Metabolic engineering of microorganisms is a key strategy in the production of commodities, and traditionally follows a synthetic biology-focused approach. While the techniques to design and build engineered microorganisms have greatly improved, cumbersome upfront characterization is still greatly required, making this process time-consuming and thus cost ineffective. Here we present an application of nanotechnology for use in metabolic engineering: using photoactivated CdSe/ZnS and CdS/ZnS core-shell quantum dots (QDs) for direct metal affinity binding and electron donation to intracellular, polyhistidine-tagged enzymes in Escherichia coli, improving enzyme catalytic activity and modulating bacterial metabolism. We exploit this metal-histidine binding and electron donation mechanism for improving metabolic flux through bottlenecks in central metabolism, improving bacterial growth. His7-tagged pentose phosphate glucose-6-phosphate dehydrogenase (G6PDH) and glycolytic phosphofructokinase-1 (PFK) enzymes were individually expressed on plasmids and the effects of light exposure, QD type and concentration, media carbon content, target protein, and histidine tagging on bacterial growth were evaluated. We found that the individual targeting of these enzymes resulted in a near 2-fold increase in E. coli growth under nutrient restricted LB media and light exposure compared to a GFP-control strain under dark conditions without QD intervention. 13C-isotope labeling and global metabolite analysis on QD-treated strains also demonstrated improved flux through each enzyme, resulting in 3.2-times more metabolites downstream of PFK in glycolysis, and 2.6-times more metabolites downstream of G6PDH in the pentose phosphate pathway compared to our control strain. Interestingly, CdSe/ZnS-associated G6PDH expression also showed 3.7-times more glutathione production, indicative of greater resistance to oxidative stress. These results clearly demonstrate the promise of these QD-enzyme biohybrids for altering cellular metabolism. The use of QDs over traditional microbe engineering is quick, efficient, and almost completely cloning-free, a first of its kind in synthetic biology and metabolic engineering.

Light-directed modulation of bacterial metabolism using cadmium quantum dots in Escherichia coli

ACS Spring Meeting 2021

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