A widespread pathway for substitution of adenine by diaminopurine in phage genomes

DNA modifications vary in form and function but generally do not alter Watson-Crick base pairing. Diaminopurine (Z) is an exception because it completely replaces adenine and forms three hydrogen bonds with thymine in cyanophage S-2L genomic DNA. However, the biosynthesis, prevalence, and importance of Z genomes remain unexplored. Here, we report a multienzyme system that supports Z-genome synthesis. We identified dozens of globally widespread phages harboring such enzymes, and we further verified the Z genome in one of these phages, Acinetobacter phage SH-Ab 15497, by using liquid chromatography with ultraviolet and mass spectrometry. The Z genome endows phages with evolutionary advantages for evading the attack of host restriction enzymes, and the characterization of its biosynthetic pathway enables Z-DNA production on a large scale for a diverse range of applications.

Liquid-Phase and Ultrahigh-Frequency-Acoustofluidics-Based Solid-Phase Synthesis of Biotin-Tagged 6′/3′-Sialyl-N-Acetylglucosamine by Sequential One-Pot Multienzyme System

6′/3′-Sialylated N-acetyllactosamine (6′/3′-SLN) is important for discrimination of the source (human or avian) of influenza virus strains. Biotinylated oligosaccharides have been widely used for analysis and quick detection. The development of efficient strategies to synthesize biotin-tagged 6′/3′-SLN have become necessary. Effective mixing is essential for enzymatic solid-phase oligosaccharide synthesis (SPOS). In the current study, newly developed technology ultrahigh-frequency-acoustofluidics (UHFA), which can provide a powerful source for efficient microfluidic mixing, solid-phase oligosaccharide synthesis and one-pot multienzyme (OPME) system, were used to develop a new strategy for oligosaccharide synthesis. Firstly, biotinylated N-acetylglucosamine was designed and chemically synthesized through traditional approaches. Secondly, biotinylated 6′- and 3′-sialyl-N-acetylglucosamines were prepared in solution through two sequential OPME modules in with a yield of ~95%. Thirdly, 6′-SLN was also prepared through UHFA-based enzymatic solid-phase synthesis on magnetic beads with a yield of 64.4%. The current strategy would be potentially used for synthesis of functional oligosaccharides.

Bioethanol Production from Food Waste Applying the Multienzyme System Produced On-Site by Fusarium oxysporum F3 and Mixed Microbial Cultures

Waste management and production of clean and affordable energy are two main challenges that our societies face. Food waste (FW), in particular, can be used as a feedstock for the production of ethanol because of its composition which is rich in cellulose, hemicellulose and starch. However, the cost of the necessary enzymes used to convert FW to ethanol remains an obstacle. The on-site production of the necessary enzymes could be a possible solution. In the present study, the multienzyme production by the fungus Fusarium oxysporum F3 under solid state cultivation using different agroindustrial residues was explored. Maximum amylase, glucoamylase, endoglucanase, b-glucosidase, cellobiohydrolase, xylanase, b-xylosidase and total cellulase titers on wheat bran (WB) were 17.8, 0.1, 65.2, 27.4, 3.5, 221.5, 0.7, 0.052 and 1.5 U/g WB respectively. F. oxysporum was used for the hydrolysis of FW and the subsequent ethanol production. To boost ethanol production, mixed F. oxysporum and S. cerevisiae cultures were also used. Bioethanol production by F. oxysporum monoculture reached 16.3 g/L (productivity 0.17 g/L/h), while that of the mixed culture was 20.6 g/L (productivity 1.0 g/L/h). Supplementation of the mixed culture with glucoamylase resulted in 30.3 g/L ethanol with a volumetric productivity of 1.4 g/L/h.

Oriented immobilization of enzyme–DNA conjugates on magnetic Janus particles for constructing a multicompartment multienzyme system with high activity and stability

An exquisitely designed multicompartment multienzyme system has been constructed by encapsulating poly-tannic acid (pTA) on the surface of enzyme–DNA conjugates, which are anchored on magnetic Janus particles.

Two pathways for thiosulfate oxidation in the alphaproteobacterial chemolithotrophParacoccus thiocyanatusSST

Chemolithotrophic bacteria oxidize various sulfur species for energy and electrons, thereby operationalizing biogeochemical sulfur cycles in nature. The best-studied pathway of bacterial sulfur-chemolithotrophy, involving direct oxidation of thiosulfate to sulfate (without any free intermediate) by the SoxXAYZBCD multienzyme system, is apparently the exclusive mechanism of thiosulfate oxidation in facultatively chemolithotrophic alphaproteobacteria. Here we explore the molecular mechanisms of sulfur oxidation in the thiosulfate- and tetrathionate-oxidizing alphaproteobacteriumParacoccus thiocyanatusSST, and compare them with the prototypical Sox process characterized inParacoccus pantotrophus. Our results revealed the unique case where, an alphaproteobacterium has Sox as its secondary pathway of thiosulfate oxidation, converting ∼10% of the thiosulfate supplied whilst 90% of the substrate is oxidized via a Tetrathionate-Intermediate pathway. Knock-out mutation, followed by the study of sulfur oxidation kinetics, showed that thiosulfate-to-tetrathionate conversion, in SST, is catalyzed by a thiosulfate dehydrogenase (TsdA) homolog that has far-higher substrate-affinity than the Sox system of this bacterium, which, remarkably, is also less efficient than theP. pantotrophusSox.soxB-deletion in SST abolished sulfate-formation from thiosulfate/tetrathionate while thiosulfate-to-tetrathionate conversion remained unperturbed. Physiological studies revealed the involvement of glutathione in SST tetrathionate oxidation. However, zero impact of the knock-out of a thiol dehydrotransferase (thdT) homolog, together with no production of sulfite as an intermediate, indicated that tetrathionate oxidation in SST is mechanistically novel, and distinct from its betaproteobacterial counterpart mediated by glutathione, ThdT, SoxBCD and sulfite:acceptor oxidoreductase. All the present findings collectively highlight extensive functional diversification of sulfur-oxidizing enzymes across phylogenetically close, as well as distant, bacteria.

Biosynthesis of Raffinose and Stachyose from Sucrose via an In Vitro Multienzyme System

ABSTRACT Herein, we present a biocatalytic method to produce raffinose and stachyose using sucrose as the substrate. An in vitro multienzyme system was developed using five enzymes, namely, sucrose synthase (SUS), UDP-glucose 4-epimerase (GalE), galactinol synthase (GS), raffinose synthase (RS), and stachyose synthase (STS), and two intermedia, namely, UDP and inositol, which can be recycled. This reaction system produced 11.1 mM raffinose using purified enzymes under optimal reaction conditions and substrate concentrations. Thereafter, a stepwise cascade reaction strategy was employed to circumvent the instability of RS and STS in this system, and a 4.2-fold increase in raffinose production was observed. The enzymatic cascade reactions were then conducted using cell extracts to avoid the need for enzyme purification and supplementation with UDP. Such modification further increased raffinose production to 86.6 mM and enabled the synthesis of 61.1 mM stachyose. The UDP turnover number reached 337. Finally, inositol in the reaction system was recycled five times, and 255.8 mM raffinose (128.9 g/liter) was obtained. IMPORTANCE Soybean oligosaccharides (SBOS) have elicited considerable attention because of their potential applications in the pharmaceutical, cosmetics, and food industries. This study demonstrates an alternative method to produce raffinose and stachyose, which are the major bioactive components of SBOS, from sucrose via an in vitro enzyme system. High concentrations of galactinol, raffinose, and stachyose were synthesized with the aid of a stepwise cascade reaction process, which can successfully address the issue of mismatched enzyme characteristics of an in vitro metabolic engineering platform. The biocatalytic approach presented in this work may enable the synthesis of other valuable galactosyl oligosaccharides, such as verbascose and higher homologs, which are difficult to obtain through plant extraction.

INVESTIGATION OF INENZYME TREATMENTS TO ASSIST EXTRACTION OF ESSENTIAL OIL FROM THE LEAVES AND BRANCHES OF CINNAMOMUM CASSIA COLLECTED IN YEN BAI PROVINCE

In order to improve the efficiency of essential oil distillation of the leaves and branchesfrom the cassia plant (Cinnamomum cassia (L.) J. Presl) growing in Yen Bai province, theeffects of enzyme treatments of the plant materials before distillation were investigated usingcrude laccase obtained from culture medium of the fungus Ganoderma lucidum and Cellic Htech2 (Novozymes, Denmark), a multienzyme system consisting of cellulase and xylanase. Theresults showed that enzyme treatments increased oil yield and shorten distillation time. The useof a mixture of both enzyme systems was more effective than using them separately. Underoptimal conditions, an increase in oil yield of 41.7 % was achieved, while distillation time wasshortened from 8 to 5 hours. The enzyme treatments did not change the qualitative compositionof the essential oils. However, significant changes in the percentages of cinnamic aldehyde(69.74 % to 85.6 %) and cinnamyl acetate (17.2 % to 1.34 %) were observed.