Investigation of protein secretion in microscale cultivation systems with novel tools
Müller, Carolin; Oldiges, Marco (Thesis advisor); Blank, Lars M. (Thesis advisor)
Aachen : RWTH Aachen University (2023)
Dissertation / PhD Thesis
Dissertation, RWTH Aachen University, 2023
Until today, it is impossible to predict a suitable signal peptide for Sec-type secretion of heterologous proteins in Gram-positive bacteria. Instead, signal peptides have to be tested for each host and target protein under process conditions. In addition, the ribosome binding site and in particular the spacer between the Shine-Dalgarno sequence and the start codon of the signal peptide can also affect protein secretion. To accelerate the identification of suitable combinations of signal peptides and target proteins, automated workflows for targeted strain construction and high-throughput screening for heterologous protein secretion in Corynebacterium glutamicum were established, which can be easily adapted to different target proteins. A plasmid library with different Bacillus subtilis signal peptides was constructed in the newly designed pPBEx2-based plasmid pCMEx12, which allows the exchange of the ribosome binding site including the spacer sequence as well as the signal peptide sequence by cassette mutagenesis. In this method, the inserts are provided as hybridized oligonucleotides that are not fully complementary, but have overhangs that can be ligated to the restriction digested backbone. For target protein secretion with pCMEx-based plasmids, a reporter gene coding for a blue chromoprotein under the control of a constitutive promoter can be exchanged with the gene of interest by Golden Gate assembly, combining restriction and ligation in a one-pot setup. Since the chromoprotein leads to blue colonies after transformation, successful cloning can be detected by a change in colony color from blue to white, in addition to a restriction enzyme digest in which the number and size of DNA fragments depend on the insert. The gene of interest is then expressed in frame with an amino-terminal signal peptide and carboxyl-terminally linked to the 11th β-sheet of the green fluorescent protein (GFP, GFP11-tag) under the control of the inducible tac promoter. The molecular cloning steps of the Golden Gate assembly, the Escherichia coli heatshock transformation, the plasmid purification and the restriction digest were automated using the Opentrons OT-2 liquid handling robot with integrated Temperature or Magnetic Module. This reduced the process time for molecular cloning to about 58% of that for the manual process. For testing cultivation workflows with online product monitoring, the C. glutamicum pPBEx2-PhoDCg-GFP enabling tightly controlled induction of GFP secretion was successfully prepared. An automated high-throughput screening workflow was developed on a Tecan Freedom EVO® robotic platform with an integrated centrifuge, microplate reader, and BioLector® for microscale cultivation with online measurement of the backscatter signal that correlates to cell dry weight. Automated preculture handling and backscatter-triggered inoculation of main cultures and induction ensure high comparability of bacterial growth. Using this workflow, suitable combinations of ribosome binding sites and B. subtilis signal peptides were identified for Fusarium solani f. sp. pisi cutinase-GFP11 secretion by C. glutamicum. Cutinase-GFP11 in the cultivation supernatant was detected 4 h after induction via activity measurement and activity-independently by assembly of the GFP11-tag with GFP1-10 in added detector solution by holo-GFP fluorescence in split GFP assay. The process time from cultivation of up to 12 different strains to detection of the target protein in the supernatant is about 1.5 days, with manual operations only required at the start of cultivation and prior to the assays. For high-throughput screening approaches, sufficient quantities of detector solution is needed. Therefore, a fed-batch cultivation process for the GFP1-10 production in laboratory-scale bioreactors was established and detector solution for up to 385 screenings in 96-well plates could be obtained. Aspects of GFP1-10 detector protein stability, storage and assay incubation conditions have been investigated. After a proof-of-concept using the secretion model protein cutinase, a B. subtilis signal peptide screening for secretion of polyethylene terephthalate degrading enzymes leaf-branch compost cutinase (LCC) and PE-H was conducted. For this, the cultivation workflow was optimized to allow the comparison of up to 24 different strains in one run. Using a process model combining Bayesian inference and Thompson sampling, the best of 24 signal peptides was identified with a probability of 80% for the PE-H and 75% for the LCC after only three batch cultivations.
- Department of Biology 
- Chair of Biotechnology