A Growth-Based, High-Throughput Selection Platform Enables Remodeling of 4-Hydroxybenzoate Hydroxylase Active Site

Maxel, Sarah; Aspacio, Derek; King, Edward J.; Zhang, Linyue; Acosta, Ana Paula; Li, Han

doi:10.1021/acscatal.0c01892

Cited by 30 publications

(35 citation statements)

References 26 publications

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“…Generally, high-throughput screening based on biosensors can be used to identify effective mutations. Maxel et al applied this selection platform to screen a NADPH-dependent-hydroxybenzoic acid hydroxylase that uses 3,4-dihydroxyphenyl acid as a substrate, and obtained variants with roughly 8-fold improved apparent catalytic efficiency (Kcat/Km) for 3,4-DHBA, compared to the wild type [ 49 , 50 ].…”

Section: Synthesis and Modification Pathways Of Melatonin In Microorganismsmentioning

confidence: 99%

Melatonin biosynthesis pathways in nature and its production in engineered microorganisms

Xie

Ding

Bai

et al. 2022

Synthetic and Systems Biotechnology

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Melatonin is a biogenic amine that can be found in plants, animals and microorganism. The metabolic pathway of melatonin is different in various organisms, and biosynthetic endogenous melatonin acts as a molecular signal and antioxidant protection against external stress. Microbial synthesis pathways of melatonin are similar to those of animals but different from those of plants. At present, the method of using microorganism fermentation to produce melatonin is gradually prevailing, and exploring the biosynthetic pathway of melatonin to modify microorganism is becoming the mainstream, which has more advantages than traditional chemical synthesis. Here, we review recent advances in the synthesis, optimization of melatonin pathway. l -tryptophan is one of the two crucial precursors for the synthesis of melatonin, which can be produced through a four-step reaction. Enzymes involved in melatonin synthesis have low specificity and catalytic efficiency. Site-directed mutation, directed evolution or promotion of cofactor synthesis can enhance enzyme activity and increase the metabolic flow to promote microbial melatonin production. On the whole, the status and bottleneck of melatonin biosynthesis can be improved to a higher level, providing an effective reference for future microbial modification.

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Section: Synthesis and Modification Pathways Of Melatonin In Microorganismsmentioning

confidence: 99%

Melatonin biosynthesis pathways in nature and its production in engineered microorganisms

Xie

Ding

Bai

et al. 2022

Synthetic and Systems Biotechnology

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“…Plasmid pLS305 was generated by the addition of mutation T415C using pLS304 as a template. Construction of strain MX203 was described previously [17].…”

Section: Media and Growthmentioning

confidence: 99%

“…Enzymes purified for residual specific activity assay (Figure 2B) were expressed in E. coli strain BW25113 [36] incubated at 30 • C after induction. Larger-scale purification for enzymes applied in follow-up specific activity assay (Figure 4) and conversion assay used E. coli strain MX203 [17] for expression and cultures were incubated at 25 • C. Expression was carried out by inoculation of 50 mL 2xYT media supplemented with 100 µg/mL spectinomycin and an overnight culture. Cells were grown at 37 • C in baffled shaking flasks and were induced at an OD 600nm of 0.6-0.8 with 0.2% arabinose.…”

Section: Media and Growthmentioning

confidence: 99%

“…Recently, we described the construction and application of an aerobic growth-based, highthroughput selection platform for engineering NADPH-dependent oxygenases [17]. This selection platform is based on the principle of cofactor redox balance [17][18][19][20][21][22]: NADPH generation is increased in the engineered E. coli strain MX203 by re-routing glucose metabolism through the pentose phosphate pathway; in addition, NADPH recycling is blocked by disrupting NADPH:quinone oxidoreductase and transhydrogenase. As a result, the cells cannot grow unless a NADPHconsuming enzyme is introduced to restore the NADPH/NADP + balance.…”

Section: Introductionmentioning

confidence: 99%

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In Vivo, High-Throughput Selection of Thermostable Cyclohexanone Monooxygenase (CHMO)

et al. 2020

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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 is restricted by poor thermostability. Limited high-throughput screening tools and challenges in rationally engineering thermostability have impeded CHMO engineering efforts. We demonstrate the application of an aerobic, high-throughput growth selection platform in Escherichia coli (strain MX203) for the discovery of thermostability enhancing mutations for CHMO. The selection employs growth for the easy readout of CHMO activity in vivo, by requiring nicotinamide adenine dinucleotide phosphate (NADPH)-consuming enzymes to restore cellular redox balance. In the presence of the native substrate cyclohexanone, variant CHMO GV (A245G-A288V) was discovered from a random mutagenesis library screened at 42 °C. This variant retained native activity, exhibited ~4.4-fold improvement in residual activity after 30 °C incubation, and demonstrated ~5-fold higher cyclohexanone conversion at 37 °C compared to the wild type. Molecular modeling indicates that CHMO GV experiences more favorable residue packing and supports additional backbone hydrogen bonding. Further rational design resulted in CHMO A245G-A288V-T415C with improved thermostability at 45 °C. Our platform for oxygenase evolution enabled the rapid engineering of protein stability critical for industrial scalability.

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“…Anaerobic fermentative growth of such mutants can be rescued by introducing pathways that reinstate fluxes from central metabolites and restore NAD + regeneration. This can provide a useful selection pressure to identify redox-active pathways, enzymes, and variants with desired properties useful for the production of products such as butanol, 2-methylpropan-1-ol, L-alanine, and 2,3-butanediol 8 – 11 (related selections can be achieved involving different mutations, NADP and/or aerobic conditions 12 – 14 ). It is difficult to discern the contributions to the selection of NAD + regeneration, relieving central metabolite accumulation, and restoring other fluxes.…”

Section: Introductionmentioning

confidence: 99%

Versatile selective evolutionary pressure using synthetic defect in universal metabolism

2021

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The non-natural needs of industrial applications often require new or improved enzymes. The structures and properties of enzymes are difficult to predict or design de novo. Instead, semi-rational approaches mimicking evolution entail diversification of parent enzymes followed by evaluation of isolated variants. Artificial selection pressures coupling desired enzyme properties to cell growth could overcome this key bottleneck, but are usually narrow in scope. Here we show diverse enzymes using the ubiquitous cofactors nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP) can substitute for defective NAD regeneration, representing a very broadly-applicable artificial selection. Inactivation of Escherichia coli genes required for anaerobic NAD regeneration causes a conditional growth defect. Cells are rescued by foreign enzymes connected to the metabolic network only via NAD or NADP, but only when their substrates are supplied. Using this principle, alcohol dehydrogenase, imine reductase and nitroreductase variants with desired selectivity modifications, and a high-performing isopropanol metabolic pathway, are isolated from libraries of millions of variants in single-round experiments with typical limited information to guide design.

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A Growth-Based, High-Throughput Selection Platform Enables Remodeling of 4-Hydroxybenzoate Hydroxylase Active Site

Cited by 30 publications

References 26 publications

Melatonin biosynthesis pathways in nature and its production in engineered microorganisms

Melatonin biosynthesis pathways in nature and its production in engineered microorganisms

In Vivo, High-Throughput Selection of Thermostable Cyclohexanone Monooxygenase (CHMO)

Versatile selective evolutionary pressure using synthetic defect in universal metabolism

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