This report describes a gut engineering technique for investigation of the functions of a specific bacterium. Native phages with host specificity can knock down the corresponding E. coli strain in the mouse gut with limited perturbation of microbial diversity and relative abundance, indicating that they, as a microbial editing tool, can effectively edit the abundance of a target bacterium.

Currently, whole-cell catalysts face challenges due to the complexity of reaction systems, although they have a cost advantage over pure enzymes. In this study, cytarabine was synthesized by purified purine phosphorylase 1 (PNP1) and uracil phosphorylase (UP), and the conversion of cytarabine from adenine arabinoside reached 72.3 ± 4.3%. However, the synthesis was unsuccessful by whole-cell catalysis due to interference from unnecessary proteins (UNPs) in cells. Thus, we carried out a large-scale gene editing involving 377 genes in the genome of Escherichia coli to reduce the negative effect of UNPs on substrate conversion and cytarabine production. Finally, the PNP1 and UP activities of the obtained mutant were increased significantly compared with the parental strain, and more importantly, the conversion rate of cytarabine by whole-cell catalysis reached 67.4 ± 2.5%. The lack of 148 proteins and downregulation of 783 proteins caused by gene editing were equivalent to partial purification of the enzymes within cells, and thus, we provided inspiration to solve the problem caused by UNP interference, which is ubiquitous in the field of whole-cell catalysis.

Enzymes with dedicated cofactor preference are essential for advanced biocatalysis and biomanufacturing, especially when employing nonnatural nicotinamide cofactors in redox reactions. However, directed evolution of an enzyme to switch its cofactor preference is often hindered by the lack of efficient and affordable method for screening as the cofactor per se or the substrate can be prohibitively expensive. Here, we developed a growth-based selection platform to identify nonnatural cofactor-dependent oxidoreductase mutants. The growth of bacteria depended on the nicotinamide cytosine dinucleotide (NCD) mediated conversion of non-metabolizable phosphite into phosphate. The strain BW14329 lacking the ability to oxidize phosphite was suitable as host, and NCD-dependent phosphite dehydrogenase (Pdh*) is essential to the selection platform. Previously confirmed NCD synthetase with NCD synthesis capacity and NCD-dependent malic enzyme were successfully identified by using the platform. The feasibility of this strategy was successfully demonstrated using derived NCD-active malic enzyme as well as for the directed evolution of NCD synthetase in Escherichia coli. A phosphite-based screening platform was built for identification of enzymes favoring nonnatural cofactor NCD. In the future, once Pdh variants favoring other biomimetic or nonnatural cofactors are available this selection platform may be readily redesigned to attain new enzyme variants with anticipated cofactor preference, providing opportunities to further expand the chemical space of redox cofactors in chemical biology and synthetic biology.

BackgroundEnzymes with dedicated cofactor preference are essential for advanced biocatalysis and biomanufacturing. However, directed evolution of an enzyme to switch its cofactor preference is often hindered by the lack of efficient and affordable method for screening as the cofactor per se or the substrate can be prohibitively expensive. Here, we developed a growth-based selection platform to identify nonnatural cofactor-dependent oxidoreductase mutants.ResultsThe growth-based selection platform was designed by coupling with nonnatural cofactor-dependent phosphite dehydrogenase (Pdh) mediated the conversion of non-metabolizable phosphite into phosphate in the culture media. Thus, Pdh variant that strongly favors nicotinamide cytosine dinucleotide (NCD), a NAD analogue, the feasibility of this strategy was successfully demonstrated using derived NCD-active malic enzyme as well as for the directed evolution of NCD synthetase in Escherichia coli.ConclusionsHere, we built a phosphite-based screening platform for identification of enzymes favoring nonnatural cofactor NCD. In the future, once Pdh variants favoring other biomimetic or nonnatural cofactors are available this selection platform may be readily redesigned to attain new enzyme variants with anticipated cofactor preference, providing opportunities to further expand the chemical space of redox cofactors in chemical biology and synthetic biology.