Biosensor-Based Optimization of Cutinase Secretion by Corynebacterium glutamicum

Abstract

The industrial microbe Corynebacterium glutamicum is gaining substantial importance as a platform host for recombinant protein secretion. We recently developed a fluorescence-based (eYFP) C. glutamicum reporter strain for the quantification of Sec-dependent protein secretion by monitoring the secretion-related stress response and now demonstrate its applicability in optimizing the secretion of the heterologous enzyme cutinase from Fusarium solani pisi. To drive secretion, either the poor-performing Pel^SP or the potent NprE^SP Sec signal peptide from Bacillus subtilis was used. To enable easy detection and quantification of the secreted cutinase we implemented the split green fluorescent protein (GFP) assay, which relies on the GFP11-tag fused to the C-terminus of the cutinase, which can complement a truncated GFP thereby reconstituting its fluorescence. The reporter strain was transformed with different mutant libraries created by error-prone PCR, which covered the region of the signal peptide and the N-terminus of the cutinase. Fluorescence-activated cell sorting (FACS) was performed to isolate cells that show increased fluorescence in response to increased protein secretion stress. Five Pel^SP variants were identified that showed a 4- to 6-fold increase in the amount and activity of the secreted cutinase (up to 4,100 U/L), whereas two improved NprE^SP variants were identified that showed a ∼35% increase in secretion, achieving ∼5,500 U/L. Most of the isolated variants carried mutations in the h-region of the signal peptide that increased its overall hydrophobicity. Using site-directed mutagenesis it was shown that the combined mutations F11I and P16S within the hydrophobic core of the Pel^SP are sufficient to boost cutinase secretion in batch cultivations to the same level as achieved by the NprE^SP. Screening of a Pel^SP mutant library in addition resulted in the identification of a cutinase variant with an increased specific activity, which was attributed to the mutation A85V located within the substrate-binding region. Taken together the biosensor-based optimization approach resulted in a substantial improvement of cutinase secretion by C. glutamicum, and therefore represents a valuable tool that can be applied to any secretory protein of interest.

Keywords: Corynebacterium glutamicum; EYFP; FACS; Sec-dependent export; fluorescence-based biosensor; protein secretion; signal peptide; split GFP.

FIGURE 1

Fluorescence-based monitoring of Sec-secretion stress in C. glutamicum and detection of secreted target protein by the split GFP assay. The overproduction of secretory proteins promotes accumulation of incompletely or misfolded proteins at the membrane-cell envelope interface. To alleviate this secretion stress, cells typically respond by the upregulation of the extra-cytoplasmic protease HtrA, which is able to degrade the misfolded proteins at the trans-side of the cytoplasmic membrane (CM). In C. glutamicum, a two-component system consisting of a sensory kinase (SK) and a response regulator (RR) is likely involved in regulation of expression of the adjacent htrA gene (Jurischka et al., 2020). In the C. glutamicum K9 biosensor strain, the htrA gene has been replaced by the eyfp gene, while leaving the htrA signal transduction pathway intact. The fluorescent reporter strain allows the monitoring of Sec-dependent secretion of recombinant proteins in a dose-dependent manner (Jurischka et al., 2020). The actual mechanism(s) and protein systems facilitating the transport of proteins across the mycolic acid outer membrane (OM) of C. glutamicum still remain an enigma. For detection of the target protein in the extracellular medium, we adapted the split GFP technology (Cabantous et al., 2005; Cabantous and Waldo, 2006) that previously had been adopted to monitor recombinant protein secretion in B. subtilis (Knapp et al., 2017). For this purpose, the eleventh β-sheet of GFP, is fused via a flexible linker to the C-terminus of the target protein (in our studies the model enzyme cutinase). The non-fluorescent detector GFP, which lacks eleventh β-sheet (GFP1-10) is produced separately in E. coli and purified from inclusion bodies. Combining the GFP11-tagged protein with the detector GFP1-10 enables reconstitution of functional fluorescent GFP. Subsequent measurement of the split GFP fluorescence allows detection and quantification of the secreted target protein. The white box indicates the signal peptide (SP); the green box indicates the GFP11-tag; SPase, signal peptidase.

FIGURE 2

Monitoring the secretion of GFP11-tagged cutinase using the C. glutamicum K9 biosensor strain and the split GFP assay. C. glutamicum K9 biosensor cells harboring pPBEx2 (EV), pPBEx2-NprE-cutinase-GFP11 or pPBEx2-Pel-cutinase-GFP11 were grown in CGXII medium containing 1% (v/v) glucose in a BioLector for 24 h at 30°C, 1,200 rpm and 85% relative humidity. Four hours after inoculation, IPTG was added to the cultures to indicated final concentrations. The fluorescence response of the biosensor cells during cultivation is shown in the upper panels as the Δspecific fluorescence over time for cells expressing NprE-cutinase-GFP11 (A) or Pel-cutinase-GFP11 (B). Cutinase-GFP11 in the respective culture supernatants after 24 h of growth was analyzed by SDS-PAGE (lower panels). The proteins were visualized by Coomassie Brilliant Blue staining. EV; C. glutamicum K9 biosensor cells harboring pPBEx2 (empty vector). The arrows indicate the position of the processed (signal peptide-less) cutinase-GFP11 (expected size 25.1 kDa). M, marker proteins with Mw indicated in kDa. (C) In parallel, culture supernatants were analyzed for cutinase activity and split GFP fluorescence. (D) Schematic view of the epPCR mutagenesis range, which spans the Pel^SP, the linker region and a substantial portion of the mature N-terminal part of the cutinase gene up to the unique internal KpnI restriction site. Positions of amino acids important for cutinase activity are indicated; i.e., the catalytic triad Ser120-Asp175-His188 and the four cysteines involved in disulfide bond formation.

FIGURE 3

Secretion performance of the variants isolated from the Pel^SP-cutinase-GFP11 mutant libraries. Plasmids were isolated from the clones isolated after FACS-based screening of the primary mutant library Pel^L1 (V1–V9) and the second-generation library Pel^L2 based on V7 (V7.1–V7.3) and then re-introduced in the C. glutamicum K9 biosensor strain. The recombinant strains were grown in CGXII medium containing 1% (v/v) glucose in a BioLector for 24 h at 30°C, 1,200 rpm and 85% relative humidity along with the control strains harboring pPBEx2 (EV), pPBEx2-NprE-cutinase-GFP11 (NprE^SP) or pPBEx2-Pel-cutinase-GFP11 (Pel^SP). Four hours after inoculation, IPTG was added (250 μM) to the cultures. At the end of the cultivation (24 h) the amount and activity of the extracellular cutinase-GFP11 was determined by (A) split GFP fluorescence and cutinase activity measurements, respectively. The secretion performance is shown relative to that of the wild-type Pel-cutinase-GFP11 (Pel^SP), which was set to 100%. (B) Complementary SDS-PAGE analysis of the extracellular levels of cutinase-GFP11 (expected size 25.1 kDa). The proteins were visualized by Coomassie Brilliant Blue staining. M, marker proteins with Mw indicated in kDa. (C) The relative specific activity for each variant was obtained by dividing the cutinase activity (%) by the split GFP fluorescence (%). The specific activity of the indicated variants relative to Pel^SP is shown in ascending order.

FIGURE 4

Secretion performance and hydrophobicity of Pel^SP variants created by site-directed mutagenesis. C. glutamicum K9 biosensor cells were transformed with pPBEx2-Pel-cutinase-GFP11 mutant plasmid encoding the different Pel^SP variants as indicated. The recombinant strains were grown in CGXII medium containing 1% (v/v) glucose in a BioLector for 24 h at 30°C, 1,200 rpm and 85% relative humidity, along with the control strains harboring pPBEx2 (EV), pPBEx2-NprE-cutinase-GFP11 (NprE^SP) and pPBEx2-Pel-cutinase-GFP11 (Pel^SP). Four hours after inoculation, IPTG was added to the cultures to a final concentration of 250 μM. (A) At the end of the cultivation (24 h), the secretion performance was assessed by measuring the split GFP fluorescence (green bars) and the cutinase activity (orange bars), respectively. The secretion performance of the different variants is shown relative to that of the Pel^SP control (100%) and is ranked according to the cutinase activity in ascending order. (B) The corresponding specific fluorescence at the end of the cultivation of the biosensor cells expressing the different variants is indicated. (C) Hydropathy plots showing the influence of the introduced single (left panel) and multiple mutations (right panel) as indicated on the overall hydrophobicity of the Pel^SP. The data were obtained by ProtScale (https://web.expasy.org) and using the hydropathy scale according to Kyte and Doolittle (1982).

FIGURE 5

Fed-batch of C. glutamicum K9 biosensor cells harboring different recombinant plasmids for secretory production of cutinase-GFP11. Biosensor cells carrying pPBEx2-NprE-cutinase-GFP11 (NprE^SP), pPBEx2-Pel-cutinase-GFP11 (Pel^SP) or the Pel^SP variants F11I, P16S, F11I/P16S or F11I/G13A/P16S were cultivated in a BioLector Pro at 30°C, 1,400 rpm, ≥30% headspace oxygen and ≥85% relative humidity. For the fed-batch process, CGXII medium with an initial glucose concentration of 5 g/L was inoculated to an OD₆₀₀ of 0.5 from the respective precultures. After 10 h, glucose was fed with a constant rate of 5.22 μL/h (equals 2.09 mg/h glucose). Regulation of pH to a set point of 6.8 was performed with 3 M KOH and was initiated 1 h after the start of the cultivation. Corresponding biomass and pH profiles are shown in Supplementary Figure S5. After 8 h of growth, cutinase-GFP11 expression was induced by the addition of IPTG (250 μM final concentration at the time of induction) and growth was continued for ∼17 h. At the end of the cultivation, the amount and activity of the secreted cutinase-GFP11 were determined by split GFP fluorescence and cutinase activity measurements, respectively.

FIGURE 6

Influence of the mutation A85V on the cutinase secretion and activity. (A) C. glutamicum K9 biosensor cells were transformed with pPBEx2-Pel-cutinase-GFP11 or pPBEx2-NprE-cutinase-GFP11 plasmid harboring either wild-type (WT) or A85V cutinase, as indicated. Biosensor cells carrying pPBEx2 (EV) served as control. The recombinant strains were grown in CGXII medium containing 1% (v/v) glucose in a BioLector for 24 h at 30°C, 1,200 rpm and 85% relative humidity. Four hours after inoculation, IPTG was added to the cultures to a final concentration of 250 μM. At the end of the cultivation (24 h), the secretion performance was assessed by measuring the split GFP fluorescence (green bars) and the cutinase activity (orange bars), respectively. (B) 3D-representation of the X-ray structure of Fusarium solani pisi cutinase (PDB: 1CEX) showing the catalytic triad Ser120-Asp175-His188 and the binding loop, which are essential for cutinase activity. The cutinase mutation A85V, which increases the enzyme activity toward pNPP, maps to the flap helix, which lines up with the substrate-binding pocket.

FIGURE 7

Secretion performance of two variants obtained from the NprE^SP-cutinase-GFP11 mutant library. Random mutagenesis of pPBEx2-NprE-cutinase-GFP11 and subsequent FACS analysis and screening of biosensor cells expressing the mutant library was performed as described for the Pel^SP-cutinase-GFP11 mutant libraries. Plasmids were isolated from promising clones and then re-introduced in the C. glutamicum K9 biosensor strain. The recombinant strains were grown in CGXII medium containing 1% (v/v) glucose in a BioLector for 24 h at 30°C, 1,200 rpm and 85% relative humidity along with the control strains harboring pPBEx2 (EV) or pPBEx2-NprE-cutinase-GFP11 (NprE^SP). Four hours after inoculation, IPTG was added to the cultures to a final concentration of 250 μM. (A) At the end of the cultivation (24 h) the amount and activity of the extracellular cutinase-GFP11 was determined by split GFP fluorescence (green bars) and cutinase activity measurements (orange bars), respectively. (B) The specific fluorescence of the corresponding biosensor cells at the end of the cultivation (24 h) is indicated.