Enhancing the expression of terminal deoxynucleotidyl transferases by N-terminal truncation.

Terminal deoxynucleotidyl transferase (TdT), a unique DNA polymerase that catalyzes the template-free incorporation of nucleotides into single-stranded DNA, has facilitated the development of various oligonucleotide-based tools and methods, especially in the field of template-free enzymatic DNA synthesis. However, expressing vertebrate-derived TdTs in Escherichia coli complicates purification and increases production costs. In this study, N-terminal truncation of TdTs was performed to improve their expression and stability. The results revealed that N-terminal truncation could enhance the expression level of six TdTs. Among the truncated mutants, N-140-ZaTdT and N-140-CpTdT, with 140 amino acids removed, exhibited an increase in protein expression, which was 9.5- and 23-fold higher than their wild-types, respectively. Importantly, the truncation preserves the catalytic function of TdT. Additionally, the Tm values of N-140-ZaTdT increased by 4.9°C. The improved expression of the truncated mutants makes them more suitable for reducing production costs and advancing enzyme engineering.

Facilitating secretory expression of apple seed ß-glucosidase in Komagataella phaffii for the efficient preparation of salidroside.

Plant-derived ß-glucosidases hold promise for glycoside biosynthesis via reverse hydrolysis because of their excellent glucose tolerance and robust stability. However, their poor heterologous expression hinders the development of large-scale production and applications. In this study, we overexpressed apple seed ß-glucosidase (ASG II) in Komagataella phaffii and enhanced its production from 289 to 4322 U L-1 through expression cassette engineering and protein engineering. Upon scaling up to a 5-L high cell-density fermentation, the resultant mutant ASG IIV80A achieved a maximum protein concentration and activity in the secreted supernatant of 2.3 g L-1 and 41.4 kU L-1, respectively. The preparative biosynthesis of salidroside by ASG IIV80A exhibited a high space-time yield of 33.1 g L-1 d-1, which is so far the highest level by plant-derived ß-glucosidase. Our work addresses the long-standing challenge of the heterologous expression of plant-derived ß-glucosidase in microorganisms and presents new avenues for the efficient production of salidroside and other natural glycosides.

Development of a Thermodynamically Favorable Multi-enzyme Cascade Reaction for Efficient Sustainable Production of ω-Amino Fatty Acids and α,ω-Diamines.

Invited for this issue's cover is the group of Huilei Yu at the East China University of Science and Technology. The image shows a sustainable biosynthesis route to nylon monomers from bio-based substrate α, ω-dicarboxylic acids. The Research Article itself is available at 10.1002/cssc.202301477.

Directed evolution of Baeyer-Villiger monooxygenase for highly secretory expressed in Pichia pastoris and efficient preparation of chiral pyrazole sulfoxide.

The methylotrophic yeast Pichia pastoris (Komagataella phaffii) is a highly distinguished expression platform for the excellent synthesis of various heterologous proteins in recent years. With the advantages of high-density fermentation, P. pastoris can produce gram amounts of recombinant proteins. While not every protein of interest can be expressed to such high titers, such as Baeyer-Villiger monooxygenase (BVMO) (AcPSMO) which is responsible for pyrazole sulfide asymmetric oxidation. In this work, an excellent yeast expression system was established to facilitate efficient AcPSMO expression, which exhibited 9.5-fold enhanced secretion. Subsequently, an ultrahigh throughput screening method based on fluorescence-activated cell sorting by fusing super folder green fluorescent protein (sfGFP) in the C-terminal of AcPSMO was developed, and directed evolution was performed. The protein expression level of the superior mutant AcPSMOP1 (S58T/T252P/E336N/H456D) reached 84.6 mg/L at 100 mL shaking flask, which was 4.7 times higher than the levels obtained with the wild-type. Finally, the optimized chassis cells were used for high-density fermentation on a 5-L scale, and AcPSMOP1 protein yield of 3.4 g/L was achieved, representing approximately 85% of the total protein secreted. By directly employing the pH-adjusted supernatant as a biocatalyst, 20 g/L pyrmetazole sulfide was completely transformed into the corresponding (S)-sulfoxide, with a 78.8% isolated yield. This work confers dramatic benefits for efficient secretion of other BVMOs in P. pastoris.

Development of a Thermodynamically Favorable Multi-enzyme Cascade Reaction for Efficient Sustainable Production of ω-Amino Fatty Acids and α,ω-Diamines.

Aliphatic ω-amino fatty acids (ω-AFAs) and α,ω-diamines (α,ω-DMs) are essential monomers for the production of nylons. Development of a sustainable biosynthesis route for ω-AFAs and α,ω-DMs is crucial in addressing the challenges posed by climate change. Herein, we constructed an unprecedented thermodynamically favorable multi-enzyme cascade (TherFavMEC) for the efficient sustainable biosynthesis of ω-AFAs and α,ω-DMs from cheap α,ω-dicarboxylic acids (α,ω-DAs). This TherFavMEC was developed by incorporating bioretrosynthesis analysis tools, reaction Gibbs free energy calculations, thermodynamic equilibrium shift strategies and cofactor (NADPH&ATP) regeneration systems. The molar yield of 6-aminohexanoic acid (6-ACA) from adipic acid (AA) was 92.3 %, while the molar yield from 6-ACA to 1,6-hexanediamine (1,6-HMD) was 96.1 %, which were significantly higher than those of previously reported routes. Furthermore, the biosynthesis of ω-AFAs and α,ω-DMs from 20.0 mM α,ω-DAs (C6-C9) was also performed, giving 11.2 mM 1,6-HMD (56.0 % yield), 14.8 mM 1,7-heptanediamine (74.0 % yield), 17.4 mM 1,8-octanediamine (87.0 % yield), and 19.7 mM 1,9-nonanediamine (98.5 % yield), respectively. The titers of 1,9-nonanediamine, 1,8-octanediamine, 1,7-heptanediamine and 1,6-HMD were improved by 328-fold, 1740-fold, 87-fold and 3.8-fold compared to previous work. Therefore, this work holds great potential for the bioproduction of ω-AFAs and α,ω-DMs.

Enzymatic synthesis of pharmacologically relevant chiral sulfoxides by improved CbBVMO variants.

Baeyer-Villiger monooxygenases (BVMOs) are able to catalyse the asymmetric oxidation of sulfides. This property has made them attractive catalysts for the synthesis of chiral sulfoxide drugs. Here, we have designed and synthesised an exhaustive combinatorial mutant library of the previously identified lansoprazole sulfide monooxygenase CbBVMOV1. From this synthetic combinatorial mutant library, the best mutant, CbBVMOV3, was selected with a specific activity of approximately 1 U mg-1 for lansoprazole sulfoxides. We then optimised the reaction conditions of a two-phase system, achieving the enzymatic asymmetric synthesis of (R)-lansoprazole in a space-time yield of 213 g L-1 d-1 and an enantiomeric excess of >99% (R) with no detectable by-products. In addition, CbBVMOV3 showed higher activity towards other prazole sulfides. These results indicate the potential application of CbBVMO in the chiral sulfoxide drug industry.

Rational Design of Taxadiene Hydroxylase by Ancestral Enzyme Construction and the Elucidation of Key Amino Acids.

Cytochrome P450 monooxygenases (CYP450s) play an important role in the biosynthesis of natural products by activating inert C-H bonds and inserting hydroxyl groups. However, the activities of most plant-derived CYP450s are extremely low, limiting the heterologous biosynthesis of natural products. Traditional enzyme engineering methods, either rational or screening-based, are not suitable for CYP450s because of the lack of crystal structures and high-throughput screening methods for this class of enzymes. CYP725A4 is the first hydroxylase involved in the biosynthesis pathway of Taxol. Its low activity, promiscuity, and multispecificity make it a bottleneck in Taxol biosynthesis. Here, we identified key amino acids that affect the in vivo activity of CYP725A4 by constructing the ancestral enzymes of CYP725A4. We obtained positive mutants that showed an improved yield of hydroxylated products based on the key amino acids identified, providing guidance for the modification of other CYP450s involved in the biosynthesis of natural products.

Improving the Enantioselectivity of CHMOBrevi1 for Asymmetric Synthesis of Podophyllotoxin Precursor.

(R)-ß-piperonyl-γ-butyrolactones are key building blocks for the synthesis of podophyllotoxin, which have demonstrated remarkable potential in cancer treatment. Baeyer-Villiger monooxygenases (BVMOs)-mediated asymmetric oxidation is a green approach to produce chiral lactones. While several BVMOs were able to oxidize the corresponding cyclobutanone, most BVMOs gave the (S) enantiomer while Cyclohexanone monooxygenase (CHMO) from Brevibacterium sp. HCU1 gave (R) enantiomer, but with a low enantioselectivity (75 % ee). In this study, we use a strategy called "focused rational iterative site-specific mutagenesis" (FRISM) at residues ranging from 6 Šfrom substrate. The mutations by using a restricted set of rationally chosen amino acids allow the formation of a small mutant library. By generating and screening less than 60 variants, we achieved a high ee of 96.8 %. Coupled with the cofactor regeneration system, 9.3 mM substrate was converted completely in a 100-mL scale reaction. Therefore, our work reveals a promising synthetic method for (R)-ß-piperonyl-γ-butyrolactone with the highest enantioselectivity, and provides a new opportunity for the chem-enzymatic synthesis of podophyllotoxin.

Engineering a Formate Dehydrogenase for NADPH Regeneration.

Nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH) constitute major hydrogen donors for oxidative/reductive bio-transformations. NAD(P)H regeneration systems coupled with formate dehydrogenases (FDHs) represent a dreamful method. However, most of the native FDHs are NAD+ -dependent and suffer from insufficient reactivity compared to other enzymatic tools, such as glucose dehydrogenase. An efficient and competitive NADP+ -utilizing FDH necessitates the availability and robustness of NADPH regeneration systems. Herein, we report the engineering of a new FDH from Candida dubliniensis (CdFDH), which showed no strict NAD+ preference by a structure-guided rational/semi-rational design. A combinatorial mutant CdFDH-M4 (D197Q/Y198R/Q199N/A372S/K371T/▵Q375/K167R/H16L/K159R) exhibited 75-fold intensification of catalytic efficiency (kcat /Km ). Moreover, CdFDH-M4 has been successfully employed in diverse asymmetric oxidative/reductive processes with cofactor total turnover numbers (TTNs) ranging from 135 to 986, making it potentially useful for NADPH-required biocatalytic transformations.

Prokaryotic expression and characterization of artificial self-sufficient CYP120A monooxygenases.

Cytochrome P450 monooxygenases CYP120As are the unique non-membrane P450s, which are extensively involved in retinoid biodegradation. As the O-functionalized 1,3,3-trimethylcyclohex-1-ene moiety exists in many bioactive compounds which could only be catalyzed by Class II P450s, exploration of the catalytic repertoire of CYP120As is therefore highly attractive. However, up to date, only one bacteriogenic candidate (CYP120A1) was demonstrated for the hydroxylation of C16 and C17 of retinoic acid, by utilizing the integral membrane protein cytochrome P450 reductase redox partner for the electron transfer. Herein, we provided an efficient prokaryotic functional expression system of CYP120As in E. coli by expression of the CYP120A1 coupled with several reductase partners. Fusion redox partners to the C-terminal of the heme-domain are also working on other CYP120A members. Among them, the fusion protein of CYP120A29 and FAD/FMN reductase from Bacillus megaterium P450BM3 (CYP101A2) showed the highest expression level. Based on the available translational fusion systems, the regioselectivity and the substrate scope of the CYP120As have also been explored. This work represents a good starting point for further expanding the catalytic potential of CYP120 family. KEY POINTS: • Characterization of CYP120As in E. coli is firstly achieved by constructing fusion proteins. • The feasibility of three P450 reductase domains to CYP120As was evaluated. • Hydroxylated products of retinoic acid by six CYP120As were sorted and analyzed.

3D Printing of Microenvironment-Specific Bioinspired and Exosome-Reinforced Hydrogel Scaffolds for Efficient Cartilage and Subchondral Bone Regeneration.

In clinical practice, repairing osteochondral defects presents a challenge due to the varying biological properties of articular cartilages and subchondral bones. Thus, elucidating how spatial microenvironment-specific biomimetic scaffolds can be used to simultaneously regenerate osteochondral tissue is an important research topic. Herein, a novel bioinspired double-network hydrogel scaffold produced via 3D printing with tissue-specific decellularized extracellular matrix (dECM) and human adipose mesenchymal stem cell (MSC)-derived exosomes is described. The bionic hydrogel scaffolds promote rat bone marrow MSC attachment, spread, migration, proliferation, and chondrogenic and osteogenic differentiation in vitro, as determined based on the sustained release of bioactive exosomes. Furthermore, the 3D-printed microenvironment-specific heterogeneous bilayer scaffolds efficiently accelerate the simultaneous regeneration of cartilage and subchondral bone tissues in a rat preclinical model. In conclusion, 3D dECM-based microenvironment-specific biomimetics encapsulated with bioactive exosomes can serve as a novel cell-free recipe for stem cell therapy when treating injured or degenerative joints. This strategy provides a promising platform for complex zonal tissue regeneration whilst holding attractive clinical translation potential.

[Construction of multi-enzyme cascade reactions and its application in the synthesis of bifunctional chemicals].

The synthesis of fine chemicals using multi-enzyme cascade reactions is a recent hot research topic in the field of biocatalysis. The traditional chemical synthesis methods were replaced by constructing in vitro multi-enzyme cascades, then the green synthesis of a variety of bifunctional chemicals can be achieved. This article summarizes the construction strategies of different types of multi-enzyme cascade reactions and their characteristics. In addition, the general methods for recruiting enzymes used in cascade reactions, as well as the regeneration of coenzyme such as NAD(P)H or ATP and their application in multi-enzyme cascade reactions are summarized. Finally, we illustrate the application of multi-enzyme cascades in the synthesis of six bifunctional chemicals, including ω-amino fatty acids, alkyl lactams, α, ω-dicarboxylic acids, α, ω-diamines, α, ω-diols, and ω-amino alcohols.

E7 Peptide Enables BMSC Adhesion and Promotes Chondrogenic Differentiation of BMSCs Via the LncRNA H19/miR675 Axis.

Therapeutic strategies based on utilizing endogenous BMSCs have been developed for the regeneration of bone, cartilage, and ligaments. We previously found that E7 peptide (EPLQLKM) could enhance BMSC homing in bio-scaffolds and, therefore, promote cartilage regeneration. However, the profile and mechanisms of E7 peptide in cartilage regeneration remain elusive. In this study, we examined the effect of E7 peptide on the BMSC phenotype, including adhesion, viability and chondrogenic differentiation, and its underlying mechanism. The konjac glucomannan microsphere (KGM), a carrier material that is free of BMSC adhesion ability, was used as the solid base of E7 peptide to better explore the independent role of E7 peptide in BMSC behavior. The results showed that E7 peptide could support BMSC adhesion and viability in a comparable manner to RGD and promote superior chondrogenic differentiation to RGD. We examined differentially expressed genes of BMSCs induced by E7 compared to RGD. Subsequently, a real-time PCR validated the significantly upregulated expression of lncRNA H19, and the knockdown of lncRNA H19 or miR675, a downstream functional unit of H19, could significantly obscure the chondrogenic differentiation induced by E7. In conclusion, this study confirmed the independent role of E7 in the adhesion and viability of BMSCs and revealed the pro-chondrogenic effect of E7 on BMSCs via the H19/miR675 axis. These results could help establish new therapeutic strategies based on employing endogenous BMSCs for cartilage tissue regeneration.

Virtual screening of carboxylic acid reductases for biocatalytic synthesis of 6-aminocaproic acid and 1,6-hexamethylenediamine.

The key precursors for nylon synthesis, that is, 6-aminocaproic acid (6-ACA) and 1,6-hexamethylenediamine (HMD), are produced from petroleum-based feedstocks. A sustainable biocatalytic alternative method from bio-based adipic acid has been demonstrated recently. However, the low efficiency and specificity of carboxylic acid reductases (CARs) used in the process hampers its further application. Herein, we describe a highly accurate protein structure prediction-based virtual screening method for the discovery of new CARs, which relies on near attack conformation frequency and the Rosetta Energy Score. Through virtual screening and functional detection, five new CARs were selected, each with a broad substrate scope and the highest activities toward various di- and ω-aminated carboxylic acids. Compared with the reported CARs, KiCAR was highly specific with regard to adipic acid without detectable activity to 6-ACA, indicating a potential for 6-ACA biosynthesis. In addition, MabCAR3 had a lower Km with regard to 6-ACA than the previously validated CAR MAB4714, resulting in twice conversion in the enzymatic cascade synthesis of HMD. The present work highlights the use of structure-based virtual screening for the rapid discovery of pertinent new biocatalysts.

A shear-thinning, ROS-scavenging hydrogel combined with dental pulp stem cells promotes spinal cord repair by inhibiting ferroptosis.

Spinal cord injury (SCI) is a serious clinical disease. Due to the deformability and fragility of the spinal cord, overly rigid hydrogels cannot be used to treat SCI. Hence, we used TPA and Laponite to develop a hydrogel with shear-thinning ability. This hydrogel exhibits good deformation, allowing it to match the physical properties of the spinal cord; additionally, this hydrogel scavenges ROS well, allowing it to inhibit the lipid peroxidation caused by ferroptosis. According to the in vivo studies, the TPA@Laponite hydrogel could synergistically inhibit ferroptosis by improving vascular function and regulating iron metabolism. In addition, dental pulp stem cells (DPSCs) were introduced into the TPA@Laponite hydrogel to regulate the ratios of excitatory and inhibitory synapses. It was shown that this combination biomaterial effectively reduced muscle spasms and promoted recovery from SCI.

Hydroxylases involved in terpenoid biosynthesis: a review.

Terpenoids are pervasive in nature and display an immense structural diversity. As the largest category of plant secondary metabolites, terpenoids have important socioeconomic value in the fields of pharmaceuticals, spices, and food manufacturing. The biosynthesis of terpenoid skeletons has made great progress, but the subsequent modifications of the terpenoid framework are poorly understood, especially for the functionalization of inert carbon skeleton usually catalyzed by hydroxylases. Hydroxylase is a class of enzymes that plays an important role in the modification of terpenoid backbone. This review article outlines the research progress in the identification, molecular modification, and functional expression of this class of enzymes in the past decade, which are profitable for the discovery, engineering, and application of more hydroxylases involved in the plant secondary metabolism.

Colorimetric High-Throughput Screening Method for Directed Evolution of Prazole Sulfide Monooxygenase.

Baeyer-Villiger monooxygenases (BVMOs) are important biocatalysts for the enzymatic synthesis of chiral sulfoxides, including chiral sulfoxide-type proton pump inhibitors for the treatment of gastrointestinal diseases. However, native BVMOs are not yet suitable for practical application due to their unsatisfactory activity and thermostability. Although protein engineering approaches can help address these issues, few feasible high-throughput methods are available for the engineering of such enzymes. Herein, a colorimetric detection method to distinguish sulfoxides from sulfides and sulfones was developed for prazole sulfide monooxygenases. Directed evolution enabled by this method has identified a prazole sulfide monooxygenase CbBVMO variant with improved activity and thermostability that catalyzes the asymmetric oxidation of lansoprazole sulfide. A 71.3 % increase in conversion and 6 °C enhancement in the melting point were achieved compared with the wild-type enzyme. This new method is feasible for high-throughput screening of prazole sulfide monooxygenase variants with improved activity, thermostability, and/or substrate specificity.

Facile Production of (+)-Aristolochene and (+)-Bicyclogermacrene in Escherichia coli Using Newly Discovered Sesquiterpene Synthases from Penicillium expansum.

Penicillium expansum, producer of a wide array of secondary metabolites, has the potential to be a source of new terpene synthases. In this work, a platform was constructed with Escherichia coli BL21(DE3) by enhancing its endogenous 2-methyl-d-erythritol-4-phosphate pathway to supply sufficient terpenoid precursors. Using this precursor-supplying platform, we discovered two sesquiterpene synthases from P. expansum: PeTS1, a new (+)-aristolochene synthase, and PeTS4, the first microbial (+)-bicyclogermacrene synthase. To enhance the sesquiterpene production by PeTS1, we employed a MBP fusion tag to improve the heterologous protein expression, resulting in the increase of aristolochene production up to 50 mg/L in a 72 h flask culture, which is the highest production reported to date. We also realized the first biosynthesis of (+)-bicyclogermacrene, achieving 188 mg/L in 72 h. This work highlights the great potential of this microbial platform for the discovery of new terpene synthases and opens new ways for the bioproduction of other valuable terpenoids.

Cryo-self-assembled silk fibroin sponge as a biodegradable platform for enzyme-responsive delivery of exosomes.

Although advances in protein assembly preparation have provided a new platform for drug delivery during tissue engineering, achieving long-term controlled exosome delivery remains a significant challenge. Diffusion-dominated exosome release using protein hydrogels results in burst release of exosomes. Here, a fibroin-based cryo-sponge was developed to provide controlled exosome release. Fibroin chains can self-assemble into silk I structures under ice-cold conditions when annealed above the glass transition temperature. Exosome release is enzyme-responsive, with rates primarily determined by enzymatic degradation of the scaffolds. In vivo experiments have demonstrated that exosomes remain in undigested sponge material for two months, superior to their retention in fibrin glue, a commonly used biomaterial in clinical practice. Fibroin cryo-sponges were implanted subcutaneously in nude mice. The exosome-containing sponge group exhibited better neovascularization and tissue ingrowth effects, demonstrating the efficacy of this exosome-encapsulating strategy by realizing sustained release and maintaining exosome bioactivity. These silk fibroin cryo-sponges containing exosomes provide a new platform for future studies of exosome therapy.

Construction of an Escherichia coli cell factory to synthesize taxadien-5α-ol, the key precursor of anti-cancer drug paclitaxel.

Paclitaxel (Taxol™), an alkaloid of diterpenoid family, is one of the most widely used anti-cancer drugs due to its effectiveness against a variety of tumors. Rather than directly extraction and chemical synthesis of paclitaxel or its intermediates from yew plants, construction of a microbial cell factory for paclitaxel biosynthesis will be more efficient and sustainable. The challenge for biosynthesis of paclitaxel lies on the insufficient precursor, such as taxadien-5α-ol. In this study, we report a recombinant Escherichia coli strain constructed with a heterologous mevalonate pathway, a taxadiene synthase from yew, and a cytochrome P450-mediated oxygenation system for the de novo production of taxadien-5α-ol, the first product of the multi-step taxadiene oxygenation metabolism. The key enzymes including taxadiene synthases and cytochrome P450 reductases were screened, and the linker for fusing taxadiene-5α-hydroxylase with its reductase partner cytochrome P450 reductase was optimized. By reducing the metabolic burden and optimizing the fermentation conditions, the final production of total oxygenated taxanes was raised up to 27 mg L-1 in a 50-mL flask cultivation, of which the yield of taxadien-5α-ol was 7.0 mg L-1, representing approximately a 12-fold and 23-fold improvements, respectively, as compared with the initial titers. The engineered MVA pathway for the overproduction of terpenoid precursors can serve as an efficient platform for the production of other valuable terpenoids.