Substantial Improvement of an Epimerase for the Synthesis of D-Allulose by Biosensor-Based High-Throughput Microdroplet Screening.

Biosynthesis of D-allulose has been achieved using ketose 3-epimerases (KEases), but its application is limited by poor catalytic performance. In this study, we redesigned a genetically encoded biosensor based on a D-allulose-responsive transcriptional regulator for real-time monitoring of D-allulose. An ultrahigh-throughput droplet-based microfluidic screening platform was further constructed by coupling with this D-allulose-detecting biosensor for the directed evolution of the KEases. Structural analysis of Sinorhizobium fredii D-allulose 3-epimerase (SfDAE) revealed that a highly flexible helix/loop region exposes or occludes the catalytic center as an essential lid conformation regulating substrate recognition. We reprogrammed SfDAE using structure-guided rational design and directed evolution, in which a mutant M3-2 was identified with 17-fold enhanced catalytic efficiency. Our research offers a paradigm for the design and optimization of a biosensor-based microdroplet screening platform.

Enhanced Thermostability of an l-Rhamnose Isomerase for d-Allose Synthesis by Computation-Based Rational Redesign of Flexible Regions.

d-Allose is a low-calorie rare sugar with great application potential in the food and pharmaceutical industries. The production of d-allose has been accomplished using l-rhamnose isomerase (L-RI), but concomitantly increasing the enzyme's stability and activity remains challenging. Here, we rationally engineered an L-RI from Clostridium stercorarium to enhance its stability by comprehensive computation-aided redesign of its flexible regions, which were successively identified using molecular dynamics simulations. The resulting combinatorial mutant M2-4 exhibited a 5.7-fold increased half-life at 75 °C while also exhibiting improved catalytic efficiency. Especially, by combining structure modeling and multiple sequence alignment, we identified an α0 region that was universal in the L-RI family and likely acted as a "helix-breaker". Truncating this region is crucial for improving the thermostability of related enzymes. Our work provides a significantly stable biocatalyst with potential for the industrial production of d-allose.

Fine-Tuning of Carbon Flux and Artificial Promoters in Bacillus subtilis Enables High-Level Biosynthesis of d-Allulose.

d-Allulose is an attractive rare sugar that can be used as a low-calorie sweetener with significant health benefits. To meet the increasing market demands, it is necessary to develop an efficient and extensive microbial fermentation platform for the synthesis of d-allulose. Here, we applied a comprehensive systematic engineering strategy in Bacillus subtilis WB600 by introducing d-allulose 3-epimerase (DAEase), combined with the deactivation of fruA, levDEFG, and gmuE, to balance the metabolic network for the efficient production of d-allulose. This resulting strain initially produced 3.24 g/L of d-allulose with a yield of 0.93 g of d-allulose/g d-fructose. We further screened and obtained a suitable dual promoter combination and performed fine-tuning of its spacer region. After 64 h of fed-batch fermentation, the optimized engineered B. subtilis produced d-allulose at titers of 74.2 g/L with a yield of 0.93 g/g and a conversion rate of 27.6%. This d-allulose production strain is a promising platform for the industrial production of rare sugar.

Engineering of Acid-Resistant d-Allulose 3-Epimerase for Functional Juice Production.

d-Allulose, a rare sugar and functional sweetener, can be biosynthesized by d-allulose 3-isomerase (DAE). However, most of the reported DAEs exhibit poor resistance under acidic conditions, which severely limited their application. Here, surface charge engineering and random mutagenesis were used to construct a mutant library of CcDAE from Clostridium cellulolyticum H10, combined with high-throughput screening to identify mutants with high activity and resistance under acidic conditions. The mutant M3 (I114R/K123E/H209R) exhibited high activity (3.36-fold of wild-type) and acid resistance (10.6-fold of wild-type) at pH 4.5. The structure-function relationship was further analyzed by molecular dynamics (MD) simulations, which indicated that M3 had a higher number of hydrogen bonds and negative surface charges than the wild type. A multienzyme cascade system including M3 was used to convert high-calorie sugars in acidic juices, and functional juices containing 7.8-15.4 g/L d-allulose were obtained. Our study broadens the manufacture of functional foods containing d-allulose.