A5034082839- Microbial Metabolic Engineering and Bioproduction
- Enzyme Catalysis and Immobilization
- Enzyme Structure and Function
- Photosynthetic Processes and Mechanisms
- Pharmacogenetics and Drug Metabolism
- Biochemical and Molecular Research
- Protein Structure and Dynamics
- Gene Regulatory Network Analysis
- Microbial metabolism and enzyme function
- Microbial Natural Products and Biosynthesis
- Electrochemical sensors and biosensors
- Bacterial Genetics and Biotechnology
- Microbial bioremediation and biosurfactants
- Metal-Catalyzed Oxygenation Mechanisms
- RNA and protein synthesis mechanisms
- Porphyrin and Phthalocyanine Chemistry
- ATP Synthase and ATPases Research
University of California, Irvine
2019-2024
Irvine University
2022
We report an aerobic, growth-based selection platform founded on NADP(H) redox balance restoration in Escherichia coli, and we demonstrate its application the high-throughput evolution of oxygenase. A single round followed by a facile growth assay enabled Pseudomonas aeruginosa 4-hydroxybenzoate hydroxylase (PobA) to efficiently hydroxylate both 4-hydroxybenzoic acid (4-HBA) 3,4-dihydroxybenzoic (3,4-DHBA), two consecutive steps gallic biosynthesis. Structural modeling suggests precise...
Noncanonical cofactors such as nicotinamide mononucleotide (NMN
Cyclohexanone monooxygenases (CHMO) consume molecular oxygen and NADPH to catalyze the valuable oxidation of cyclic ketones. However, CHMO usage is restricted by poor stability stringent specificity for NADPH. Efforts engineer have been limited sensitivity enzyme perturbations in conformational dynamics long-range interactions that cannot be predicted. We demonstrate an aerobic, high-throughput growth selection platform Escherichia coli oxygenase evolution based on NADH redox balance....
Noncanonical cofactor biomimetics (NCBs) such as nicotinamide mononucleotide (NMN
Directed evolution methods based on high-throughput growth selection enable efficient discovery of enzymes with improved function in vivo. High-throughput is particularly useful when engineering oxygenases, which are sensitive to structural perturbations and prone uncoupled activity. In this work, we combine the principle that reactive oxygen species (ROS) produced by oxygenase activity detrimental cell fitness a redox balance-based method for enables concurrent advancement catalytic...
Cyclohexanone monooxygenase (CHMO) from Acinetobacter sp. NCIMB 9871 is characterized as having wide substrate versatility for the biooxidation of (cyclic) ketones into esters and lactones with high stereospecificity. Despite industrial potential, CHMO usage restricted by poor thermostability. Limited high-throughput screening tools challenges in rationally engineering thermostability have impeded efforts. We demonstrate application an aerobic, growth selection platform Escherichia coli...
The future of biomanufacturing is dependent on rewiring biological systems to establish an alternative approach our current chemical industries. However, a key limitation in that desired processes must rely the same two redox cofactors as natural metabolism, nicotinamide adenine dinucleotide (phosphate) NAD(P)+, shuttle electrons energy. Thus, competition resources with reactions within host cells nearly unavoidable. One strategy overcome cofactor resource implementation third, noncanonical...
ABSTRACT We report an aerobic, growth-based selection platform founded on NADP(H) redox balance restoration in Escherichia coli , and demonstrate its application high-throughput evolution of oxygenase. A single round enabled Pseudomonas aeruginoasa 4-hydroxybenzoate hydroxylase (PobA) to accept 3,4-dihydroxybenzoic acid efficiently, essential step toward gallic biosynthesis. The best variant DA015 exhibited more than 5-fold higher catalytic efficiency compared previously engineered enzymes....
ABSTRACT Cyclohexanone monooxygenases (CHMO) consume molecular oxygen and NADPH to catalyze the valuable oxidation of cyclic ketones. However, CHMO usage is restricted by poor thermostability stringent specificity for NADPH. Efforts engineer have been limited sensitivity enzyme perturbations in conformational dynamics long-range interactions that cannot be predicted. We demonstrate a pair aerobic, high-throughput growth selection platforms Escherichia coli oxygenase evolution, based on or...