Project description:Living cells possess membraneless organelles formed by liquid-liquid phase separation. With the aim of better understanding the general functions of membraneless microcompartments, this paper constructs acellular multicompartment reaction systems using an aqueous multiphase system. Membraneless coacervate droplets are placed within a molecularly crowded environment, where a larger dextran (DEX) droplet is submerged in a polyethylene glycol (PEG) solution. The coacervate droplets are capable of sequestering reagents and enzymes with a long retention time, and demonstrate multistep cascading reactions through the liquid-liquid interfaces. The ability to change phase dynamics is also demonstrated through salt-mediated dissolution of coacervate droplets, which leads to the release and mixing of separately sequestered reagents and enzymes. Finally, as phase-separated materials in membraneless organelles are often substrates and substrate analogues for the enzymes sequestered or excluded in the organelles, this paper explores the interaction between DEX and dextranase, an enzyme that hydrolyzes DEX. The results reveal that dextranase suffers from substrate inhibition when partitioned directly in a DEX phase but that this inhibition can be mitigated and reactions greatly accelerated by compartmentalization of dextranase inside a coacervate droplet that is adjacent to, but phase-separated from, the DEX phase. The insight that compartmentalization of enzymes can accelerate reactions by mitigating substrate inhibition is particularly novel and is an example where artificial membraneless organelle-like systems may provide new insights into physiological cell functions.
Project description:Compartmentalized microfluidic devices with immobilized catalysts are a valuable tool for overcoming the incompatibility challenge in (bio) catalytic cascade reactions and high-throughput screening of multiple reaction parameters. To achieve flow control in microfluidics, stimuli-responsive hydrogel microvalves were previously introduced. However, an application of this valve concept for the control of multistep reactions was not yet shown. To fill this gap, we show the integration of thermoresponsive poly(N-isopropylacrylamide) (PNiPAAm) microvalves (diameter: 500 and 600 µm) into PDMS-on-glass microfluidic devices for the control of parallelized enzyme-catalyzed cascade reactions. As a proof-of-principle, the biocatalysts glucose oxidase (GOx), horseradish peroxidase (HRP) and myoglobin (Myo) were immobilized in photopatterned hydrogel dot arrays (diameter of the dots: 350 µm, amount of enzymes: 0.13-2.3 µg) within three compartments of the device. Switching of the microvalves was achieved within 4 to 6 s and thereby the fluid pathway of the enzyme substrate solution (5 mmol/L) in the device was determined. Consequently, either the enzyme cascade reaction GOx-HRP or GOx-Myo was performed and continuously quantified by ultraviolet-visible (UV-Vis) spectroscopy. The functionality of the microvalves was shown in four hourly switching cycles and visualized by the path-dependent substrate conversion.
Project description:BackgroundChitin, the main form of aminated polysaccharide in nature, is a biocompatible, polycationic, and antimicrobial biopolymer used extensively in industrial processes. Despite the abundance of chitin, applications thereof are hampered by difficulties in feedstock harvesting and limited structural versatility. To address these problems, we proposed a two-step cascade employing carbohydrate oxidoreductases and amine transaminases for plant polysaccharide aminations via one-pot reactions. Using a galactose oxidase from Fusarium graminearum for oxidation, this study compared the performance of CvATA (from Chromobacterium violaceum) and SpATA (from Silicibacter pomeroyi) on a range of oxidized carbohydrates with various structures and sizes. Using a rational enzyme engineering approach, four point mutations were introduced on the SpATA surface, and their effects on enzyme activity were evaluated.ResultsHerein, a quantitative colorimetric assay was developed to enable simple and accurate time-course measurement of the yield of transamination reactions. With higher operational stability, SpATA produced higher product yields in 36 h reactions despite its lower initial activity. Successful amination of oxidized galactomannan by SpATA was confirmed using a deuterium labeling method; higher aminated carbohydrate yields achieved with SpATA compared to CvATA were verified using HPLC and XPS. By balancing the oxidase and transaminase loadings, improved operating conditions were identified where the side product formation was largely suppressed without negatively impacting the product yield. SpATA mutants with multiple alanine substitutions besides E407A showed improved product yield. The E407A mutation reduced SpATA activity substantially, supporting its predicted role in maintaining the dimeric enzyme structure.ConclusionsUsing oxidase-amine transaminase cascades, the study demonstrated a fully enzymatic route to polysaccharide amination. Although the activity of SpATA may be further improved via enzyme engineering, the low operational stability of characterized amine transaminases, as a result of low retention of PMP cofactors, was identified as a key factor limiting the yield of the designed cascade. To increase the process feasibility, future efforts to engineer improved SpATA variants should focus on improving the cofactor affinity, and thus the operational stability of the enzyme.
Project description:Amino alcohols are versatile compounds with a wide range of applications. For instance, they have been used as chiral scaffolds in organic synthesis. Their synthesis by conventional organic chemistry often requires tedious multi-step synthesis processes, with difficult control of the stereochemical outcome. We present a protocol to enzymatically synthetize amino alcohols starting from the readily available L-lysine in 48 h. This protocol combines two chemical reactions that are very difficult to conduct by conventional organic synthesis. In the first step, the regio- and diastereoselective oxidation of an unactivated C-H bond of the lysine side-chain is catalyzed by a dioxygenase; a second regio- and diastereoselective oxidation catalyzed by a regiodivergent dioxygenase can lead to the formation of the 1,2-diols. In the last step, the carboxylic group of the alpha amino acid is cleaved by a pyridoxal-phosphate (PLP) decarboxylase (DC). This decarboxylative step only affects the alpha carbon of the amino acid, retaining the hydroxy-substituted stereogenic center in a beta/gamma position. The resulting amino alcohols are therefore optically enriched. The protocol was successfully applied to the semipreparative-scale synthesis of four amino alcohols. Monitoring of the reactions was conducted by high performance liquid chromatography (HPLC) after derivatization by 1-fluoro-2,4-dinitrobenzene. Straightforward purification by solid-phase extraction (SPE) afforded the amino alcohols with excellent yields (93% to >95%).
Project description:mRNAs are emerging modalities for vaccination and protein replacement therapy. Increasing the amount of protein produced by stabilizing the transcript or enhancing translation without eliciting a strong immune response are major steps towards overcoming the present limitations and improving their therapeutic potential. The 5' cap is a hallmark of mRNAs and non-natural modifications can alter the properties of the entire transcript selectively. Here, we developed a versatile enzymatic cascade for regioselective benzylation of various biomolecules and applied it for post-synthetic modification of mRNA at the 5' cap to demonstrate its potential. Starting from six synthetic methionine analogues bearing (hetero-)benzyl groups, S-adenosyl-l-methionine analogues are formed and utilized for N7G-cap modification of mRNAs. This post-synthetic enzymatic modification exclusively modifies mRNAs at the terminal N7G, producing mRNAs with functional 5' caps. It avoids the wrong orientation of the 5' cap-a problem in common co-transcriptional capping. In the case of the 4-chlorobenzyl group, protein production was increased to 139% during in vitro translation and to 128-150% in four different cell lines. This 5' cap modification did not activate cytosolic pathogen recognition receptors TLR3, TLR7 or TLR8 significantly more than control mRNAs, underlining its potential to contribute to the development of future mRNA therapeutics.
Project description:Microfluidic flow reactors permit the implementation of sensitive biocatalysts in polymeric environments (e.g., hydrogel dots), mimicking nature through the use of diverse microstructures within defined confinements. However, establishing complex hybrid structures to mimic biological processes and functions under continuous flow with optimal utilization of all components involved in the reaction process represents a significant scientific challenge. To achieve spatial, chemical, and temporal control for any microfluidic application, compartmentalization is required, as well as the unification of different sensitive compartments in the reaction chamber for the microfluidic flow design. This study presents a self-regulating microfluidic system fabricated by a sequential photostructuring process with an intermediate chemical process step to realize pH-sensitive hybrid structures for the fabrication of a microfluidic double chamber reactor for controlled enzymatic cascade reaction (ECR). The key point is the adaptation and retention of the function of pH-responsive horseradish peroxidase-loaded polymersomes in a microfluidic chip under continuous flow. ECR is successfully triggered and controlled by an interplay between glucose oxidase-converted glucose, the membrane state of pH-responsive polymersomes, and other parameters (e.g., flow rate and fluid composition). This study establishes a promising noninvasive regulatory platform for extended spatio-chemical control of current and future ECR and other cascade reaction systems.
Project description:d-pantolactone is an intermediate in the synthesis of d-pantothenic acid, which is known as vitamin B5. The commercial synthesis of d-pantolactone is carried out through the selective resolution of dl-pantolactone catalyzed by lactone hydrolase. In contrast to a kinetic resolution approach, the deracemization of dl-pantolactone is a simpler, greener, and more sustainable way to obtain d-pantolactone with high optical purity. Herein, an efficient three-enzyme cascade was developed for the deracemization of dl-pantolactone, using l-pantolactone dehydrogenase from Amycolatopsis methanolica (AmeLPLDH), conjugated polyketone reductase from Zygosaccharomyces parabailii (ZpaCPR), and glucose dehydrogenase from Bacillus subtilis (BsGDH). The AmeLPLDH was used to catalyze the dehydrogenated l-pantolactone into ketopantolactone; the ZpaCPR was used to further catalyze the ketopantolactone into d-pantolactone; and glucose dehydrogenase together with glucose fulfilled the function of coenzyme regeneration. All three enzymes were co-expressed in E. coli strain BL21(DE3), which served as the whole-cell biocatalyst. Under optimized conditions, 36 h deracemization of 1.25 M dl-pantolactone d-pantolactone led to an e.e.p value of 98.6%, corresponding to productivity of 107.7 g/(l·d).
Project description:Among the human milk oligosaccharides (HMOs), one of the most abundant oligosaccharides and has great benefits for human health is 3'-sialyllactose (3'-SL). Given its important physiological functions and the lack of cost-effective production processes, we constructed an in vitro multi-enzymatic cofactor recycling system for the biosynthesis of 3'-SL from a low-cost substrate. First, we constructed the biosynthetic pathway and increased the solubility of cytidine monophosphate kinase (CMK) with chaperones. We subsequently identified that β-galactosidase (lacZ) affects the yield of 3'-SL, and hence with the lacZ gene knocked out, a 3.3-fold increase in the production of 3'-SL was observed. Further, temperature, pH, polyphosphate concentration, and concentration of divalent metal ions for 3'-SL production were optimized. Finally, an efficient biotransformation system was established under the optimized conditions. The maximum production of 3'-SL reached 38.7 mM, and a molar yield of 97.1% from N-acetylneuraminic acid (NeuAc, sialic acid, SA) was obtained. The results demonstrate that the multi-enzymatic cascade biosynthetic pathway with cofactor regeneration holds promise as an industrial strategy for producing 3'-SL.
Project description:A stereoselective three-enzyme cascade for synthesis of diasteromerically pure ?-oxyfunctionalized ?-amino acids was developed. By coupling a dynamic kinetic resolution (DKR) using an N-acylamino acid racemase (NAAAR) and an L-selective aminoacylase from Geobacillus thermoglucosidasius with a stereoselective isoleucine dioxygenase from Bacillus thuringiensis, diastereomerically pure oxidized amino acids were produced from racemic N-acetylamino acids. The three enzymes differed in their optimal temperature and pH-spectra. Their different metal cofactor dependencies led to inhibitory effects. Under optimized conditions, racemic N-acetylmethionine was quantitatively converted into L-methionine-(S)-sulfoxide with 97% yield and 95% de. The combination of these three different biocatalysts allowed the direct synthesis of diastereopure oxyfunctionalized amino acids from inexpensive racemic starting material.
Project description:Arabinoside and derived nucleoside analogs, a family of nucleoside analogs, exhibit diverse typically biological activities and are widely used as antibacterial, antiviral, anti-inflammatory, antitumor, and other drugs in clinical and preclinical trials. Although with a long and rich history in the field of medicinal chemistry, the biosynthesis of arabinoside has only been sporadically designed and studied, and it remains a challenge. Here, we constructed an in vitro multi-enzymatic cascade for the biosynthesis of arabinosides. This artificial biosystem was systematically optimized, involving an exquisite pathway design, NADP+ regeneration, meticulous enzyme selection, optimization of the key enzyme dosage, and the concentration of inorganic phosphate. Under the optimized conditions, we achieved 0.37 mM of vidarabine from 5 mM of sucrose and 2 mM of adenine, representing 18.7% of the theoretical yield. Furthermore, this biosystem also has the capability to produce other arabinosides, such as spongouridine, arabinofuranosylguanine, hypoxanthine arabinofuranoside, fludarabine, and 2-methoxyadenine arabinofuranoside, from sucrose, and corresponding nucleobase by introducing different nucleoside phosphorylases. Overall, our biosynthesis approach provides a pathway for the biosynthesis of arabinose-derived nucleoside analogs, offering potential applications in the pharmaceutical industry.