Scalable, two-stage, autoinduction of recombinant protein expression in E. coli utilizing phosphate depletion

Abstract

We report the scalable production of recombinant proteins in Escherichia coli, reliant on tightly controlled autoinduction, triggered by phosphate depletion in the stationary phase. The method, reliant on engineered strains and plasmids, enables improved protein expression across scales. Expression levels using this approach have reached as high as 55% of the total cellular protein. The initial use of the method in instrumented fed-batch fermentations enables cell densities of ∼30 gCDW/L and protein titers up to 8.1 ± 0.7 g/L (∼270 mg/gCDW). The process has also been adapted to an optimized autoinduction media, enabling routine batch production at culture volumes of 20 μl (384-well plates), 100 μl (96-well plates), 20 ml, and 100 ml. In batch cultures, cell densities routinely reach ∼5-7 gCDW/L, offering protein titers above 2 g/L. The methodology has been validated with a set of diverse heterologous proteins and is of general use for the facile optimization of routine protein expression from high throughput screens to fed-batch fermentation.

Keywords: autoinduction; phosphate depletion; protein expression; stationary phase.

FIGURE 1

Growth and byproduct formation of Escherichia coli strains in minimal media fermentations. Biomass levels and residual glucose concentration (blue) as a function of time for (a) BL21(DE3)pLys, (b) BW25113, (c) BWapldf, and (d) DLF_R002, respectively. (e) Distribution of glucose utilized during growth in minimal medium fermentations for BW25113 (white), BL21(DE3) pLys (light gray), BWapldf (dark gray), and DLF_R002 (black). Results are averages of duplicate fermentations. CO₂ was explicitly measured via off-gas analysis for strain BW25113, BWapldf, and DLF_R002. In the case of BL21(DE3) pLys, CO₂ is included in unknown products required to account for glucose consumption. OD, optical density

FIGURE 2

Autoinduction of GPFuv expression in 1 L bioreactors with (a) FGM10 minimal media and host strain DLF_R002 (triplicates, solid line, open symbols) and DLF_R003 (DLF_R002, ΔompT; single run, dotted line, filled squares) bearing plasmid pHCKan-yibDp-GFPuv. (b) FGM30 minimal media and host strain DLF_R003 (triplicates) bearing plasmid pHCKan-yibDp-GFPuv. Optical density (black lines) and GFPuv were measured over time. Shaded area is standard error of triplicate growth profiles. X’s, triangles, circles, and squares are normalized GFPuv fluorescence units, each symbol corresponding to a single fermentation. Green line is the best fit of the expression profiles. OD, optical density

FIGURE 3

Media development using design of experiment (DoE) methodology. 212 Media formulations were evaluated for autoinduction based on phosphate depletion, each comprising different “levels” of casamino acids, yeast extract, trace metals (TM mix), calcium sulfate (CaSO₄), magnesium sulfate (MgSO₄), iron(II) sulfate (FeSO₄), ammonium sulfate ((NH₄)₂SO₄), and citric acid. (a) Upper panel: GFP (green bars) and OD_{600 nm} (gray bars) rank ordered plot for all media formulations. Standard deviations are from triplicate experiments. Lower panel: Nutrient concentration levels for all media (refer to Supporting Information Materials Section 4, for specific concentrations for each level). Strain DLF_R002 with plasmid pHCKan-yibDp-GFPuv was used for all experiments. (b) GFP fluorescence (green line), phosphate levels (black circles) and OD_{600 nm} (black line) for strain DLF_R002 with plasmid pHCKan-yibDp-GFPuv in media #36 (autoinduction broth) media. Standard deviations (shaded regions) are from triplicate experiments. OD, optical density

FIGURE 4

Head to head comparison of autoinduction via phosphate depletion with pET-based expression in BL21(DE3). (a) pET-based mCherry expression in BL21(DE3) with pLysS. mCherry (red lines) and biomass levels (OD_{600 nm}, black lines) over time. Solid lines: lactose-based autoinduction. Dashed lines: IPTG induction in LB media. (b) yibDp-based mCherry expression in DLF_R002 in AB media mCherry (red lines) and biomass levels (OD_{600 nm}, black lines). (c) Cytometry of induced populations (gray: empty vector control, red: pET-mCherry in BL21(DE3) + pLysS, green: yibDp-mCherry in DLF_R002). (d) Expression of the Lon substrate (GFP-β20-cp6) in BL21(DE3) and DLF_R002. Normalized fluorescence is relative fluorescence normalized to OD. Black line: GFP control (non-Lon substrate) in DLF_R002. Red line: BL21(DE3) expressing GFP-β20-cp6. Green line: DLF_R002 expressing GFP-β20-cp6. Shaded areas are standard deviations of at least three replicates. AB, autoinduction broth; IPTG, isopropyl β-D-1-thiogalactopyranoside; LB, Luria broth; OD, optical density

FIGURE 5

Optimization of autoinduction in batch cultures at various scales. Impact of various fill volumes on expression in AB. Varying fill volumes in 384- and 96-well plates as well as 250 ml baffled Erlenmeyer and 2.8 L Fernbach flasks. When using 384-well plates, 0.05% polypropylene glycol (2,000 MW) was added to the media. DLF_R002 with plasmid pHCKan-yibDp-GFPuv was used for all experiments

FIGURE 6

Autoinduction in AB in 96-well plates for a diverse set of recombinant proteins including: GFPuv, mCherry, AlaDh* (a mutant alanine dehydrogenase), Nef (HIV-1 Nef protein), hGLYAT2 (human glycine acyltransferase-2 an N-terminal chitin binding tag), cimA3.7 (a mutant citramalate synthase), GST, mdlC (benzoylformate decarboxylase), matB (malonyl-CoA synthetase), and SBS (bornyl-diphosphate synthase with a C-terminal mCherry tag). Percent of total expression is given for three replicates. Refer to Figure S7 for an example SDS-PAGE result. AB, autoinduction broth; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis