Oxygen dependence of metabolic fluxes and energy generation of CEN.PK113-1A-2

T, b) in the TCA cycle and c) from G6P to glycolysis and PPP in CEN.PK113-1A in glucose-limited chemostats, at D = 0.1 h, in 20.9%, 2.8%, 1.0%, 0.5% and 0.0% oxygen of the chemostat inlet gas. Replicate experiments are indicated with I and II.<p><b>Copyright information:</b></p><p>Taken from "Oxygen dependence of metabolic fluxes and energy generation of CEN.PK113-1A"</p><p>http://www.biomedcentral.com/1752-0509/2/60</p><p>BMC Systems Biology 2008;2():60-60.</p><p>Published online 9 Jul 2008</p><p>PMCID:PMC2507709.</p><p></p

Oxygen dependence of metabolic fluxes and energy generation of CEN.PK113-1A-0

T, b) in the TCA cycle and c) from G6P to glycolysis and PPP in CEN.PK113-1A in glucose-limited chemostats, at D = 0.1 h, in 20.9%, 2.8%, 1.0%, 0.5% and 0.0% oxygen of the chemostat inlet gas. Replicate experiments are indicated with I and II.<p><b>Copyright information:</b></p><p>Taken from "Oxygen dependence of metabolic fluxes and energy generation of CEN.PK113-1A"</p><p>http://www.biomedcentral.com/1752-0509/2/60</p><p>BMC Systems Biology 2008;2():60-60.</p><p>Published online 9 Jul 2008</p><p>PMCID:PMC2507709.</p><p></p

Oxygen dependence of metabolic fluxes and energy generation of CEN.PK113-1A-1

He C-metabolic flux analysis for determination of net fluxes in different oxygenation conditions. The cytosolic and mitochondrial compartments and glycolytic, pentose phosphate, TCA cycle and fermentative pathways were included in the model. The fluxes are presented as net fluxes and the directions of the arrows represent the directions of positive net fluxes. The compounds consumed or produced by external fluxes are denoted with a subscript ext. The anabolic reactions from metabolic intermediates to biosynthesis are represented by small arrows.<p><b>Copyright information:</b></p><p>Taken from "Oxygen dependence of metabolic fluxes and energy generation of CEN.PK113-1A"</p><p>http://www.biomedcentral.com/1752-0509/2/60</p><p>BMC Systems Biology 2008;2():60-60.</p><p>Published online 9 Jul 2008</p><p>PMCID:PMC2507709.</p><p></p

MAB02 zymogram activity of protease deletion strain supernatants.

<p>White regions on the blue stained gel indicate an area of protease activity against MAB02. The major activity occurs between 65–90 kDa and a smaller protease activity occurs at 28 kDa. Culture supernatant samples from day 7 were diluted 1:2 in sodium citrate buffer (50 mM, pH 5.5) and 30 μl was loaded into a 12% zymogram SDS PAGE gel containing MAB02. The M124 parental strain, M181 (Δ<i>pep1</i>), M219 (Δ<i>pep1</i>Δ<i>tsp1</i>), and M277 (Δ<i>pep1</i>Δ<i>tsp1</i>Δ<i>slp1</i>) supernatants were assayed.</p

Total protease activity of protease deletion culture supernatants.

<p>The relative ability of each culture supernatant to degrade succinylated casein is shown. The supernatants were diluted to 2 mg/ml total protein in 50 mM sodium citrate, pH 5.5 before being assayed. 50 μl of diluted supernatant was loaded into a 96 well plate and 50 μl of succinylated casein was added to begin the reaction. The M181 (Δ1), M219 (Δ2), M277 (Δ3) strain supernatants display less protease activity compared to the M124 control strain.</p

Strain list.

<p>Strain list.</p

Protease deletion strains grow faster than parental M124 strain.

<p>Parallel fermentation data (DASGIP Bioreactor System) showing the growth curves for the M124 strain (wt) and the M277 (Δ3), M307 (Δ4), M369 (Δ5), M396 (Δ6), and M486 (Δ7) multiple deletion strains in complex media. Growth was monitored over 3 days following the Aber Futura biomass signal (capacitance) and CO₂ transfer rates. (Note: M277 CO₂ transfer rates are not displayed due to defective off gas monitoring for this single reactor.)</p

Inhibitor treatments stabilized antibody heavy chain.

<p>Immunoblot showing the rituximab heavy chain fragments created by M44 supernatant proteases. The fed batch supernatant was diluted to 6 mg/ml in sodium citrate buffer pH 5.5 and incubated with 0.05 μg/μl of rituximab, 100 μM chymostatin, 1 mg/ml SBTI, or a combination of both inhibitors at 37°C for up to 19 hours.</p

Aminobenzamidine purified serine proteases degraded antibody.

<p>Fig 4A. Proteases from the aminobenzamidine affinity purification fraction #4 degraded human IgG. Samples were taken at 0 min and 20 hours after incubating at pH 5.5 and 37°C. The IgG control was antibody in buffer incubated for 20 hours. Immunoblotting was done to detect the heavy and light chain of the nonreduced IgG. The full length antibody runs around 150 kDa. Fig 4B. Zymogram analysis of MAB02 antibody protease degradation from aminobenzamidine column purified fractions (F2-F4) and from M44 supernatant samples. Purified samples were incubated with and without PMSF serine protease inhibitor at pH 5.5. White band indicate degradation of MAB02 antibody. Protease activity came from 65 and 29 kDa, as the arrows indicate.</p

Consecutive deletion of proteases resulted in dramatic reduction in protease activity.

<p>Normalized protease activity data from culture supernatants from each of the protease deletion supernatants and the parent strain M124. Protease activity is against a casein substrate. The 6 protease deletion strain has only 6% of the wild type parent strain and the 7 protease deletion strain protease activity went down to 4% based upon casein substrate activity. Wild-type (M124), Δ1 (M181), Δ2 (M219), Δ3 (M277), Δ4 (M307), Δ5 (M369), Δ6 (M396), and Δ7 (M486).</p