MATERIALS AND METHODS
Strain, Plasmid, and Media
The competent cell line HMS174 (DE3) was purchased through Novagen (EMD Chemicals, Darmstadt, Germany) and transformed with
a pET-29a plasmid containing a recombinant gene of interest. The transformation was performed using the heat-shock method.
Inoculants and fermentations were grown in 2XYS media (16 g yeast extract, 10 g soytone, 5 g NaCl per L) with kanamycin (Sigma,
St. Louis, MO) used as the antibiotic selection marker. Fermentation was carried out in 2.5 L vessels using a BioFlo 3000
controller (New Brunswick Scientific, Edison, NJ). The inoculating volume was 10% of the final fermentation volume. The dissolved
oxygen (DO) level was held at 40% by sparging with 1 vvm of air and agitation was maintained at 500 rpms. If the DO fell below
40%, the air sparge was mixed with pure oxygen as needed to maintain 40% DO. NH4OH and H2SO4 were used to maintain the pH at 7. Temperature was maintained at 37 °C. The growing culture was fed 40% glucose,
and +20 g/L NaPO4 in 2XYS after the optical density of the culture reached 1–2 (A600). Feed rates were constant throughout
culture growth and induction.
Analysis
Fermentation samples (20 mL) were taken every hour. Optical density (OD) was determined and 1 mL of culture was microfuged
for 1 min at 14,000 rpm (Heraeus, DJB Labcare, UK). The corresponding pellet was used to determine dry cell weight (DCW) and
the supernatant was analyzed for residual acetate, glucose, and phosphate concentrations. Pellets were dried in a rotovap
for 2 h at medium heat. Growth curves were created from OD and DCW changes over time for both cultures. Pellets also were
recovered to monitor induction of recombinant protein. The cell pellets were solubilized with a reducing sample buffer and
run on an SDS-PAGE gel. The gel was then stained with Simply Blue (Invitrogen, Carlsbad, CA) and destained with water. The
supernatants were analyzed using a Nova-Bio 300 bioanalyzer (Nova Biomedical, Waltham, MA). Final wet cell pellets were weighed
for cell mass/L generated for both cultures. Inclusion bodies were isolated from both cultures by lysing cells in a microfluidizer
at 18,000 psi and centrifuging inclusion bodies from cell lysate. Inclusion bodies were washed twice with 20% isopropyl alcohol
(IPA). Their respective yields per gm of wet cell paste were calculated.
RESULTS
Lack of Phosphate in Feed Substantially Slows Growth of Culture
Figure 1a
|
To evaluate the metabolic needs of a high cell density culture, phosphate was added to the 40% glucose feed for one of the
cultures at 20 gm/L. Although the glucose feed rate of 0.16 g/min was the same for both cultures, the culture without phosphate
feed had approximately half the growth rate (1.5 h) of the culture grown with phosphate in the feed (2.7 h). Furthermore,
the cultures with and without phosphate feed metabolized 99% and 74% of the added glucose, respectively, while the optical
densities for each culture reached 15 (–PO4) and 27 (+PO4) (Figure 1A). Recovered wet cell weights for the harvested cell
pellets correlated well with final optical densities.
Substantial Acetate Accumulation Absent in the Culture Without Phosphate Feed
Figure 1b
|
Interestingly, the culture without phosphate feed showed a substantial residual glucose accumulation, but acetate levels were
depressed (0.3 g/L). This may have something to do with the need for phosphate, in the form of phosphoenolpyruvate, to actively
shuttle glucose from the outside to the inside of the cell.5 Accordingly, the culture with added phosphate showed no accumulation of glucose, with a much higher amount of acetate production
per liter (1 g/L). In previous experiments under this feed strategy, acetate production was 5 g/L with no negative effects
on cell growth or recombinant protein production (data not shown).
Figure 2
|
The buildup of acetate is typical of a fast growing culture being fed glucose.6,7,8 This suggests that the culture without phosphate feed may have switched to the glyoxylate shunt pathway (a 2-carbon source
such as acetate) in the absence of an efficient metabolic state for the 6-carbon molecule glucose (Figure 1B). As shown in
Figure 2A, the acetate concentration in the glucose culture without phosphate feed plateaus at ~4 h, whereas the culture with
added phosphate feed continues to increase three-fold until about hour 5. After induction, the acetate levels decrease dramatically
in the phosphate fed-culture, indicating the increased need for an additional carbon source and the ability of this strain
to use acetate efficiently. The culture without phosphate feed maintained its extracellular acetate concentration while also
maintaining the induction fidelity of the recombinant protein product.
|
