دورية أكاديمية
Debottlenecking 4-hydroxybenzoate hydroxylation in Pseudomonas putida KT2440 improves muconate productivity from p-coumarate.
| العنوان: | Debottlenecking 4-hydroxybenzoate hydroxylation in Pseudomonas putida KT2440 improves muconate productivity from p-coumarate. |
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| المؤلفون: | Kuatsjah E; Renewable Resources and Enabling Sciences Center, National Renewable Energy Laboratory, Golden, CO, 80401, USA; Center for Bioenergy Innovation, Oak Ridge National Laboratory, Oak Ridge, TN, 37830, USA., Johnson CW; Renewable Resources and Enabling Sciences Center, National Renewable Energy Laboratory, Golden, CO, 80401, USA., Salvachúa D; Renewable Resources and Enabling Sciences Center, National Renewable Energy Laboratory, Golden, CO, 80401, USA., Werner AZ; Renewable Resources and Enabling Sciences Center, National Renewable Energy Laboratory, Golden, CO, 80401, USA; Center for Bioenergy Innovation, Oak Ridge National Laboratory, Oak Ridge, TN, 37830, USA., Zahn M; Centre for Enzyme Innovation, School of Biological Sciences, Institute of Biological and Biomedical Sciences, University of Portsmouth, Portsmouth, PO1 2DY, UK., Szostkiewicz CJ; Renewable Resources and Enabling Sciences Center, National Renewable Energy Laboratory, Golden, CO, 80401, USA., Singer CA; Renewable Resources and Enabling Sciences Center, National Renewable Energy Laboratory, Golden, CO, 80401, USA., Dominick G; Renewable Resources and Enabling Sciences Center, National Renewable Energy Laboratory, Golden, CO, 80401, USA., Okekeogbu I; Biosciences Division, Oak Ridge National Laboratory, 1 Bethel Valley Rd, Oak Ridge, TN, 37830, USA., Haugen SJ; Renewable Resources and Enabling Sciences Center, National Renewable Energy Laboratory, Golden, CO, 80401, USA., Woodworth SP; Renewable Resources and Enabling Sciences Center, National Renewable Energy Laboratory, Golden, CO, 80401, USA., Ramirez KJ; Renewable Resources and Enabling Sciences Center, National Renewable Energy Laboratory, Golden, CO, 80401, USA., Giannone RJ; Center for Bioenergy Innovation, Oak Ridge National Laboratory, Oak Ridge, TN, 37830, USA; Biosciences Division, Oak Ridge National Laboratory, 1 Bethel Valley Rd, Oak Ridge, TN, 37830, USA., Hettich RL; Center for Bioenergy Innovation, Oak Ridge National Laboratory, Oak Ridge, TN, 37830, USA; Biosciences Division, Oak Ridge National Laboratory, 1 Bethel Valley Rd, Oak Ridge, TN, 37830, USA., McGeehan JE; Centre for Enzyme Innovation, School of Biological Sciences, Institute of Biological and Biomedical Sciences, University of Portsmouth, Portsmouth, PO1 2DY, UK., Beckham GT; Renewable Resources and Enabling Sciences Center, National Renewable Energy Laboratory, Golden, CO, 80401, USA; Center for Bioenergy Innovation, Oak Ridge National Laboratory, Oak Ridge, TN, 37830, USA. Electronic address: gregg.beckham@nrel.gov. |
| المصدر: | Metabolic engineering [Metab Eng] 2022 Mar; Vol. 70, pp. 31-42. Date of Electronic Publication: 2022 Jan 01. |
| نوع المنشور: | Journal Article; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, Non-P.H.S. |
| اللغة: | English |
| بيانات الدورية: | Publisher: Academic Press Country of Publication: Belgium NLM ID: 9815657 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1096-7184 (Electronic) Linking ISSN: 10967176 NLM ISO Abbreviation: Metab Eng Subsets: MEDLINE |
| أسماء مطبوعة: | Original Publication: Brugge, Belgium ; Orlando, FL : Academic Press, c1999- |
| مواضيع طبية MeSH: | Pseudomonas putida*/genetics , Pseudomonas putida*/metabolism, Hydroxybenzoates/metabolism ; Hydroxylation ; Parabens |
| مستخلص: | The transformation of 4-hydroxybenzoate (4-HBA) to protocatechuate (PCA) is catalyzed by flavoprotein oxygenases known as para-hydroxybenzoate-3-hydroxylases (PHBHs). In Pseudomonas putida KT2440 (P. putida) strains engineered to convert lignin-related aromatic compounds to muconic acid (MA), PHBH activity is rate-limiting, as indicated by the accumulation of 4-HBA, which ultimately limits MA productivity. Here, we hypothesized that replacement of PobA, the native P. putida PHBH, with PraI, a PHBH from Paenibacillus sp. JJ-1b with a broader nicotinamide cofactor preference, could alleviate this bottleneck. Biochemical assays confirmed the strict preference of NADPH for PobA, while PraI can utilize either NADH or NADPH. Kinetic assays demonstrated that both PobA and PraI can utilize NADPH with comparable catalytic efficiency and that PraI also efficiently utilizes NADH at roughly half the catalytic efficiency. The X-ray crystal structure of PraI was solved and revealed absolute conservation of the active site architecture to other PHBH structures despite their differing cofactor preferences. To understand the effect in vivo, we compared three P. putida strains engineered to produce MA from p-coumarate (pCA), showing that expression of praI leads to lower 4-HBA accumulation and decreased NADP + /NADPH ratios relative to strains harboring pobA, indicative of a relieved 4-HBA bottleneck due to increased NADPH availability. In bioreactor cultivations, a strain exclusively expressing praI achieved a titer of 40 g/L MA at 100% molar yield and a productivity of 0.5 g/L/h. Overall, this study demonstrates the benefit of sampling readily available natural enzyme diversity for debottlenecking metabolic flux in an engineered strain for microbial conversion of lignin-derived compounds to value-added products. (Copyright © 2022 International Metabolic Engineering Society. All rights reserved.) |
| فهرسة مساهمة: | Keywords: Aromatic catabolism; Flavoprotein; Metabolic engineering; Muconic acid; Paenibacillus sp. JJ-1b; Para-hydroxybenzoate hydroxylase; PobA; PraI; Pseudomonas putida KT2440 |
| المشرفين على المادة: | 0 (Hydroxybenzoates) 0 (Parabens) JG8Z55Y12H (4-hydroxybenzoic acid) |
| تواريخ الأحداث: | Date Created: 20220104 Date Completed: 20220314 Latest Revision: 20220314 |
| رمز التحديث: | 20221213 |
| DOI: | 10.1016/j.ymben.2021.12.010 |
| PMID: | 34982998 |
| قاعدة البيانات: | MEDLINE |
Full Text Finder | تدمد: | 1096-7184 |
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| DOI: | 10.1016/j.ymben.2021.12.010 |