Metabolic engineering of type II methanotroph,Methylosinus trichosporium OB3b,for production of 3-hydroxypropionic acid from methane via a malonyl-CoA reductase-dependent pathway |
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| Affiliation: | 1. Department of Chemical Engineering, Kyung Hee University, Gyeonggi-do, 17104, Republic of Korea;2. Department of Chemical and Biomolecular Engineering, Sogang University, Seoul, 04107, Republic of Korea;1. State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China;2. Shanghai Collaborative Innovation Center for Biomanufacturing Technology, 130 Meilong Road, Shanghai 200237, China;1. Department of Civil and Environmental Engineering, Stanford University, Stanford, CA 94305, USA;2. Department of Chemistry, Stanford University, Stanford, CA 94305, USA;3. Woods Institute for the Environment, Stanford, CA 94305, USA;4. William and Cloy Codiga Resource Recovery Center, Stanford, CA 94305, USA;1. Department of Chemical and Biomolecular Engineering, Rice University, Houston 77030, United States;2. Department of BioSciences, Rice University, Houston 77030, United States |
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| Abstract: | We engineered a type II methanotroph, Methylosinus trichosporium OB3b, for 3-hydroxypropionic acid (3HP) production by reconstructing malonyl-CoA pathway through heterologous expression of Chloroflexus aurantiacus malonyl-CoA reductase (MCR), a bifunctional enzyme. Two strategies were designed and implemented to increase the malonyl-CoA pool and thus, increase in 3HP production. First, we engineered the supply of malonyl-CoA precursors by overexpressing endogenous acetyl-CoA carboxylase (ACC), substantially enhancing the production of 3HP. Overexpression of biotin protein ligase (BPL) and malic enzyme (NADP+-ME) led to a ∼22.7% and ∼34.5% increase, respectively, in 3HP titer in ACC-overexpressing cells. Also, the acetyl-CoA carboxylation bypass route was reconstructed to improve 3HP productivity. Co-expression of methylmalonyl-CoA carboxyltransferase (MMC) of Propionibacterium freudenreichii and phosphoenolpyruvate carboxylase (PEPC), which provides the MMC precursor, further improved the 3HP titer. The highest 3HP production of 49 mg/L in the OB3b-MCRMP strain overexpressing MCR, MMC and PEPC resulted in a 2.4-fold improvement of titer compared with that in the only MCR-overexpressing strain. Finally, we could obtain 60.59 mg/L of 3HP in 42 h using the OB3b-MCRMP strain through bioreactor operation, with a 6.36-fold increase of volumetric productivity compared than that in the flask cultures. This work demonstrates metabolic engineering of type II methanotrophs, opening the door for using type II methanotrophs as cell factories for biochemical production along with mitigation of greenhouse gases. |
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| Keywords: | Methane 3-hydroxypropionic acid Malonyl-CoA reductase Acetyl-CoA carboxylase Acetyl-CoA carboxylation bypass |
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