Improving Squalene Production by Enhancing the NADPH/NADP Ratio, Modifying the Isoprenoid-feeding Module and Blocking the Menaquinone Pathway in
Overview
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Background: Squalene is currently used widely in the food, cosmetics, and medicine industries. It could also replace petroleum as a raw material for fuels. Microbial fermentation processes for squalene production have been emerging over recent years. In this study, to study the squalene-producing potential of (, we employed several increasing strategies for systematic metabolic engineering. These include the expression of human truncated squalene synthase, the overexpression of rate-limiting enzymes in isoprenoid pathway, the modification of isoprenoid-feeding module and the blocking of menaquinone pathway.
Results: Herein, human truncated squalene synthase was engineered in to create a squalene-producing bacterial strain. To increase squalene yield, we employed several metabolic engineering strategies. A fivefold squalene titer increase was achieved by expressing rate-limiting enzymes (IDI, DXS, and FPS) involved in the isoprenoid pathway. Pyridine nucleotide transhydrogenase (UdhA) was then expressed to improve the cellular NADPH/NADP ratio, resulting in a 59% increase in squalene titer. The Embden-Meyerhof pathway (EMP) was replaced with the Entner-Doudoroff pathway (EDP) and pentose phosphate pathway (PPP) to feed the isoprenoid pathway, along with the overexpression of and genes which encode rate-limiting enzymes in the EDP and PPP, leading to a 104% squalene content increase. Based on the blocking of menaquinone pathway, a further 17.7% increase in squalene content was achieved. Squalene content reached a final 28.5 mg/g DCW and 52.1 mg/L.
Conclusions: This study provided novel strategies for improving squalene yield and demonstrated the potential of producing squalene by .
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