Automated chemoenzymatic modular synthesis of human milk oligosaccharides on a digital microfluidic platform.

Glycans, along with proteins, nucleic acids, and lipids, constitute the four fundamental classes of biomacromolecules found in living organisms. Generally, glycans are attached to proteins or lipids to form glycoconjugates that perform critical roles in various biological processes. Automatic synthesis of glycans is essential for investigation into structure-function relationships of glycans. In this study, we presented a method that integrated magnetic bead-based manipulation and modular chemoenzymatic synthesis of human milk oligosaccharides (HMOs), on a DMF (Digital Microfluidics) platform. On the DMF platform, enzymatic modular reactions were conducted in solution, and purification of products or intermediates was achieved by using DEAE magnetic beads, circumventing the intricate steps required for traditional solid-phase synthesis. With this approach, we have successfully synthesized eleven HMOs with highest yields of up to >90% on the DMF platform. This study would not only lay the foundation for OPME synthesis of glycans on the DMF platform, but also set the stage for developing automated enzymatic glycan synthesizers based on the DMF platform.

Digital microfluidics-engaged automated enzymatic degradation and synthesis of oligosaccharides.

Glycans are an important group of natural biopolymers, which not only play the role of a major biological energy resource but also as signaling molecules. As a result, structural characterization or sequencing of glycans, as well as targeted synthesis of glycans, is of great interest for understanding their structure-function relationship. However, this generally involves tedious manual operations and high reagent consumptions, which are the main technical bottlenecks retarding the advances of both automatic glycan sequencing and synthesis. Until now, automated enzymatic glycan sequencers or synthesizers are still not available on the market. In this study, to promote the development of automation in glycan sequencing or synthesis, first, programmed degradation and synthesis of glycans catalyzed by enzymes were successfully conducted on a digital microfluidic (DMF) device by using microdroplets as microreactors. In order to develop automatic glycan synthesizers and sequencers, a strategy integrating enzymatic oligosaccharide degradation or synthesis and magnetic manipulation to realize the separation and purification process after enzymatic reactions was designed and performed on DMF. An automatic process for enzymatic degradation of tetra-N-acetyl chitotetraose was achieved. Furthermore, the two-step enzymatic synthesis of lacto-N-tetraose was successfully and efficiently completed on the DMF platform. This work demonstrated here would open the door to further develop automatic enzymatic glycan synthesizers or sequencers based on DMF.

A novel decrystallizing protein CxEXL22 from Arthrobotrys sp. CX1 capable of synergistically hydrolyzing cellulose with cellulases.

A novel expansin-like protein (CxEXL22) has been identified and characterized from newly isolated Arthrobotrys sp. CX1 that can cause cellulose decrystallization. Unlike previously reported expansin-like proteins from microbes, CxEXL22 has a parallel ß-sheet domain at the N terminal, containing many hydrophobic residues to form the hydrophobic surface as part of the groove. The direct phylogenetic relationship implied the genetic transfers occurred from nematode to nematicidal fungal Arthrobotrys sp. CX1. CxEXL22 showed strong activity for the hydrolysis of hydrogen bonds between cellulose molecules, especially when highly crystalline cellulose was used as substrate. The hydrolysis efficiency of Avicel was increased 7.9-fold after pretreating with CxEXL22. The rupture characterization of crystalline region indicated that CxEXL22 strongly binds cellulose and breaks up hydrogen bonds in the crystalline regions of cellulose to split cellulose chains, causing significant depolymerization to expose much more microfibrils and enhances cellulose accessibility.

Perturbation of formate pathway and NADH pathway acting on the biohydrogen production.

The formate pathway and NADH pathway as two common hydrogen-producing metabolic pathways have been well characterized to understand and improve biohydrogen production. These two pathways have been thought to be separate and have been independently investigated. However, in this study, perturbation of genes (hycA, fdhF, fhlA, ldhA, nuoB, hybO, fdh1, narP, and ppk) in Enterobacter aerogenes related to the formate pathway or NADH pathway revealed that these two pathways affected each other. Further metabolic analysis suggested that a linear relationship existed between the relative change of hydrogen yield in the formate pathway or NADH pathway and the relative change of NADH yield or ATP yield. Thus, this finding provides new insight into the role of cellular reducing power and energy level in the hydrogen metabolism. It also establishes a rationale for improving hydrogen production from a global perspective.