Microbial DNA polymerases and proteases for molecular applications

Abstract

Enzymes serve as biological catalysts in a wide range of applications, from household products to industrial processes, research, diagnostics and forensic science. They accelerate reactions with high specificity under mild reaction conditions and are essential for life. Advances in recombinant DNA technology have enabled the use of microorganisms as efficient cell factories for production of heterologous enzymes. Proteases and DNA polymerases are important enzymes in biotechnology due to their roles in protein degradation and DNA synthesis, respectively. In this thesis work, I focused on challenges related to the production of microbial proteases and DNA polymerases. It involved exploring host interactions between yeast and recombinant bacterial proteases and developing a simplified protocol for in-house production of DNA polymerases. Additionally, this thesis also focused on characterizing polymerase performance, including error types and rates during in vitro DNA amplification.

Bacterial proteases were fused to Green Fluorescent Protein (GFP) and produced in the yeast Saccharomyces cerevisiae. Both plasmid-based and integration-based systems were evaluated for use in protease production. The protein production after induction with galactose was followed on a single-cell level using flow cytometry (FCM), enabled by the GFP fusion. The plasmid-based strains resulted in population heterogeneity with two subpopulations, where approximately half of the cells did not show fluorescence levels above the autofluorescence. On the other hand, the integration-based strains resulted in homogenous populations where almost all the cells produced GFP fusions that resulted in fluorescence above the autofluorescence level. IdeS, a cysteine protease with a unique substrate specificity towards human IgG, was successfully produced as detected by FCM, Western blot and an activity assay. BdpK, a broad-spectrum serine protease, was produced and fluorescence was detected. However, no protease activity was observed, likely due to the formation of inclusion bodies. SpeB, another cysteine protease with a broad substrate scope, posed a substantial fitness burden on the yeast cells, even before induction. This indicates leakiness of the promoter used. Compartmentalization of the proteases to the peroxisome was successful and could potentially be used as a strategy to protect the host cells from the proteolytic activity.

A simplified protocol for in-house production of DNA polymerases was developed, which only requires readily available laboratory reagents, for use in the event of supply shortage or disruptions. The recombinant polymerases were successfully produced in the Escherichia coli strain Rosetta (DE3) pLysS and purified using Immobilized Metal Affinity Chromatography (IMAC) and gravity flow. The produced DNA polymerases were then shown to be compatible with several molecular biology techniques, including PCR, colony PCR, quantitative PCR (qPCR) and library preparation for sequencing. Comparable performance was achieved across the polymerase variants produced.

The effects of polymerase characteristics on amplicon yield and PCR error formation in Short Tandem Repeat (STR) analysis, including single-base substitutions and stutter artefacts, were studied using the SiMSen-Seq library preparation method. Unique Molecular Identifiers (UMIs) were applied in the first barcoding PCR, which was then followed by a second PCR (adaptor PCR), enabling detailed analysis of polymerase-introduced errors. Six DNA polymerases with different characteristics were used. The SiMSen-Seq library preparation was successful for all polymerases when applied in the adaptor PCR. DNA polymerases with both proofreading activity and DNA-binding domains produced high STR amplicon yield and lower levels of base substitutions compared to DNA polymerases lacking these domains. Stutter levels were not clearly connected with proofreading or DNA-binding properties.

This thesis demonstrates that alternative microbial hosts and simplified production workflows can be efficiently used for the recombinant production of both proteases and DNA polymerases, with specific strategies to mitigate host stress and optimize yield. The simplified polymerase production protocol enables accessible and reliable enzyme synthesis using standard laboratory resources. Furthermore, detailed characterization of polymerase performance in STR analysis highlights the importance of enzyme properties in minimizing PCR errors.

• author: Lindh, Tova
• supervisor: Johannes Hedman; Magnus Carlquist; Mattias Collin; Rolf Lood; Maja Sidstedt
• opponent: Dr. Dyrberg Andersen, Jeppe, University of Copenhagen, Denmark.
• organization: Division of Biotechnology and Applied Microbiology
• publishing date: 2025
• type: Thesis
• publication status: published
• subject: Microbiology; Molecular Biology; Forensic Science; Diagnostic Biotechnology
• keywords: DNA polymerase, protease, recombinant protein, GFP, flow cytometry, PCR, qPCR, sequencing, production protocol, Saccharomyces cerevisiae, BdpK, IdeS, SpeB, Escherichia coli, Taq Polymerase, Pfu polymerase, Sso7d, fidelity, Processivity, STR markers, base substitutions, stutter artefacts
• pages: 127 pages
• defense location: Lecture Hall KC:B, Kemicentrum, Naturvetarvägen 22, Faculty of Engineering LTH, Lund University, Lund.
• defense date: 2025-12-12 09:00:00
• ISBN: ISBN 978-91-8096-143-1
• ISBN: ISBN 978-91-8096-142-4
• project: Flow cytometry analysis of proteases of bacterial origin fused with GFP to follow expression in Saccharomyces cerevisiae
• language: English
• LU publication?: yes
• id: 1d6f96ad-321d-4d18-a06a-d0e32e7e5fe6
• date added to LUP: 2025-11-17 12:01:57
• date last changed: 2025-11-18 10:54:28