop/sample-technologies/protein-sample-technologies/expression-kits-and-vectors/

Some Commonly Used E. coli Strains

AG1AB1157B2155BL21BL21(AI)BL21(DE3)BL21 (DE3) pLysSBNN93BNN97BW26434, CGSC Strain # 7658C600C600 hflA150 (Y1073, BNN102)CSH50D1210DB3.1DH1DH5αDH5α Turbo (NEB)DH10B (Invitrogen)DH12S (Invitrogen)DM1 (Invitrogen)E. cloni(r) 5alpha (Lucigen)E. cloni(r) 10G (Lucigen)E. cloni(r) 10GF' (Lucigen)E. coli K12 ER2738 (NEB)ER2566 (NEB)ER2267 (NEB)HB101HMS174(DE3)High-Control(tm) BL21(DE3) (Lucigen)High-Control(tm) 10G (Lucigen)

IJ1126IJ1127JM83JM101JM103JM105JM106JM107JM108JM109JM109(DE3)JM110JM2.300LE392M15 (Qiagen)Mach1MC1061MC4100MFDpirMG1655OmniMAX2OverExpress(tm)C41(DE3) (Lucigen)OverExpress(tm)C41(DE3)pLysS (Lucigen)OverExpress(tm)C43(DE3) (Lucigen)OverExpress(tm)C43(DE3)pLysS (Lucigen)Rosetta(DE3)pLysSRosetta-gami(DE3)pLysSRR1RV308STBL2 (Invitrogen)STBL3 (Invitrogen)

STBL4SURE (Stratagene)SURE2 (Stratagene)TG1 (Lucigen)TOP10 (Invitrogen)Top10F' (Invitrogen)W3110W3110 (λ857S7)WM3064XL1-Blue (Stratagene)XL1-Blue MRF' (Stratagene)XL2-Blue (Stratagene)XL2-Blue MRF' (Stratagene)XL1-Red (Stratagene)XL10-Gold (Stratagene)XL10-Gold KanR (Stratagene)

http://openwetware.org/wiki/E._coli_genotypes

http://www.thermofisher.com/order/catalog/product/K456001

https://www.promega.com/products/vectors/bacterial-expression-vectors/

https://www.qiagen.com/us/shop/sample-technologies/protein-sample-technologies/expression-kits-and-vectors/



BL21(DE3) E.coli Strain• BL21 cells are deficient in the Lon protease which degrades many foreign proteins.

• Another gene missing from the genome 21 is the one coding for the outer membrane protease OmpT, whose function is to degrade extracellular proteins. The liberated amino acids are then taken up by the cell. This is problematic in the expression of a recombinant protein as, after cell lysis, OmpT may digest it.

• Plasmid loss is prevented thanks to the hsdSB mutation which disrupts DNA methylation and degradation.

• When the genes for the desired recombinant protein placed under a T7 promoter, then T7 RNAP needs to be provided by the host cell. In the popular BL21(DE3) strain, the λDE3 prophage was inserted in the chromosome of BL21 and contains the T7 RNAP gene under the lacUV5 promoter.

• The BL21(DE3) and its derivatives are by far the most used strains for protein expression.

Rosado, G. L. and E. A. Ceccarelli (2014). "Recombinant protein expression in Escherichia coli: advances and challenges." Frontiers in microbiology 5: 172.



E.coli Genome-scale Reconstructions• Escherichia coli 042

• Escherichia coli 536

• Escherichia coli 55989

• Escherichia coli ABU 83972

• Escherichia coli APEC O1

• Escherichia coli ATCC 8739

• Escherichia coli B str. REL606

• Escherichia coli BL21(DE3) AM946981

• Escherichia coli BL21(DE3) BL21-Gold(DE3)pLysS AG

• Escherichia coli BL21(DE3) CP001509

• Escherichia coli BW2952

• Escherichia coli CFT073

• Escherichia coli DH1

• Escherichia coli DH1 ME8569

• Escherichia coli E24377A

• Escherichia coli ED1a

• Escherichia coli ETEC H10407

• Escherichia coli HS

• Escherichia coli IAI1

• Escherichia coli IAI39

• Escherichia coli IHE3034

• Escherichia coli KO11FL

• Escherichia coli LF82

• Escherichia coli NA114

• Escherichia coli O103:H2 str. 12009

• Escherichia coli O111:H- str. 11128

• Escherichia coli O127:H6 str. E2348/69

• Escherichia coli O157:H7 EDL933

• Escherichia coli O157:H7 str. EC4115

• Escherichia coli O157:H7 str. Sakai

• Escherichia coli O157:H7 str. TW14359

• Escherichia coli O26:H11 str. 11368

• Escherichia coli O55:H7 str. CB9615

• Escherichia coli O83:H1 str. NRG 857C

• Escherichia coli S88

• Escherichia coli SE11

• Escherichia coli SE15

• Escherichia coli SMS-3-5

• Escherichia coli str. K-12 substr. DH10B

• Escherichia coli str. K-12 substr. MG1655

• Escherichia coli str. K-12 substr. W3110

• Escherichia coli UM146

• Escherichia coli UMN026

• Escherichia coli UMNK88

• Escherichia coli UTI89

• Escherichia coli W

• Escherichia coli W CP002185

• Escherichia coli K-12 MG1655

Monk, J. M., P. Charusanti, et al. (2013). Proceedings of the National Academy of Sciences of the United States of America 110(50): 20338-20343.

Strain used at USU










E.coli K-12 substr. MG1655 Model(iJO1366)

similar to

Escherichia coli BL21(DE3)(iECD_1391)



3gp (Glycine)

pyr (Alanine)

akg (Proline, Glutamine)

AlanineProline

Glycine

Glutamine

iJO1366 Model

Metabolic Precursors

pyr

akg

glu-L

3gp



Glycine Superpathway Biosynthesis

http://biocyc.org/ECOLI/NEW-IMAGE?type=PATHWAY&object=SER-GLYSYN-PWY

L-serine is self-limiting

When L-serine is transported into the cell, the serine/glycine pathway can be shutdown Feedback Inhibition



Glycine Pathway: L-threonine Degradation II & IV

http://biocyc.org/ECOLI/NEW-IMAGE?type=PATHWAY&object=THREONINE-DEG2-PWY

The second route threonine degradation is catabolized by threonine dehydrogenase (EC 1.1.1.103) to form 2-amino-3-oxobutanoate, which is mainly cleaved by 2-amino-3-ketobutyrate CoA ligase, forming glycine and acetyl-CoA. The 2-amino-3-oxobutanoate can also be spontaneously converted to aminoacetone, which may be further metabolized to methylglyoxal (see L-threonine degradation III (to methylglyoxal)). A fourth route has been demonstrated in several bacteria and fungi. This route is based on the enzyme low-specificity L-threonine aldolase (EC 4.1.2.5), which cleaves threonine directly into glycine and acetaldehyde.

Degradation Pathway II Degradation Pathway IV

http://biocyc.org/ECOLI/NEW-IMAGE?type=PATHWAY&object=PWY-5436



Glycine & L-serine Transporters

ytfD, dagA, CycA APC transporter

http://biocyc.org/gene?orgid=ECOLI&id=CYCA-MONOMER#tab=RXNS

ygjU, SstT DAACS transporter

http://biocyc.org/gene?orgid=ECOLI&id=YGJU-MONOMER#tab=RXNS

http://biocyc.org/gene?orgid=ECOLI&id=TDCC-MONOMER#tab=RXNS

serine / threonine:H+ symporter TdcC

Reduces protomotive force Increases cytoplasmic Na+ concentration



http://biocyc.org/ECOLI/NEW-IMAGE?type=PATHWAY&object=PWY0-1061

L-Alanine Biosynthesis Pathways

L-alanine is an essential component of protein and peptidoglycan. Only about 10 percent of the total alanine synthesized flows into peptidoglycan.

At least three pathways (alanine biosynthesis I, alanine biosynthesis II, and alanine biosynthesis III) contribute to the synthesis of alanine. Alanine biosynthesis I is established only by existence of the relevant enzymes. Its contribution to alanine synthesis remains speculative because alanine auxotrophs have not yet been isolated. Because alanine, but not valine, represses AvtA, its primary purpose is probably synthesis of L-alanine. Existence of the Alanine biosyntheis II pathways rests on the evidence that glutamate-pyruvate aminotransferase acitivity is found in crude cell extracts; the enzymes has not been purified nor have mutant alleles of its designated encoding gene (alaB) been isolated. The conversion can also be mediated as a side reaction of alanine racemase. The alanine biosyntheis III pathway, mediated by cysteine desulfurase activity, which is required to donate sulfane sulfur for the synthesis of Fe-S clusters, thiamine, thionucleosides in tRNAs, biotin, lipoic acid, and molybdopterin, probably contributes only a minor amount of the cell's alanine requirement, as judged by the cell's total requirement for sulfane sulfur



L-Alanine Transporter Pathways

http://biocyc.org/ECOLI/NEW-IMAGE?type=REACTION&object=RXN0-5202

CycA is a transporter involved in the uptake of glycine, serine and alanine. CycA is a member of the APC superfamily of amino acid transporters and is likely to function as a proton/serine, alanine or glycine symporter.

L-alanine

L-alanine

H+

H+

cycA

H+ symporter

PeriplasmInner Membrane

Cytosol

L-alanine

L-alanine ATPH2OADPH+ Pi

L-alanine transport via ABC system

L-alanine

L-alanine

Na

Na

Na symporter

YaaJlivF

YaaJ may function as a sodium/alanine symporter

http://biocyc.org/gene?orgid=ECOLI&id=EG11555http://biocyc.org/gene?orgid=ECOLI&id=EG10536#


Requires energy, ATP, for transport



Regulatory Issues – Proline BiosynthesisRegulatory constraints are not included in the constraint-based reconstructions.

http://biocyc.org/ECOLI/NEW-IMAGE?type=PATHWAY&object=PROSYN-PWY&detail-level=2

Proline is self-limiting When L-proline is transported into the cell, the serine/glycine pathway can be shutdown

Feedback Inhibition



L-proline Transporters

proline ABC transporter = [ProV]2[ProW]2[ProX]

http://biocyc.org/ECOLI/NEW-IMAGE?type=ENZYME&object=ABC-26-CPLX

proline:Na+ symporter

http://biocyc.org/gene?orgid=ECOLI&id=PUTP-MONOMER#tab=RXNS

http://biocyc.org/gene?orgid=ECOLI&id=PROP-MONOMER#tab=RXNS

L-proline:H+ symporter (osmolyte:H+ symporter ProP)


Requires Energy



Glutamine Biosynthesis Pathway

glnHPQ

http://biocyc.org/ECOLI/NEW-IMAGE?type=REACTION&object=ABC-12-RXN

The GlnHPQ high-affinity glutamine transport system is a member of the ATP-Binding Cassette (ABC) Superfamily of transporters. Based on sequence similarity, GlnH is the periplasmic glutamine-binding protein, GlnQ is the ATP-binding component, and GlnP is the membrane component of the ABC transporter

Rxn name Rxn description Formula SubsystemGLNS glutamine synthetase atp[c] + nh4[c] + glu-L[c] -> h[c] + adp[c] + gln-L[c] + pi[c] Glutamate MetabolismGLNabcpp L-glutamine transport via ABC system (periplasm) atp[c] + gln-L[p] + h2o[c] -> h[c] + adp[c] + gln-L[c] + pi[c] Transport, Inner Membrane

Requires Energy










Quadrupled MaSp2 Amino Acid Sequence

KLHMTGPGQQGPGGYGPGQQGPSGPGSAAAAAAAAGPGQQGPGGYGPGQQGPGGYGPGQQGPSGPGSAAAAAAAAGPGGYGPGQQGPGGYGPGQQGPGGYGPGQQGPSGPGSAAAAAAAAGPGQQGPGGYGPGQQGPGGYGPGQQGPSGPGSAAAAAAAAGPGQQGPGGYGPGQQGPSGPGSAAAAAAAAGPGQQGPGGYGPGQQGPGGYGPGQQGPSGPGSAAAAAAAAGPGGYGPGQQGPGGYGPGQQGPGGYGPGQQGPSGPGSAAAAAAAAGPGQQGPGGYGPGQQGPGGYGPGQQGPSGPGSAAAAAAAAGPGQQGPGGYGPGQQGPSGPGSAAAAAAAASGGDKLHMTGPGQQGPGGYGPGQQGPSGPGSAAAAAAAAGPGQQGPGGYGPGQQGPGGYGPGQQGPSGPGSAAAAAAAAGPGGYGPGQQGPGGYGPGQQGPGGYGPGQQGPSGPGSAAAAAAAAGPGQQGPGGYGPGQQGPGGYGPGQQGPSGPGSAAAAAAAAGPGQQGPGGYGPGQQGPSGPGSAAAAAAAAGPGQQGPGGYGPGQQGPGGYGPGQQGPSGPGSAAAAAAAAGPGGYGPGQQGPGGYGPGQQGPGGYGPGQQGPSGPGSAAAAAAAAGPGQQGPGGYGPGQQGPGGYGPGQQGPSGPGSAAAAAAAAGPGQQGPGGYGPGQQGPSGPGSAAAAAAAASGGDKLHMTGPGQQGPGGYGPGQQGPSGPGSAAAAAAAAGPGQQGPGGYGPGQQGPGGYGPGQQGPSGPGSAAAAAAAAGPGGYGPGQQGPGGYGPGQQGPGGYGPGQQGPSGPGSAAAAAAAAGPGQQGPGGYGPGQQGPGGYGPGQQGPSGPGSAAAAAAAAGPGQQGPGGYGPGQQGPSGPGSAAAAAAAAGPGQQGPGGYGPGQQGPGGYGPGQQGPSGPGSAAAAAAAAGPGGYGPGQQGPGGYGPGQQGPGGYGPGQQGPSGPGSAAAAAAAAGPGQQGPGGYGPGQQGPGGYGPGQQGPSGPGSAAAAAAAAGPGQQGPGGYGPGQQGPSGPGSAAAAAAAASGGDKLHMTGPGQQGPGGYGPGQQGPSGPGSAAAAAAAAGPGQQGPGGYGPGQQGPGGYGPGQQGPSGPGSAAAAAAAAGPGGYGPGQQGPGGYGPGQQGPGGYGPGQQGPSGPGSAAAAAAAAGPGQQGPGGYGPGQQGPGGYGPGQQGPSGPGSAAAAAAAAGPGQQGPGGYGPGQQGPSGPGSAAAAAAAAGPGQQGPGGYGPGQQGPGGYGPGQQGPSGPGSAAAAAAAAGPGGYGPGQQGPGGYGPGQQGPGGYGPGQQGPSGPGSAAAAAAAAGPGQQGPGGYGPGQQGPGGYGPGQQGPSGPGSAAAAAAAAGPGQQGPGGYGPGQQGPSGPGSAAAAAAAASGGD

• MaSp2 spider silk amino acid sequence (349 Amino acids with MW of 29.5 Daltons) multiplied 4 times (1396 Amino acids with MW of 118 kDa)

• MaSp2 spider silk Cobra reaction (1396 * 4.306 mol ATP/mol amino acid)model = addReaction(model,‘MaSp2',‘288 ala-L[c] + 4 asp-L[c] + 192 gln-L[c] + 512 gly[c] + 4 his-L[c] + 4 lys-L[c] + 4 met-L[c] + 236 pro-L[c] + 76 ser-L[c] + 4 thr-L[c] + 68 tyr-L[c] + 6011.12 atp[c] + 6011.12 h2o[c] -> masp2[c] + 6011.12 adp[c] + 6011.12 h[c] + 6011.12 pi[c]');










Components Produced by Double Plasmid System

Plasmid Nucleotides

Lac Repressor(154 kDa) MaSp2 Spider Silk Protein

(118 kDa)

Chloramphenicol Marker (25.6 kDa)

Glycine X, Y, Z tRNA

Kanamycin Marker(29 kDa)

Proline L, M tRNA Glycine T tRNA



MaSp2 pET19KT Vector

• MaSp2 primary vector

10,182 bp

• MaSp2 Protein

4103 bp

1396 amino acids

MW of 118K Daltons

• Kanamycin Marker

1049 bp

264 amino acids

MW of 29K Daltons

pET19KT [MaSp2]32.xdna

http://www.snapgene.com/products/snapgene_viewer/



MaSp2 4GPP Vector(Secondary Plasmid)

• CmR chloramphenicol resistance taken

from the pACYC184 vector

• glyVXY genes

glyV, glyX, glyY genes for glycine tRNA

• GlyT, ProL and ProM sequence taken

from the pET19SX vector

glyT gene for glycine tRNA

proM, proL genes for proline tRNA

4GPP Vector-HSH.dna

http://www.snapgene.com/products/snapgene_viewer/



MaSp2 Spider Silk Plasmid Code% Add plasmid DNA nucleotides reaction

• model=addReaction(model,'PLASMID','5029.91 dgtp[c] + 5029.91 dctp[c] + 5029.91 datp[c] + 5029.91 dttp[c] -> plasmid[c]');model = changeRxnBounds(model,'PLASMID',0.63e-9,'l');model = addDemandReaction(model,'plasmid[c]');

% Add kanamycin resistance model=addReaction(model,'KanR','35 ala-L[c] + 19 arg-L[c] + 3 asn-L[c] + 25 asp-L[c] + 5 cys-L[c] + 11 gln-L[c] + 18 glu-L[c] + 21 gly[c] + 7 his-L[c] + 10 ile-L[c] + 32 leu-L[c] + 4 lys-L[c] + 6 met-L[c] + 11 phe-L[c] + 11 pro-L[c] + 11 ser-L[c] + 10 thr-L[c] + 5 trp-L[c] + 4 tyr-L[c] + 16 val-L[c] + 1136.8 atp[c] + 1136.8 h2o[c] -> kanr[c] + 1136.8 adp[c] + 1136.8 h[c] + 1136.8 pi[c]');model = changeRxnBounds(model,'KanR',0.000569,'l');model = addDemandReaction(model,'kanr[c]');

% Add plasmid2 DNA nucleotides reaction (4GPP)model = addReaction(model,'PLASMID2','2593.76 dgtp[c] + 2593.76 dctp[c] + 2532.24 datp[c] + 2532.24 dttp[c] -> plasmid2[c]');model = changeRxnBounds(model,'PLASMID2',0.63e-9,'l');model = addDemandReaction(model,'plasmid2[c]');

% Add Chloramphenicol resistance model = addReaction(model,'CmR','15 ala-L[c] + 5 arg-L[c] + 10 asn-L[c] + 12 asp-L[c] + 5 cys-L[c] + 13 gln-L[c] + 12 glu-L[c] + 10 gly[c] + 12 his-L[c] + 9 ile-L[c] + 13 leu-L[c] + 12 lys-L[c] + 9 met-L[c] + 20 phe-L[c] + 7 pro-L[c] + 10 ser-L[c] + 13 thr-L[c] + 5 trp-L[c] + 11 tyr-L[c] + 16 val-L[c] + 1136.8 atp[c] + 1136.8 h2o[c] -> cmr[c] + 943 adp[c] + 943 h[c] + 943 pi[c]');model = changeRxnBounds(model,'CmR',0.000643,'l');model = addDemandReaction(model,'cmr[c]');

% Add MaSp2 reactionmodel = addReaction(model,'MaSp2','288 ala-L[c] + 4 asp-L[c] + 192 gln-L[c] + 512 gly[c] + 4 his-L[c] + 4 lys-L[c] + 4 met-L[c] + 236 pro-L[c] + 76 ser-L[c] + 4 thr-L[c] + 68 tyr-L[c] + 6011.12 atp[c] + 6011.12 h2o[c] -> masp2[c] + 6011.12 adp[c] + 6011.12 h[c] + 6011.12 pi[c]');

% Add MaSp2 demand reactionmodel = addDemandReaction(model,'masp2[c]'); %'DM_masp2[c]'model = changeRxnBounds(model,'MaSp2',0.00233,'l');










Maximum MaSp2 Production Assumptions• Cell optimized for growth

• Assume minimal mediumUnlimited uptake of NH4, SO4, O2, and Pi

Free exchange of H+, H2O, and CO2

Unlimited uptake allowed for mineralsUnlimited secretion allowed for all carbon sources, minerals and amino acids No uptake allowed for any other carbon sources or amino acidsMaximum glucose uptake of -10 mmol/gDW-hMaximum oxygen uptake of -20 mmol/gDW-h

• No internal limits on internal enzyme capacity

• No upper limits on cell concentration in fermentation process

• No consideration for toxicity or other environmental triggers



Phenotype Phase Plane Analysis

Phase 1

Phase 3

Phase 2

Phase 4

Phase 5

No growth; not enough glucose

Growth is limited by excess oxygen; not enough glucose to reduce all the oxygen and produce biomass optimally

Not enough oxygen to fully oxidize glucose; acetate produced through fermentation

Not enough oxygen to fully oxidize glucose; acetate and formate are produced and secreted

Not enough oxygen to fully oxidize glucose; acetate, formate and ethanol are produced and secreted.

AerobicGlucoseBioMassPPP.m



Production Envelope & MaSp2 Phenotype Phase Plane Analysis (Glucose)

MaSp2_PPP_2Plasmid_iECD_1391.mMaSp2_ProductionEnvelope_iECD_1391_2Plasmid.m

EX_glc(e) ≥ -10; EX_o2(e) ≥ -20;

All cell resources are focused on bioproduct production;

All cell resources are focused on cell growth;

0.00233 mmol/gDW∙h



Metabolic Load of Plasmids – Cell Growth Phenotype Phase Plane(Without MaSp2 Production)

MaSp2_PPP_2Plasmid_iECD_1391.m

Acetate, Formate, GlycolateAcetate, Formate

AcetateFormateGlycolateEthanol

Targeted Operating Point



Cell Growth Rate with Metabolic Load(2 Plasmid System with Glucose)

MaSp2 > 0.006 mmol/gDW⋅h(708 mg/gDW⋅h)


MaSp2 > 0.00233 mmol/gDW⋅h(275 mg/gDW⋅h)*


Note the impact of increased flux through the MaSp2 reaction (larger copy number and stronger promoters)

EX_glc(e) ≥ -10; EX_o2(e) ≥ -20; *Protein production = Flux-rate ⋅ MW



Practical Cytoplasmic Recombinant Protein Production Limits• Maximum biomass density

An upper limit for cell concentration in a large scale fermenter is less than 100 gDW/L Limited by nutrient management, dissolved oxygen gradients, non-limiting glucose concentration References

Choi, J. H., K. C. Keum, et al. (2006). "Production of recombinant proteins by high cell density culture of Escherichia coli." Chemical Engineering Science 61(3): 876-885

Shiloach, J. and R. Fass (2005). "Growing E. coli to high cell density--a historical perspective on method development." Biotechnol Adv 23(5): 345-357

• Protein production limit (Total protein = 550 mg per gram dry weight biomass) An upper limit for the production of a recombinant protein is less than 50% of the total protein (275

mg/gDW) Example: Assuming a 100 gDW/L fermentation, the upper limit of cytoplasmic recombinant protein is

(100 gDW/L)(0.275 g/gDW) = 27.5 g/L! References

Rosano, G. L. and E. A. Ceccarelli (2014). "Recombinant protein expression in Escherichia coli: advances and challenges." Front Microbiol 5: 172.



MaSp2 Production: Upper Limit (EX_glc(e) ≥ -10, OD600 = 100, 64 Steps, 48 MaSp2 Samples)

At low protein expression there will be a high growth rate which will allow the biomass to reach it’s upper limit of 100 g/L quickly.

The upper limit of biomass concentration is set at 100 g/L

The upper limit of the cytoplasmic protein limit is set at 50% of the total protein dry weight

Increased protein expression reduces growth rate which prevents the biomass concentration from reaching the upper limit

Longer growth time at low protein expression still allows the upper limit of protein accumulation

Limited by short time to reach the maximum protein upper limit

Limited by short time to reach the biomass concentration upper limit

MaSp2_Dynamic_BL21_maxProd_100Limit_2Plasmid_ProteinLimit.m



At low protein expression it will take a longer time to reach the upper cytoplasmic protein limit of 50% of the total protein dry weight. At high expression it can reach

the upper limit quickly

Constraints

EX_glc(e) ≥ -10; EX_o2(e) ≥ -20;

Induction at OD600 = 100(47 g/L)

Max Biomass Conc.= 100 g/L

Max MaSp2 Protein= 50% Total Protein

Max Prod Time= 4 hours

Time step = 0.0625 s

Minimal Media

BL21 (iECD_1391)



Peak MaSp2 Production - GlucoseEX_glc(e) ≥ -10; EX_o2(e) ≥ -20; initBiomass = 47 gDW/L (OD600 = 100); timeStep = 0.0195 h; nSteps = 64

26.79 g/L

MaSp2 = 0.002622 mmol∙gDW-1 ∙ h-1

MaSp2_Dynamic_Production_BL21_2Plasmid.m

MaSp2 = 0.002622


Maximum Production Time =1.25 h









Constraints

EX_glc(e) ≥ -10; EX_o2(e) ≥ -20;

Induction at OD600 = 30(14.1 g/L)





Minimal Media

BL21 (iECD_1391)


The upper limit is set at 100 g/L




At low protein expression it will take a longer time to reach the upper cytoplasmic protein limit of 50% of the total protein dry weight. At high expression it can reach

the upper limit quickly




Production Envelope & MaSp2 Phenotype Phase Plane Analysis (Low Glucose)

MaSp2_PPP_2Plasmid_iECD_1391.mMaSp2_ProductionEnvelope_iECD_1391_2Plasmid.m

EX_glc(e) ≥ -5; EX_o2(e) ≥ -20;

All cell resources are focused on bioproduct production;

All cell resources are focused on cell growth;



Cell Growth Rate with Metabolic Load(2 Plasmid System with Low Glucose)

MaSp2 > 0.00233 mmol/gDW⋅h(275 mg/gDW⋅h) *



MaSp2 > 0.0038 mmol/gDW⋅h(448 mg/gDW⋅h) *

Note the impact of increased flux through the MaSp2 reaction (larger copy number and stronger promoters)

EX_glc(e) ≥ -5; EX_o2(e) ≥ -20; *Protein production = Flux-rate ⋅ MW





ConstraintsEX_glc(e) ≥ -5; EX_o2(e) ≥ -20;





Time step = 0.0625/4 s

Number of steps= 64*4

Minimal Media

BL21 (iECD_1391)

2.625 h

0.8438

14.01

27.29

0.00126










MaSp2 Production Envelope & Phenotype Phase Plane Analysis (Glycerol)

MaSp2_PPP_2Plasmids_iECD_1391_Glycerol.mMaSp2_ProductionEnvelope_iECD_1391_2Plasmid_Glycerol.m

EX_glyc(e) ≥ -10; EX_o2(e) ≥ -20; 0.004637

0.5071

0.00233 mmol/gDW∙h



Maximum Growth-Rate Using Glycerol for MaSp2 Production




MaSp2_PPP_2Plasmids_iECD_1391_Glycerol.m



MaSp2 Production: Upper Limit (EX_glyc(e) ≥ -10, OD600 = 100, 64 Steps, 48 MaSp2 Samples)




MaSp2_Dynamic_BL21_maxProd_100Limit_2Plasmid_PL_Glycerol.m



At low protein expression there

will be a high growth rate which will allow the biomass to reach it’s upper limit of 100 g/L quickly.




At low protein expression it will take a longer time to reach the upper cytoplasmic protein limit of 50% of the total protein dry weight. At high

expression it can reach the upper

limit quickly

Constraints

EX_glyc(e) ≥ -10; EX_o2(e) ≥ -20;






Minimal Media

BL21 (iECD_1391)



Peak MaSp2 Production - GlycerolEX_glyc(e) ≥ -10; EX_o2(e) ≥ -20; initBiomass = 47 gDW/L (OD600 = 100); timeStep = 0.03418 h; nSteps = 64


26.61 g/L

MaSp2 = 0.0015 mmol∙gDW-1 ∙ h-1

MaSp2_Dynamic_Production_BL21_2Plasmid_Glycerol.m

MaSp2 = 0.0015

Maximum Production Time =2.188 h




Due to the slower growth rate of glycerol the maximum biomass concentration never reaches 100 g/L in the maximum production time of 4 hours


Increased protein expression reduces growth rate which prevents the biomass concentration from reaching

the upper limit


Limited by the 4 hours maximum production time



At low protein expression it will take a longer time to reach the upper cytoplasmic protein limit of 50% of the total protein dry weight. At high

expression it can reach the upper

limit quickly Limited by the 4 hours maximum production time


Constraints

EX_glyc(e) ≥ -10; EX_o2(e) ≥ -20;

Induction at OD600 = 30(14.1 g/L)





Minimal Media

BL21 (iECD_1391)



MaSp2 Production Envelope & Phenotype Phase Plane Analysis (Low Glycerol)

MaSp2_PPP_2Plasmids_iECD_1391_Glycerol.mMaSp2_ProductionEnvelope_iECD_1391_2Plasmid_Glycerol.m

EX_glyc(e) ≥ -5; EX_o2(e) ≥ -20; 0.002007

0.2189







Constraints

EX_glyc(e) ≥ -5; EX_o2(e) ≥ -20;






Minimal Media

BL21 (iECD_1391)





At low protein expression it will take a longer time to reach the upper cytoplasmic protein limit of 50% of the total protein dry weight.












Nutrient Limits in Production • E. coli growth is inhibited when the following nutrients are present above certain concentrations

(shown in brackets) glucose (50 g/L), ammonia (3 g/L), iron (1.15 g/L), magnesium (8.7 g/L), phosphorous (10 g/L) and zinc (0.038 g/L)

• References Lee, S. Y. (1996). "High cell-density culture of Escherichia coli." Trends Biotechnol 14(3): 98-

105. Riesenberg, D. (1991). "High-cell-density cultivation of Escherichia coli." Current opinion in

biotechnology 2(3): 380-384.










Stringent Response• The stringent response, also called stringent control, is a stress response of bacteria in reaction to amino-acid starvation, fatty acid

limitation, iron limitation, heat shock and other stress conditions. The stringent response is signaled by the alarmone (p)ppGpp, and modulates transcription of up to 1/3 of all genes in the cell. This in turn causes the cell to divert resources away from growth and division and toward amino acid synthesis in order to promote survival until nutrient conditions improve.

• In Escherichia coli (p)ppGpp production is mediated by the ribosomal protein L11 (rplK resp. relC) and the ribosome-associated (p)ppGppsynthetase I, RelA; deacylated tRNA bound in the ribosomal A-site is the primary induction signal. RelA converts GTP and ATP into pppGpp by adding the pyrophosphate from ATP onto the 3' carbon of the ribose in GTP releasing AMP. pppGpp is converted to ppGpp by the gpp gene product, releasing Pi. ppGpp is converted to GDP by the spoT gene product, releasing pyrophosphate (PPi). GDP is converted to GTP by the ndk gene product. Nucleoside triphosphate (NTP) provides the Pi, and is converted to Nucleoside diphosphate (NDP).

• During the stringent response, (p)ppGpp accumulation affects the resource-consuming cell processes replication, transcription, and translation. (p)ppGpp is thought to bind RNA polymerase and alter the transcriptional profile, decreasing the synthesis of translational machinery (such as rRNA and tRNA), and increasing the transcription of biosynthetic genes. Additionally, the initiation of new rounds of replication is inhibited and the cell cycle arrests until nutrient conditions improve. Translational GTPases involved in protein biosynthesis are also affected by ppGpp, with Initiation Factor 2 (IF2) being the main target.

• Chemical reaction catalyzed by RelA: ATP + GTP → AMP + pppGpp

• Chemical reaction catalyzed by SpoT: ppGpp → GDP + PPi or pppGpp -> GTP + PPi



The (p)ppGpp-mediated Stringent Response

The (p)ppGpp-mediated stringent response. (A) Low amino acid concentrations lead to decreased charging of the corresponding tRNAs. (B) The translational machinery depends on the translocation along the mRNA whereby a new acetylated-tRNA is positioned in the ribosome. Whenever an uncharged tRNA binds to the ribosome, the elongation of the polypeptide chain is stalled. (C) The stringent factor RelA is then activated in the presence of the ribosomal protein L11, catalyzing the synthesis of (p)ppGpp nucleotides. (D) These nucleotides bind directly to the RNA polymerase and affect the binding abilities of sigma factors to the core RNA polymerase. (E) The cofactor DksA also binds to the RNA polymerase and augments the (p)ppGpp regulation of the transcription initiation at certain σ70-dependent promoters, functioning both as negative and positive regulators. (F) These regulators change the gene expression: (i) decreasing the transcription activity of genes involved in translational activities; (ii) and increasing the transcription of stress-related operons and genes encoding for enzymes needed for the synthesis and the transport of amino acids

http://microbialinformaticsj.biomedcentral.com/articles/10.1186/2042-5783-1-14



Events Associated with the Stringent ResponseAmino Acid Depletion

Block Protein Synthesis Inhibition of DNA initiation

(p)ppGpp

Inhibition of Other Operons

Inhibition of tRNA Synthesis

Inhibition of rRNA Synthesis

Inhibition of Protein Chain

Elongation

Direct Stimulation of Biosynthetic and Catabolic Operons

Inhibition of R-Protein Synthesis

Increased Fidelityof Protein Synthesis

The series of events ensuing after amino acid restriction on bacteria. The solid arrows indicate processes known on good evidence to result from ppGpp accumulation. The dashed arrows depict more speculative relations

M Schaechter et al, Microbe, ASM Press, 2006, p. 244










Opportunities to Understand the MaSp2 Production Issues• Exponential growth

Verify growth phenotype using transcriptomics & proteomics Sample points: OD600 = 20, 40, 60, 80, 100 Include metabolites associated with glycolysis, TCA, anaplerotic, key amino acids (glycine,

proline, alanine, glutamine, & glutamate), cofactors (ATP, ADP, NADH, NADPH, FADH)• Induction

Understand the phenotype changes that occur at induction using transcriptomics & proteomics Sample points: -10 min, +10 min, +20 min, +30 min, +60 min Include metabolites associated with glycolysis, TCA, anaplerotic, key amino acids (glycine,

proline, alanine, glutamine, & glutamate), cofactors (ATP, ADP, NADH, NADPH, FADH)• MaSp2 Production

Understand the phenotypes during MaSp2 production using transcriptomics & proteomics Sample points: +1 hr, +2 hr, +3 hr, +4 hr, +5 hr Include metabolites associated with glycolysis, TCA, anaplerotic, key amino acids (glycine,

proline, alanine, glutamine, & glutamate), cofactors (ATP, ADP, NADH, NADPH, FADH)



Future Modeling Work

• Can cofactor balancing improve MaSp2 production?

Cofactors include ATP,ADP, NAD, NADH, NADP, NADPH

• What is the regulating structure for the key amino acids?

• What is the regulating structure for amino acid transporters?

Role of catabolite repression

• Can reducing carbon uptake increase MaSp2 production?

• What is the impact of the other stress responses on MaSp2 production?

• What are the key metabolic pool concentrations at induction and during MaSp2 production?

• Is there a better strain?

Secretion?



EXTRAS



Induction Response“The use of inducible expression systems and the moment of induction of the recombinant expression have been pointed as important factors to minimize the metabolic burden. The physiological state of the culture at the time of induction can affect the metabolic response to the recombinant process and, more importantly, its efficiency.

If the induction is performed at the mid-logarithmic growth phase, cells can provide sufficient levels of energy and metabolic precursors for the recombinant biosynthesis, since the cell is at its maximum catabolic capacity.

However, if induced at the late-logarithmic or stationary phase there is a higher cell density for product formation, but the metabolic state of the cells is unfavourable and the presence of stress-related proteins like proteases can reduce the yield of foreign proteins.”

Decline PhaseDeath Phase

Log (Exponential) Phase

Lag Phase

Cell

Den

sity

(OD

)Time (hours)

Stationary Phase

Acceleration Phase

Purpose: Identify phenotypes that can exist in exponential growth phase

Carneiro, S., E. C. Ferreira, et al. (2013). "Metabolic responses to recombinant bioprocesses in Escherichia coli." J Biotechnol 164(3): 396-408.

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