Biology subjects

Aspacio, D.

Publications and source records attributed to Aspacio, D..

4 recordsLinked to original sources

bioRxiv2023

Shifting Redox Reaction Equilibria on Demand Using an Orthogonal Redox Cofactor

Natural metabolism relies on chemical compartmentalization of two redox cofactors, NAD+ and NADP+, to orchestrate life-essential redox reaction directions. However, in whole cells the reliance on these canonical cofactors limits flexible control of redox reaction direction as these reactions are permanently tied to catabolism or anabolism. In cell-free systems, NADP+ is too expensive in large scale. We have previously reported the use of nicotinamide mononucleotide, (NMN+) as a low-cost, noncanonical redox cofactor capable of specific electron delivery to diverse chemistries. Here, we present Nox Ortho, an NMNH-specific water-forming oxidase, that completes the toolkit to modulate NMNH/NMN+ ratio. This work uncovers an enzyme design principle that succeeds in parallel engineering of six butanediol dehydrogenases as NMN(H)-orthogonal biocatalysts consistently with a 103 - 106 -fold cofactor specificity switch from NAD(P)+ to NMN+. We combine these to produce chiral-pure 2,3-butanediol (Bdo) isomers without interference from NAD(H) or NADP(H) in vitro and in E. coli cells. We establish that NMN(H) can be held at a distinct redox ratio on demand, decoupled from both NAD(H) and NADP(H) redox ratios in vitro and in vivo.

synthetic biology↗

bioRxiv2020

Pairing two growth-based, high-throughput selections to fine tune conformational dynamics in oxygenase engineering

Cyclohexanone monooxygenases (CHMO) consume molecular oxygen and NADPH to catalyze the valuable oxidation of cyclic ketones. However, CHMO usage is restricted by poor thermostability and stringent specificity for NADPH. Efforts to engineer CHMO have been limited by the sensitivity of the enzyme to perturbations in conformational dynamics and long-range interactions that cannot be predicted. We demonstrate a pair of aerobic, high-throughput growth selection platforms in Escherichia coli for oxygenase evolution, based on NADPH or NADH redox balance. We utilize the NADPH-dependent selection in the directed evolution of thermostable CHMO and discover the variant CHMO GV (A245G-A288V) with a 2.7-fold improvement in residual activity compared to the wild type after 40 {degrees}C incubation. Addition of a previously reported mutation resulted in A245G-A288V-T415C which has further improved thermostability at 45 {degrees}C. We apply the NADH-dependent selection to alter the cofactor specificity of CHMO to accept NADH, a less expensive cofactor than NADPH. We identified the variant CHMO DTNP (S208D-K326T-K349N-L143P) with a 21-fold cofactor specificity switch from NADPH to NADH compared to the wild type. Molecular modeling indicates that CHMO GV experiences more favorable residue packing and backbone torsions, and CHMO DTNP activity is driven by cooperative fine-tuning of cofactor contacts. Our introduced tools for oxygenase evolution enable the rapid engineering of properties critical to industrial scalability.

bioengineering↗

bioRxiv2020

A growth-based, high-throughput selection platform enables remodeling of 4-hydroxybenzoate hydroxylase active site

We report an aerobic, growth-based selection platform founded on NADP(H) redox balance restoration in Escherichia coli, and demonstrate its application in high-throughput evolution of oxygenase. A single round of selection enabled Pseudomonas aeruginoasa 4-hydroxybenzoate hydroxylase (PobA) to accept 3,4-dihydroxybenzoic acid efficiently, an essential step toward gallic acid biosynthesis. The best variant DA015 exhibited more than 5-fold higher catalytic efficiency compared to previously engineered enzymes. Structural modeling suggests precise re-organization of active site hydrogen bond network, which is difficult to obtain without deep navigation of combinatorial sequence space. We envision universal application of this selection platform in engineering NADPH-dependent oxidoreductases.

bioengineering↗

bioRxiv2020

Metabolic engineering of Escherichia coli for optimized biosynthesis of nicotinamide mononucleotide, a noncanonical redox cofactor

Background Noncanonical redox cofactors are emerging as important tools in cell-free biosynthesis to increase the economic viability, to enable exquisite control, and to expand the range of chemistries accessible. However, these noncanonical redox cofactors need to be biologically synthesized to achieve full integration with renewable biomanufacturing processes.Results In this work, we engineered Escherichia coli cells to biosynthesize the noncanonical cofactor nicotinamide mononucleotide (NMN+), which has been efficiently used in cell-free biosynthesis. First, we developed a growth-based screening platform to identify effective NMN+ biosynthetic pathways in E. coli. Second, we explored various pathway combinations and host gene disruption to achieve an intracellular level of ~1.5 mM NMN+, a 130-fold increase over the cell’s basal level, in the best strain, which features a previously uncharacterized nicotinamide phosphoribosyltransferase (NadV) from Ralstonia solanacearum. Last, we revealed mechanisms through which NMN+ accumulation impacts E. coli cell fitness, which sheds light on future work aiming to improve the production of this noncanonical redox cofactor.Conclusion These results further the understanding of effective production and integration of NMN+ into E. coli. This may enable the implementation of NMN+-directed biocatalysis without the need for exogenous cofactor supply.Competing Interest StatementThe authors have declared no competing interest.AbbreviationsNAD+nicotinamide adenine dinucleotideNADP+nicotinamide adenine dinucleotide phosphateP2NA+3-carbomoyl-1-phenethylpyridin-1-ium chlorideNMN+nicotinamide mononucleotideNadVnicotinamide phosphoribosyltransferasesNadE*nicotinamide mononucleotide synthasePncCnicotinamide mononucleotideNaMN+nicotinic acid mononucleotideNRnicotinamide ribosidePnuCnicotinamide riboside transporterPnuC*mutant nicotinamide riboside transporterNrk1nicotinamide riboside kinase from Saccharomyces cerevisiaeNadRnicotinamide riboside kinase (Salmonella enterica)NAnicotinamideLC-MSliquid chromatography-mass spectrometryNaADnicotinic acid adenine dinucleotidePCRpolymerase chain reactionIPTGisopropyl-β-D-thiogalactopyranosider.p.m.rotations per minuteView Full Text

synthetic biology↗