Engineering Substrate Promiscuity of Nucleoside Phosphorylase Via an Insertions-Deletions Strategy.

Nucleoside phosphorylases (NPs) are the key enzymes in the nucleoside metabolism pathway and are widely employed for the synthesis of nucleoside analogs, which are difficult to access via conventional synthetic methods. NPs are generally classified as purine nucleoside phosphorylase (PNP) and pyrimidine or uridine nucleoside phosphorylase (PyNP/UP), based on their substrate preference. Here, based on the evolutionary information on the NP-I family, we adopted an insertions-deletions (InDels) strategy to engineer the substrate promiscuity of nucleoside phosphorylase AmPNPΔS2V102 K, which exhibits both PNP and UP activities from a trimeric PNP (AmPNP) of Aneurinibacillus migulanus. Furthermore, the AmPNPΔS2V102 K exerted phosphorylation activities toward arabinose nucleoside, fluorosyl nucleoside, and dideoxyribose, thereby broadening the unnatural-ribose nucleoside substrate spectrum of AmPNP. Finally, six purine nucleoside analogues were successfully synthesized, using the engineered AmPNPΔS2V102 K instead of the traditional "two-enzymes PNP/UP" approach. These results provide deep insights into the catalytic mechanisms of the PNP and demonstrate the benefits of using the InDels strategy to achieve substrate promiscuity in an enzyme, as well as broadening the substrate spectrum of the enzyme.

A high-fidelity, dual site-specific integration system in CHO cells by a Bxb1 recombinase.

Site-specific integration (SSI) via recombinase mediated cassette exchange (RMCE) has shown advantages over random integration methods for expression of biotherapeutics. As an extension of our previous work developing SSI host cells, we developed a dual-site SSI system having two independent integration sites at different genomic loci, each containing a unique landing pad (LP). This system was leveraged to generate and compare two RMCE hosts, one (dFRT) compatible with the Flp recombinase, the other (dBxb1) compatible with the Bxb1 recombinase. Our comparison demonstrated that the dBxb1 host was able to generate stable transfectant pools in a shorter time frame, and cells within the dBxb1 transfectant pools were more phenotypically and genotypically stable. We further improved process performance of the dBxb1 host, resulting in desired fed batch performance attributes. Clones derived from this improved host (referred as 41L-11) maintained stable expression profiles over extended generations. While the data represents a significant improvement in the efficiency of our cell line development process, the dual LP architecture also affords a high degree of flexibility for development of complex protein modalities.

Evolving the 3-O/6-O regiospecificity of a microbial glycosyltransferase for efficient production of ginsenoside Rh1 and unnatural ginsenoside.

Glycosyltransferase is a popular and promising enzyme to produce high-value-added natural products. Rare ginsenoside Rh1 and unnatural ginsenoside 3ß-O-Glc-PPT are promising candidates for drugs. Herein, the microbial glycosyltransferase UGTBL1 was able to catalyze the 20(S)-protopanaxatriol (PPT) 3-O/6-O-glycosylation with poor 6-O-regiospecificity. A structure-guided strategy of mutations involving loop engineering, PSPG motif evolution, and access tunnel engineering was proposed to engineer the enzyme UGTBL1. The variant I62R/M320H/P321Y/N170A from protein engineering achieved a great improvement in 6-O regioselectivity which increased from 10.98 % (WT) to 96.26 % and a booming conversion of 95.57 % for ginsenoside Rh1. A single mutant M320W showed an improved 3-O regioselectivity of 84.83 % and an increased conversion of 98.13 % for the 3ß-O-glc-PPT product. Molecular docking and molecular dynamics (MD) simulations were performed to elucidate the possible molecular basis of the regiospecificity and catalytic activity. The unprecedented high titer of ginsenoside Rh1 (20.48 g/L) and 3ß-O-Glc-PPT (18.04 g/L) was attained with high regioselectivity and yields using fed-batch cascade reactions from UDPG recycle, which was the highest yield reported to date. This work could provide an efficient and cost-effective approach to the valuable ginsenosides.

Engineering the ß-Fructofuranosidase Fru6 with Promoted Transfructosylating Capacity for Fructooligosaccharide Production.

Levan-type fructooligosaccharides (FOS) exhibit enhanced health-promoting prebiotic effects on gut microbiota. The wild type (WT) of ß-fructofuranosidase Fru6 could mainly yield 6-ketose. Semirational design and mutagenesis of Fru6 were exploited to promote the transfructosylating capacity for FOS. The promising variants not only improved the formation of 6-kestose but also newly produced tetrasaccharides of 6,6-nystose and 1,6-nystose (a new type of FOS), and combinatorial mutation boosted the production of 6-kestose and tetrasaccharides (39.9 g/L 6,6-nystose and 4.6 g/L 1,6-nystose). Molecular docking and molecular dynamics (MD) simulation confirmed that the mutated positions reshaped the pocket of Fru6 to accommodate bulky 6-kestose in a reactive conformation with better accessibility for tetrasaccharides formation. Using favored conditions, the variant S165A/H357A could yield 6-kestose up to 335 g/L, and tetrasaccharides (6,6-nystose and 1,6-nystose) reached a high level of 121.1 g/L (134.5 times of the mutant S423A). The ß-(2,6)-linked FOS may show the potential application for the prebiotic ingredients.

Personalized and Programmable Microneedle Dressing for Promoting Wound Healing.

Microneedle (MN) dressings, with the ability of transdermal drug delivery, have played an essential role in the field of wound healing. However, patients may still feel uncomfortable when sensitive unhealing wounds are pieced by strong needles. Here, inspired by the structure of mosquito mouthparts, which possess a fixation part and a liquid-transferring part, we present a novel MN wound dressing with superfine needle tips, personalized pattern design, programmable needle length, and multiple mechanical strengths for intelligent and painless drug delivery. By simply stretching the silicone rubber (Ecoflex) molds before engraving, superfine MNs can be formed in the restored molds. Meanwhile, by utilizing intelligent image recognition, precise treatment for irregular wounds is achieved. Notably, combined with temperature-responsive N-isopropylacrylamide (NIPAM) hydrogel and inverse opal (IO) photonic crystals (PCs), a controllable drug release system has been achieved on MN dressings. Moreover, the performance of the MN dressing in facilitating wound recovery has been demonstrated by full-thickness skin wounds of a mouse model. These results indicate that novel personalized and programmable MN wound dressings are of considerable value in the field of wound management.

Highly Regioselective and Efficient Biosynthesis of Polydatin by an Engineered UGTBL1-AtSuSy Cascade Reaction.

Polydatin, resveratrol-3-O-ß-glucoside, possesses various biological activities. However, the regioselective glucosylation of resveratrol by UDP-glycosyltransferases (UGTs) constitutes a persistent problem. In this study, semi-rational design and iterative combinatorial mutagenesis were carried out to screen the mutants of UGTBL1 and the high specificity with the glycosylation of the 3-OH group of resveratrol was explored. The triple mutant I62G/M112D/K143G exhibited near-perfect control of polydatin synthesis (regioselectivity ∼ 99%), and the ratio of polydatin to resveratrol-4'-O-ß-glucoside was finally enhanced by 786-fold. Molecular docking revealed that the mutant could form three H-bonds between 3-, 5-, and 4'-OH groups of resveratrol and the residues around the active center, resulting in the oriented-binding of resveratrol. Furthermore, UGTBL1 mutant coupling sucrose synthase AtSuSy can synthesize polydatin at an unprecedented high titer of 10.33 g/L, together with efficient UDPG regeneration (RCmax = 54). This study provides an efficient approach for the regioselective biosynthesis of polydatin.

Specific immobilization of lipase on functionalized 3D printing scaffolds via enhanced hydrophobic interaction for efficient resolution of racemic 1-indanol.

Lipase immobilization with hydrophobic interaction is of interesting exploration, and some functionalized groups on supports are special for activity increasing. To achieved a good performance of cost-effective immobilization on macro-supports for feasible usage and recycle, eco-friendly PLA-based 3D printing macro-scaffolds with fabrication was designed, and phenyl groups with different length of linkers and combined two kinds of groups were anchored for lipase YCJ01 binding with improving payload, the highest enzyme expression of 2227.5 U/g, activity recovery of 137.3%, and increasing specific activity of 815.9 U/mg were attained by using PLA@AMTS-C7-Ph/PLA@AMTS-C9-Ph scaffolds as carries. The immobilized lipase YCJ01 on bifunctionalized 3D printing scaffolds was further applied to the efficient resolution of racemic 1-indanol (267 mM) with high stereoselectivity using a binary solvent system. The immobilized lipase YCJ01 could control the over transesterification of (S)-1-indanol and exhibit good operational stability of repetitive usage for 9 cycles. This is beneficial to obtain the high enantiomerical pure product by feasible separation of immobilized biocatalyst without rigorous operation.

Combing multiple site-directed mutagenesis of penicillin G acylase from Achromobacter xylosoxidans PX02 with improved catalytic properties for cefamandole synthesis.

Penicillin G acylase (PGA) was an important biocatalyst for enzymatic production of second-generation cephalosporin. PGA from Achromobacter xylosoxidans PX02 (AxPGA) showed relatively lower identity to EcPGA (54.9% in α subunit and 51.7% in ß subunit), which could synthesize cefamandole in the kinetically controlled N-acylation (kcNa). Semi-rational design of AxPGA and "small and smart" mutant libraries were developed with minimal screening to improve cefamandole production. A triple mutant αR141A/αF142I/ßF24G by combining the mutational sites (ßF24, αR141, and αF142) from different subunits of AxPGA showed better performance in cefamandole production, with 4.2-fold of improvement in the (kcat/Km)AD value for activated acyl donor (R)-Methyl mandelate. Meanwhile, the (kcat/Km)Ps value for cefamandole by mutant αR141A/αF142I/ßF24G was sharply dropped by 25.5 times, indicating its highly synthetic activity and extremely low hydrolysis of cefamandole. Strikingly, the triple mutant αR141A/αF142I/ßF24G could form cefamandole with a yield of 85% at an economical substrate ratio (acyl donor/nucleophile) of 1.3:1 (82% at 1.1:1), which advanced the greener and more sustainable process of cefamandole production than the wild type. Furtherly, the improved synthetic ability and lower hydrolysis of cefamandole by mutant were rationalized using molecular docking.

Modern evolution of paper-based analytical devices for wearable use: from disorder to order.

Paper devices have attracted great attention for their rapid development in multiple fields, such as life sciences, biochemistry, and materials science. When manufacturing paper chips, flexible materials, such as cellulose paper or other porous flexible membranes, can offer several advantages in terms of their flexibility, lightweight, low cost, safety and wearability. However, traditional cellulose paper sheets with chaotic cellulose fiber constitutions do not have special structures and optical characteristics, leading to poor repeatability and low sensitivity during biochemical sensing, limiting their wide application. Recent evidence showed that the addition of ordered structure provides a promising method for manufacturing intelligent flexible devices, making traditional flexible devices with multiple functions (microfluidics, motion detection and optical display). There is an urgent need for an overall summary of the evolution of paper devices so that readers can fully understand the field. Hence, in this review, we summarized the latest developments in intelligent paper devices, starting with the fabrication of paper and smart flexible paper devices, in the fields of biology, chemistry, electronics, etc. First, we outlined the manufacturing methods and applications of both traditional cellulose paper devices and modern smart devices based on pseudopaper (order paper). Then, considering different materials, such as cellulose, nitrocellulose, nature sourced photonic crystals (photonic crystals sourced from nature directly) and artificial photonic crystals, we summarized a new type of smart flexible device containing an ordered structure. Next, the applications of paper devices in biochemical sensing, wearable sensing, and cross-scale sensing were discussed. Finally, we summarized the development direction of this field. The aim of this review is to take an integral cognition approach to the development of smart flexible paper devices in multiple fields and promote communications between materials science, biology, chemistry and electrical science.

Efficient synthesis of ß-lactam antibiotics with in situ product removal by a newly isolated penicillin G acylase.

A penicillin G acylase (PGA) from Achromobacter xylosoxidans PX02 was newly isolated, and site-directed mutagenesis at three important positions αR141, αF142, ßF24 was carried out for improving the enzymatic synthesis of ß-lactam antibiotics. The efficient mutant ßF24A was selected, and the (Ps/Ph)ini (ratio between the initial rate of synthesis and hydrolysis of the activated acyl donor) dramatically increased from 1.42-1.50 to 23.8-24.1 by means of the optimization of reaction conditions. Interestingly, the efficient enzymatic synthesis of ampicillin (99.1% conversion) and amoxicillin (98.7% conversion) from a high concentration (600 mM) of substrate 6-APA in the low acyl donor/nucleus ratio (1.1:1) resulted in a large amount of products precipitation from aqueous reaction solution. Meanwhile, the by-product D-phenylglycine was hardly precipitated, and 93.5% yield of precipitated ampicillin (561 mM) and 94.6% yield of precipitated amoxicillin (568 mM) were achieved with high purity (99%), which significantly simplified the downstream purification. This was the first study to achieve efficient ß-lactam antibiotics synthesis process with in situ product removal, with barely any by-product formation. The effect enzymatic synthesis of antibiotics in aqueous reaction solution with in situ product removal provides a promising model for the industrial semi-synthesis of ß-lactam antibiotics.

Sharpening upconversion nanoparticles to reduce surface quenching.

Lanthanide-doped upconversion nanocrystals with different surface sharpness properties were synthesized to study the effects of surface sharpness on optical performance. Nanocrystals with hexagonal sharp surfaces showed a significant reduction in the number of surface defects and increased the luminescence intensity by 2.5 times compared to nanocrystals with spherical surfaces.

Emerging paper microfluidic devices.

Paper has unique advantages over other materials, including low cost, flexibility, porosity, and self-driven liquid pumping, thus making it widely used in various fields in biology, chemistry, physics and materials science. Recently, many multifunctional and highly integrated membrane-based devices have been achieved with the rapid development of membrane-building materials such as paper and pseudo-paper. Therefore, the rigid boundary between paper and other membranes has become blurred; paper can be considered a flexible membrane, and membranes with appropriately flexible or porous structures can also be defined as paper. Paper can manipulate liquids and respond photoelectrically to external objects to be measured, making it suitable for (bio)chemical sensing (chromatographic analysis, electrochemical analysis and wearable sensing). This review focuses on the development of microfluidic devices built with both traditional paper and other flexible membranes, including fabrication, (bio)chemical sensing, microfluidics manipulation and multiple applications.

Development of sugarcane resource for efficient fermentation of exopolysaccharide by using a novel strain of Kosakonia cowanii LT-1.

This work focuses on the development of non-food fermentation for the cost-effective biosynthesis of exopolysaccharide (EPS) by using a new strain of Kosakonia cowanii LT-1. This novel strain more efficiently utilizes sucrose for EPS production than other glycosyl donors. Comparative transcriptomic analysis is used to understand EPS synthesis promotion and the effects of sucrose on EPS biosynthesis. We speculate that ATP-binding cassette transporter, phosphotransferase, and two-component systems may be the most essential factors for EPS biosynthesis. The enhanced oxidative phosphorylation increases the synthesis rate of ATP to satisfy the energy demands for EPS production with sucrose as the substrate. Sugarcane juice, a cheap raw material, could improve the EPS yield in batch fermentation and achieve approximately 29.66% cost savings for substrate. Our work presents a promising non-food fermentation approach for the synthesis of high-value industrial products.

Comparison of Integrases Identifies Bxb1-GA Mutant as the Most Efficient Site-Specific Integrase System in Mammalian Cells.

Phage-derived integrases can catalyze irreversible, site-specific integration of transgenic payloads into a chromosomal locus, resulting in mammalian cells that stably express transgenes or circuits of interest. Previous studies have demonstrated high-efficiency integration by the Bxb1 integrase in mammalian cells. Here, we show that a point mutation (Bxb1-GA) in Bxb1 target sites significantly increases Bxb1-mediated integration efficiency at the Rosa26 locus in Chinese hamster ovary cells, resulting in the highest integration efficiency reported with a site-specific integrase in mammalian cells. Bxb1-GA point mutant sites do not cross-react with Bxb1 wild-type sites, enabling their use in applications that require orthogonal pairs of target sites. In comparison, we test the efficiency and orthogonality of ϕC31 and Wß integrases, and show that Wß has an integration efficiency between those of Bxb1-GA and wild-type Bxb1. Our data present a toolbox of integrases for inserting payloads such as gene circuits or therapeutic transgenes into mammalian cell lines.

Efficient Synthesis of Purine Nucleoside Analogs by a New Trimeric Purine Nucleoside Phosphorylase from Aneurinibacillus migulanus AM007.

Purine nucleoside phosphorylases (PNPs) are promising biocatalysts for the synthesis of purine nucleoside analogs. Although a number of PNPs have been reported, the development of highly efficient enzymes for industrial applications is still in high demand. Herein, a new trimeric purine nucleoside phosphorylase (AmPNP) from Aneurinibacillus migulanus AM007 was cloned and heterologously expressed in Escherichia coli BL21(DE3). The AmPNP showed good thermostability and a broad range of pH stability. The enzyme was thermostable below 55 °C for 12 h (retaining nearly 100% of its initial activity), and retained nearly 100% of the initial activity in alkaline buffer systems (pH 7.0-9.0) at 60 °C for 2 h. Then, a one-pot, two-enzyme mode of transglycosylation reaction was successfully constructed by combining pyrimidine nucleoside phosphorylase (BbPyNP) derived from Brevibacillus borstelensis LK01 and AmPNP for the production of purine nucleoside analogs. Conversions of 2,6-diaminopurine ribonucleoside (1), 2-amino-6-chloropurine ribonucleoside (2), and 6-thioguanine ribonucleoside (3) synthesized still reached >90% on the higher concentrations of substrates (pentofuranosyl donor: purine base; 20:10 mM) with a low enzyme ratio of BbPyNP: AmPNP (2:20 µg/mL). Thus, the new trimeric AmPNP is a promising biocatalyst for industrial production of purine nucleoside analogs.

Efficient Synthesis of Crocins from Crocetin by a Microbial Glycosyltransferase from Bacillus subtilis 168.

Crocins are the most important active ingredient found in Crocus sativus, a well-known "plant gold". The glycosyltransferase-catalyzed glycosylation of crocetin is the last step of biosynthesizing crocins and contributes to their structural diversity. Crocin biosynthesis is now hampered by the lack of efficient glycosyltransferases with activity toward crocetin. In this study, two microbial glycosyltransferases (Bs-GT and Bc-GTA) were successfully mined based on the comprehensive analysis of the PSPG motif and the N-terminal motif of the target plant-derived UGT75L6 and Cs-GT2. Bs-GT from Bacillus subtilis 168, an enzyme with a higher activity of glycosylation toward crocetin than that of Bc-GTA, was characterized. The efficient synthesis of crocins from crocetin catalyzed by microbial GT (Bs-GT) was first reported with a high molecular conversion rate of 81.9%, resulting in the production of 476.8 mg/L of crocins. The glycosylation of crocetin on its carboxyl groups by Bs-GT specifically produced crocin-5 and crocin-3, the important rare crocins.

ß-Galactosidase BMG without galactose and glucose inhibition: Secretory expression in Bacillus subtilis and for synthesis of oligosaccharide.

ß-Galactosidases can catalyze the hydrolysis of lactose and the synthesis of galacto-oligosaccharides (GOS) from lactose. The catalytic activity of ß-galactosidases is usually inhibited by galactose and glucose, which hampers the complete hydrolysis of lactose in food products. In this report, a ß-galactosidase (denoted as BMG) from Bacillus megaterium YZ08 without the inhibition by galactose and glucose is the first to our knowledge reported. Efficient secretory expression of BMG was successfully achieved in Bacillus subtilis WB800. With the increasing of galactose and glucose, the activity of BMG dramatically enhanced. With addition of 0.6 M galactose or 1 M glucose, the initial activity to oNPG hydrolysis was increased by 2.1-fold and 4.3-fold, respectively. Moreover, the synthetic rate of galacto-oligosaccharides was enhanced by 1.1-fold and 1.9-fold with the addition of 0.7 M galactose or glucose, respectively. The GOS (278 g/L) from 600 g/L lactose by BMG were efficiently produced within 12 h, and the substrate lactose was completely used up. These results suggest that BMG shows a potential application in the food industry.

Fitting replacement of signal peptide for highly efficient expression of three penicillin G acylases in E. coli.

High level expression of penicillin G acylase (PGA) in Escherichia coli is generally constricted by a complex maturation process and multiple limiting steps. In this study, three PGAs isolated from Providencia rettgeri (PrPGA), Alcaligenes faecalis (AfPGA), and Achromobacter xylosoxidans (AxPGA) were efficiently expressed in E. coli by replacing with applicable signal peptide. Different bottlenecks of the expression process were analyzed for PrPGA, AfPGA, and AxPGA. Subsequently, five efficient signal peptides, including OmpA, pelB, Lpp, PhoA, and MalE, were used to replace the original signal peptides of the PGAs. With respect to AfPGA and AxPGA, translocation was the primary limitation, and the use of pelB signal peptide effectively overcame this barrier. For PrPGA, which was almost not expressed in wild type, the translation initiation efficiency was optimized by replacing with MalE signal peptide. In addition, low temperature (20 °C) slowed down the transcription and translation, thereby facilitating the posttranslational process and preventing the formation of inclusion bodies. Furthermore, combined induction with IPTG and arabinose not only enhanced the cell density but also remarkably improved the expression of PGAs. Final specific activities of the three PGAs reached 2100 (PrPGA), 9200 (AfPGA), and 1400 (AxPGA) U/L/OD600, respectively. This simple and robust strategy by fitting replacement of signal peptide might dramatically improve the expression of PGAs from various bacteria, which was significant in the production of many valuable ß-lactam antibiotics.

Switching glycosyltransferase UGTBL1 regioselectivity toward polydatin synthesis using a semi-rational design.

The 62nd residue of glycosyltransferase UGTBL1 was identified as a "hotspot" for glycosylation at 3-OH of resveratrol. Via semi-rational design including structure-guided alanine scanning and saturation mutations, the mutation I62G significantly switched the regioselectivity from 4'-OH to 3-OH of resveratrol and mainly produced polydatin (87.7%), a therapeutic natural product.