Metabolic engineering of Deinococcus radiodurans for pinene production from glycerol

Abstract

Background The objective of this work was to engineer Deinococcus radiodurans R1 as a microbial cell factory for the production of pinene, a monoterpene molecule prominently used for the production of fragrances, pharmaceutical products, and jet engine biofuels. Our objective was to produce pinene from glycerol, an abundant by-product of various industries. Results To enable pinene production in D. radiodurans, we expressed the pinene synthase from Abies grandis, the geranyl pyrophosphate (GPP) synthase from Escherichia coli, and overexpressed the native 1-deoxy-d-xylulose 5-phosphate synthase. Further, we disrupted the deinoxanthin pathway competing for the substrate GPP by either inactivating the gene dr0862, encoding phytoene synthase, or substituting the native GPP synthase with that of E. coli. These manipulations resulted in a D. radiodurans strain capable of producing 3.2 ± 0.2 mg/L pinene in a minimal medium supplemented with glycerol, with a yield of 0.13 ± 0.04 mg/g glycerol in shake flask cultures. Additionally, our results indicated a higher tolerance of D. radiodurans towards pinene as compared to E. coli. Conclusions In this study, we successfully engineered the extremophile bacterium D. radiodurans to produce pinene. This is the first study demonstrating the use of D. radiodurans as a cell factory for the production of terpenoid molecules. Besides, its high resistance to pinene makes D. radiodurans a suitable host for further engineering efforts to increase pinene titer as well as a candidate for the production of the other terpenoid molecules.

Recent Advances and Multiple Strategies of Monoterpenoid Overproduction in Saccharomyces cerevisiae and Yarrowia lipolytica

“…92,113 Additionally, various microorganisms have been utilized for heterologous pinene synthesis, including purple nonsulfur photosynthetic bacteria, D. radiodurans and Candida glycerinogenes. 17,114,115 The highest reported yield of pinene to date was achieved in E. coli. Niu et al initially enhanced pinene tolerance to 2.0% through adaptive laboratory evolution.…”

“…Subsequently, they employed CRISPR/Cas9 technology to screen for the truncated α-Pinene synthase variant and further optimized the coordinated expression of different key enzymes using protein tags. Finally, they attained the highest reported titer of 36.6 mg/L of α-Pinene using soybean oil as the sole carbon source in 2021. , Additionally, various microorganisms have been utilized for heterologous pinene synthesis, including purple nonsulfur photosynthetic bacteria, D. radiodurans and Candida glycerinogenes . ,, The highest reported yield of pinene to date was achieved in E. coli . Niu et al initially enhanced pinene tolerance to 2.0% through adaptive laboratory evolution.…”

Sustainable cycloaliphatic polyurethanes: from synthesis to applications

“…Pinene can also be found in other plants or can be biosynthesized from engineered microbial communities from glycerol, which can be obtained after plastic waste recycling (chemical or biological). 268 Various applications have been developed from neat pinene in medicine, fuel production and flavor or fragrance. 269 Chemical modifications.…”

Toward improved terpenoids biosynthesis: strategies to enhance the capabilities of cell factories

“…Pathway engineering via deletion of competing pathways in the central carbon pathway for acetyl-CoA accumulation while increasing the availability of NADPH has also proven positive as seen in β-carotene production (Wu et al 2020b ). Also, a reduction in lipid biosynthesis through the inactivation of diacylglycerol acyltransferases produced 22.8 g/L of β-farnesene in Y. lipolytica (Shi et al 2021a ), whereas inactivation of phytoene synthase ( dr0862 ) in Deinococcus radiodurans yielded 3.2 ± 0.2 mg/L of pinene (Helalat et al 2021 ). Optimization of fermentation parameters, fermentation medium, and terpenoid pathway could improve biomass production while increasing the synthesis of target metabolites (Dai et al 2021 ; Liu et al 2020c , d ; Lv et al 2020 ; Walls et al 2020 ).…”