AUTHOR=Germann Anna T. , Nakielski Andreas , Dietsch Maximilian , Petzel Tim , Moser Daniel , Triesch Sebastian , Westhoff Philipp , Axmann Ilka M. 

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

JOURNAL=Frontiers in Plant Science

VOLUME=14

YEAR=2023

URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2023.1024981

DOI=10.3389/fpls.2023.1024981

ISSN=1664-462X

ABSTRACT=<p>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. <italic>Synechocystis</italic> sp. PCC 6803 natively produces squalene from CO<sub>2</sub> through the MEP pathway. Based on the predictions of a constraint-based metabolic model, we took a systematic overexpression approach to quantify native <italic>Synechocystis</italic> gene’s impact on squalene production in a squalene-hopene cyclase gene knock-out strain (Δ<italic>shc</italic>). Our <italic>in silico</italic> analysis revealed an increased flux through the Calvin-Benson-Bassham cycle in the Δ<italic>shc</italic> 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 <italic>Synechocystis</italic> Δ<italic>shc</italic> under the control of the rhamnose-inducible promoter P<sub>rha</sub>. Squalene production was increased in an inducer concentration dependent manner through the overexpression of most predicted genes, which are genes of the MEP pathway, <italic>ispH</italic>, <italic>ispE</italic>, and <italic>idi</italic>, leading to the greatest improvements. Moreover, we were able to overexpress the native squalene synthase gene (<italic>sqs)</italic> in <italic>Synechocystis</italic> Δ<italic>shc</italic>, which reached the highest production titer of 13.72 mg l<sup>-1</sup> reported for squalene in <italic>Synechocystis</italic> sp. PCC 6803 so far, thereby providing a promising and sustainable platform for triterpene production.</p>