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The biosynthetic efficiency of curcumin, a highly bioactive compound from the plant Curcuma longa, needs to be improved. In this study, we performed host cell and biosynthetic pathway engineering to improve curcumin biosynthesis. Using in vivo-directed evolution, the expression level of curcuminoid synthase (CUS), the rate-limiting enzyme in the curcumin biosynthetic pathway, was significantly improved. Furthermore, as curcumin is a highly hydrophobic compound, two cell membrane engineering strategies were applied to optimize the biosynthetic efficiency. Curcumin storage was increased by overexpression of monoglucosyldiacylglycerol synthase from Acholeplasma laidlawii, which optimized the cell membrane morphology. Furthermore, unsaturated fatty acid supplementation was used to enhance membrane fluidity, which greatly ameliorated the damaging effect of curcumin on the cell membrane. These two strategies enhanced curcumin biosynthesis and demonstrated an additive effect.

Overexpressing key enzymes of biosynthetic pathways for overproduction of value-added products usually imposes metabolic burdens on cells, which can be circumvented by improving the key enzyme activities. p-Coumarate: CoA ligase (4CL) is a critical enzyme in the phenylpropanoid pathway that synthesizes various natural products. To screen for 4CL with improved activity, a biosensor of resveratrol whose biosynthetic pathway involves 4CL was designed by engineering the TtgR regulatory protein. The biosensor exhibited good specificity and robustness, allowing rapid and sensitive selection of resveratrol hyper-producers. A 4CL variant with improved activity was selected from a 4CL mutagenesis library constructed in the resveratrol biosynthetic pathway in Escherichia coli. This mutant led to increased production of not only resveratrol but also the flavonoid naringenin, when introduced in their corresponding biosynthetic pathways. These findings demonstrate the feasibility of improving key enzyme activities in important biosynthetic pathways with the aid of designed biosensors of pathway products.

To mimic the delicately regulated metabolism in nature for improved efficiency, artificial and customized regulatory components for dynamically controlling metabolic networks in multiple layers are essential in laboratory engineering. For this purpose, a novel regulatory component for controlling vanillin biosynthetic pathway was developed through directed evolution, which was responsive to both the product vanillin and substrate ferulic acid, with different capacities. This regulatory component facilitated pathway expression via dynamic control of the intracellular substrate and product concentrations. As vanillin is an antimicrobial compound, low pathway expression and vanillin formation levels enabled better cell growth at an early stage, and the product feedback-activated pathway expression at later stages significantly improved biosynthesis efficiency. This novel multiple-layer dynamic control was demonstrated effective in managing the trade-off between cell growth and production, leading to improved cell growth and vanillin production compared to the conventional or quorum-sensing promoter-controlled pathway. The multiple-layer dynamic control enabled by designed regulatory components responsive to multiple signals shows potential for wide applications in addition to the dynamic controls based on biosynthetic intermediate sensing and quorum sensing reported to date.

In this study the repressor of Escherichia coli lac operon, LacI, has been engineered for altered effector specificity. A LacI saturation mutagenesis library was subjected to Fluorescence Activated Cell Sorting (FACS) dual screening. Mutant LacI-L5 was selected and it is specifically induced by lactulose but not by other disaccharides tested (lactose, epilactose, maltose, sucrose, cellobiose and melibiose). LacI-L5 has been successfully used to construct a whole-cell lactulose biosensor which was then applied in directed evolution of cellobiose 2-epimerase (C2E) for elevated lactulose production. The mutant C2E enzyme with ~32-fold enhanced expression level was selected, demonstrating the high efficiency of the lactulose biosensor. LacI-L5 can also be used as a novel regulatory tool. This work explores the potential of engineering LacI for customized molecular biosensors which can be applied in practice.