A systematic overexpression approach reveals native targets to increase squalene production in Synechocystis sp. PCC 6803

Anna T Germann; Andreas Nakielski; Maximilian Dietsch; Tim Petzel; Daniel Moser; Sebastian Triesch; Philipp Westhoff; Ilka M Axmann

doi:10.3389/fpls.2023.1024981

A systematic overexpression approach reveals native targets to increase squalene production in Synechocystis sp. PCC 6803

Front Plant Sci. 2023 May 30:14:1024981. doi: 10.3389/fpls.2023.1024981. eCollection 2023.

Authors

Anna T Germann¹, Andreas Nakielski¹, Maximilian Dietsch¹, Tim Petzel¹, Daniel Moser², Sebastian Triesch³, Philipp Westhoff⁴, Ilka M Axmann¹

Affiliations

¹ Institute for Synthetic Microbiology, Department of Biology, Heinrich Heine University Düsseldorf, Düsseldorf, Germany.
² Institute for Plant Sciences and Cluster of Excellence on Plant Sciences (CEPLAS), University of Cologne, Cologne, Germany.
³ Institute of Plant Biochemistry, Cluster of Excellence on Plant Science (CEPLAS), Heinrich Heine University, Düsseldorf, Germany.
⁴ Plant Metabolism and Metabolomics Laboratory, Cluster of Excellence on Plant Sciences (CEPLAS), Heinrich Heine University Düsseldorf, Düsseldorf, Germany.

Abstract

Cyanobacteria are a promising platform for the production of the triterpene squalene (C30), a precursor for all plant and animal sterols, and a highly attractive intermediate towards triterpenoids, a large group of secondary plant metabolites. Synechocystis sp. PCC 6803 natively produces squalene from CO₂ through the MEP pathway. Based on the predictions of a constraint-based metabolic model, we took a systematic overexpression approach to quantify native Synechocystis gene's impact on squalene production in a squalene-hopene cyclase gene knock-out strain (Δshc). Our in silico analysis revealed an increased flux through the Calvin-Benson-Bassham cycle in the Δshc mutant compared to the wildtype, including the pentose phosphate pathway, as well as lower glycolysis, while the tricarboxylic acid cycle predicted to be downregulated. Further, all enzymes of the MEP pathway and terpenoid synthesis, as well as enzymes from the central carbon metabolism, Gap2, Tpi and PyrK, were predicted to positively contribute to squalene production upon their overexpression. Each identified target gene was integrated into the genome of Synechocystis Δshc under the control of the rhamnose-inducible promoter P_rha. Squalene production was increased in an inducer concentration dependent manner through the overexpression of most predicted genes, which are genes of the MEP pathway, ispH, ispE, and idi, leading to the greatest improvements. Moreover, we were able to overexpress the native squalene synthase gene (sqs) in Synechocystis Δshc, which reached the highest production titer of 13.72 mg l^-1 reported for squalene in Synechocystis sp. PCC 6803 so far, thereby providing a promising and sustainable platform for triterpene production.

Keywords: FBA; MEP pathway; Synechocystis; metabolic engineering; metabolic modeling; squalene.

Grants and funding

This work was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany’s Excellence Strategy – EXC-2048/1 - project ID 390686111, and further, by project ID 391465903/GRK 2466.