Combining Protein Phase Separation and Bio-orthogonal Linking to Coimmobilize Enzymes for Cascade Biocatalysis.

The designed and ordered co-immobilization of multiple enzymes for vectorial biocatalysis is challenging. Here, a combination of protein phase separation and bioorthogonal linking is used to generate a zeolitic imidazole framework (ZIF-8) containing co-immobilized enzymes. Zn2+ ions induce the clustering of minimal protein modules, such as 6-His tag, proline-rich motif (PRM) and SRC homology 3 (SH3) domains, and allow for phase separation of the coupled aldoketoreductase (AKR) and alcohol dehydrogenase (ADH) at low concentrations. This is achieved by fusing SpyCatcher and PRM-SH3-6His peptide fragments to the C and N termini of AKR, respectively, and the SpyTag to ADH. Addition of 2-methylimidazole results in droplet formation and enables in situ spatial embedding the recombinant AKR and ADH to generate the cascade biocalysis system encapsulated in ZIF-8 (AAE@ZIF). In synthesizing (S)-1-(2-chlorophenyl) ethanol, ater 6 cycles, the yield can still reach 91%, with 99.99% enantiomeric excess (ee) value for each cycle. However, the yield could only reach 72.9% when traditionally encapsulated AKR and ADH in ZIF-8 are used. Thus, this work demonstrates that a combination of protein phase separation and bio-orthogonal linking enables the in situ creation of a stable and spatially organized bi-enzyme system with enhanced channeling effects in ZIF-8.

Precision Engineering of the Co-immobilization of Enzymes for Cascade Biocatalysis.

The design and orderly layered co-immobilization of multiple enzymes on resin particles remain challenging. In this study, the SpyTag/SpyCatcher binding pair was fused to the N-terminus of an alcohol dehydrogenase (ADH) and an aldo-keto reductase (AKR), respectively. A non-canonical amino acid (ncAA), p-azido-L-phenylalanine (p-AzF), as the anchor for covalent bonding enzymes, was genetically inserted into preselected sites in the AKR and ADH. Employing the two bioorthogonal counterparts of SpyTag/SpyCatcher and azide-alkyne cycloaddition for the immobilization of AKR and ADH enabled sequential dual-enzyme coating on porous microspheres. The ordered dual-enzyme reactor was subsequently used to synthesize (S)-1-(2-chlorophenyl)ethanol asymmetrically from the corresponding prochiral ketone, enabling the in situ regeneration of NADPH. The reactor exhibited a high catalytic conversion of 74 % and good reproducibility, retaining 80 % of its initial activity after six cycles. The product had 99.9 % ee, which that was maintained in each cycle. Additionally, the double-layer immobilization method significantly increased the enzyme loading capacity, which was approximately 1.7 times greater than that of traditional single-layer immobilization. More importantly, it simultaneously enabled both the purification and immobilization of multiple enzymes on carriers, thus providing a convenient approach to facilitate cascade biocatalysis.

Engineered aldoxime dehydratase to enable the chemoenzymatic conversion of benzyl amines to aromatic nitriles.

A chemoenzymatic strategy has been implemented to synthesize nitriles from benzyl amines under mild conditions. Aldoxime dehydratase (Oxd) plays a decisive role to convert aldoximes into corresponding nitriles. However, natural Oxds commonly exhibit extremely low catalytic capacity toward benzaldehyde oximes. Here, we engineered the OxdF1 from Pseudomonas putida F1 to enhance its catalytic efficiency toward benzaldehyde oximes by a semi-rational design strategy. The protein structure-based CAVER analysis indicates that M29, A147, F306, and L318 are located adjacent to the substrate tunnel entrance of OxdF1, which were responsible for the transportation of substrate into the active site. After two rounds of mutagenesis, the maximum activities of the mutants L318F and L318F/F306Y were 2.6 and 2.8 U/mg respectively, which were significantly higher than the wild OxdF1 of 0.7 U/mg. Meanwhile, the lipase type B from Candida antarctica was functionally expressed in Escherichia coli cells to selectively oxidize benzyl amines to aldoximes using urea-hydrogen peroxide adduct (UHP) as an oxidant in ethyl acetate. To merge the oxidation and dehydration reactions, a reductive extraction solution was added to remove the residue UHP, which is critical to eliminate its inhibition on the Oxd activity. Consequently, nine benzyl amines were efficiently converted into corresponding nitriles by the chemoenzymatic sequence.

Enzymatic properties of a non-classical aldoxime dehydratase capable of producing alkyl and arylalkyl nitriles.

Nitriles are of significant interest in the flavor and fragrance industries with potential application in cosmetics due to their higher stability than analogous aldehydes. However, the traditional methods to prepare nitriles need toxic reagents and hash conditions. This work aimed to develop a chemoenzymatic strategy to synthesize nitriles from natural aldehydes with aldoxime as the intermediate. A non-classical aldoxime dehydratase (Oxd) was discovered from the fungus Aspergillus ibericus (OxdAsp) to catalyze the dehydration of aldoximes to corresponding nitriles under mild conditions. The amino acid sequence of OxdAsp exhibits an approximately 20% identity with bacterial Oxds. OxdAsp contains a heme prosthetic group bound with the axial H287 in the catalytic pocket. The structure models of OxdAsp with substrates suggest that its catalytic triad is Y138-R141-E192, which is different from the classically bacterial Oxds of His-Arg-Ser/Thr. The catalytic mechanism of OxdAsp was proposed based on the mutagenesis of key residues. The hydroxyl group of the substrate is fixed by E192 to increase its basicity. Y138 acts as a general acid-based catalyst, and its phenolic proton is polarized by the adjacent R141. The protonated Y138 would donate a proton to the hydroxyl group of the substrate and eliminate a water molecule from aldoxime to produce nitrile. The recombinant OxdAsp can efficiently dehydrate citronellal oxime and cinnamaldoxime to citronellyl nitrile and cinnamonitrile in aqueous media, which are applied as fragrance ingredients in the food and cosmetic fields. KEY POINTS: • A novel aldoxime dehydratase from the Aspergillus genus was first characterized as a heme-binding protein. • The catalytic mechanism was predicted based on the molecular interactions of the catalytic pocket with the substrate. • A chemoenzymatic strategy was developed to synthesize nitriles from natural aldehydes with aldoxime as the intermediate.

Putting precision and elegance in enzyme immobilisation with bio-orthogonal chemistry.

The covalent immobilisation of enzymes generally involves the use of highly reactive crosslinkers, such as glutaraldehyde, to couple enzyme molecules to each other or to carriers through, for example, the free amino groups of lysine residues, on the enzyme surface. Unfortunately, such methods suffer from a lack of precision. Random formation of covalent linkages with reactive functional groups in the enzyme leads to disruption of the three dimensional structure and accompanying activity losses. This review focuses on recent advances in the use of bio-orthogonal chemistry in conjunction with rec-DNA to affect highly precise immobilisation of enzymes. In this way, cost-effective combination of production, purification and immobilisation of an enzyme is achieved, in a single unit operation with a high degree of precision. Various bio-orthogonal techniques for putting this precision and elegance into enzyme immobilisation are elaborated. These include, for example, fusing (grafting) peptide or protein tags to the target enzyme that enable its immobilisation in cell lysate or incorporating non-standard amino acids that enable the application of bio-orthogonal chemistry.

Enhancement of Monascus yellow pigments production by activating the cAMP signalling pathway in Monascus purpureus HJ11.

BACKGROUND: Monascus azaphilone pigments (MonAzPs), which were produced by Monascus species, have been used as important food colorant and food supplements for more than one billion people during their daily life. Moreover, MonAzPs recently have received more attention because of their diverse physiological activities. However, the high microbial production of MonAzPs is still not always guaranteed. Herein, the aim of this study was to develop an efficient biotechnological process for MonAzPs production. RESULTS: In this study, exogenous cyclic adenosine monophosphate (cAMP) treatment not only induced MonAzPs production, but also stimulated the expression of a cAMP phosphodiesterase gene, named as mrPDE, in M. purpureus HJ11. Subsequently, MrPDE was identified as a cAMP phosphodiesterase by in vitro enzymatic reaction with purified enzyme. Further, a gene knockout mutant of mrPDE was constructed to verify the activation of cAMP signalling pathway. Deletion of mrPDE in M. purpureus HJ11 improved cAMP concentration by 378% and enhanced PKA kinase activity 1.5-fold, indicating that activation of cAMP signalling pathway was achieved. The ΔmrPDE strain produced MonAzPs at 8563 U/g, with a 2.3-fold increase compared with the WT strain. Moreover, the NAPDH/NADP+ ratio of the ΔmrPDE strain was obviously higher than that of the wild type strain, which led to a higher proportion of yellow MonAzPs. With fed-batch fermentation of the ΔmrPDE strain, the production and yield of MonAzPs achieved 332.1 U/mL and 8739 U/g. CONCLUSIONS: A engineered M. purpureus strain for high MonAzPs production was successfully developed by activating the cAMP signalling pathway. This study not only describes a novel strategy for development of MonAzPs-producing strain, but also provides a roadmap for engineering efforts towards the production of secondary metabolism in other filamentous fungi.

Evidence for the participation of an extra α-helix at ß-subunit surface in the thermal stability of Co-type nitrile hydratase.

Nitrile hydratase (NHase) has attracted considerable attention since it can efficiently catalyze the hydration of nitriles to valuable amides. However, the poor stability of NHase is one of the main drawbacks in the industrial application. In this study, we compared the structural difference between Fe-type and Co-type NHase and found that an extra α helix existed at the ß-subunit surface of Co-type NHase (defined as the ß-6th helix). Then, the effects of the ß-6th helix were investigated on the thermal stability and the catalytic kinetics of a Co-type NHase from Aurantimonas manganoxydans ATCC BAA-1229 (NHase1229). When the ß-6th helix was deleted or disrupted, the thermal stability of NHase1229 was reduced to 17.6 and 12.9% of that of wild NHase1229, respectively. Thus, the ß-6th helix is important for the thermal stability of Co-type NHase. Based on the structural characteristics of Co-type NHase, the ß-6th helix may be interacted with another helix at the α-subunit (defined as the α-2nd helix) by hydrophobic network just as a "magnetic suction buckle" on the enzyme surface to stabilize the binding of α- and ß-subunits. The ß-6th helix is located at the mouth of the substrate and product tunnel, so it plays crucial roles in catalytic process. Furthermore, the ß-6th helix in NHase1229 was swapped with a thermophilic NHase fragment from Pseudonocardia thermophila JCM3095 (NHase1229-Swap). The thermal stability of NHase1229-Swap was significantly improved, and the half-life was approximately 2.4-fold at 40 °C than that of the wild NHase1229. The knowledge is useful for improving the stability of NHases by restriction fragment swapping.

Isolation an Aldehyde Dehydrogenase Gene from Metagenomics Based on Semi-nest Touch-Down PCR.

Culture-independent approaches to analyze metagenome are practical choices for rapid exploring useful genes. The mg-MSDH gene, acquired from the hot spring metagenomic, was retrieved full lengths of functional gene using semi-nest touch-down PCR. Two pairs of degenerate primers were used to separate seven conserve partial sequences by semi-nest touch-down PCR. One of them showed similarity with aldehyde dehydrogenase was used as a target fragment for isolating full-length sequence. The full-length mg-MSDH sequence contained a 1,473 bp coding sequence encoding a 490-amino-acid polypeptide and assigned an accession number JQ715422 in Genbank. The upstream sequences TAGGAG of the start codon (GTG), suggested that was a ribosome binding site. The coding sequence of mg-MSDH was ligated to pET-303 vector and the reconstructive plasmid was successfully overexpressed in E. coli. The purified recombinant mg-MSDH enzyme showed propionaldehyde oxidative activity of 3.0 U mg(-1) at 37 °C.

Targeting estrogen mediated CYP4F2/CYP4F11-20-HETE metabolic disorder decelerates tumorigenesis in ER+ breast cancer.

Purpose: As the most common subset of breast cancer (BC), estrogen receptor positive (ER+) BC accounting for 80% of cases, has become a global public health concern. The female hormone estrogen (E2) unequivocally drives ER + breast malignancies. The reasons that estrogen affects BC development has long been considered, yet further study remains to be conducted of the molecular events in the E2-estrogen receptor α (ERα) signaling pathway in ER + BC progression, especially lipid metabolism, so providing more options for tailored and individualized therapy. Our aim is to find out new targets and clinical biomarkers for ER + breast cancer treatment from the perspective of lipid metabolism. Methods: Lipid metabolomics profiling was used to examine the membrane phospholipid stimulated by E2. Clinical BC samples were used to assess the association of CYP4F2, CYP4F11 expression with clinicopathological characteristics and patient outcomes. Some inhibitors of main enzymes in AA metabolism were used combined with E2 to assess roles of CYP4F2/CYP4F11 in the progression of ER + BC. CYP4F2, CYP4F11 overexpression and knockdown BC cell lines were employed to examine the effects of CYP4F2, CYP4F11 on cellular proliferation, apoptosis and tumor growth. Western blotting, qPCR, Immunohistochemical staining and flow cytometry were also conducted to determine the underlying mechanisms related to CYP4F2, CYP4F11 function. Results: The activation of the CYP450 signaling pathway in arachidonic acid metabolism contributed to ER + BC tumorigenesis. In ER + BC, CYP4F2 and CYP4F11 overexpression induced by E2 could promote cancer cell proliferation and resistance to apoptosis by producing the metabolite 20-HETE and activating the antiapoptotic protein Bcl-2. CYP4F2 and CYP4F11 elevation correlates with poorer overall survival and disease-free survival in ER + BC patients. Conclusion: CYP4F2, CYP4F11 and their metabolite 20-HETE could serve as effective prognostic markers and attractive therapeutic targets for novel anticancer drug development about ER + BC.

Addition of Co(2+) to culture medium decides the functional expression of a recombinant nitrile hydratase in Escherichia coli.

A nitrile hydratase (NHase) gene from Aurantimonas manganoxydans, cloned and expressed in Escherichia coli, gave an enzyme that efficiently hydrated 3-cyanopyridine to nicotinamide with high thermal stability. We have now found that adding Co(2+) at 0.1 mM to LB medium was essential for production of an active enzyme. However, ≥0.3 mM Co(2+) inhibited the growth of host cells in LB medium and decreased the production of the recombinant NHase. Furthermore, ß-mercaptoethanol promoted regeneration of the Co(2+)-defective apoenzyme in vitro possibly by breaking a key disulfide bond thereby promoting the incorporation of Co(2+) into the apoenzyme.

Discovery of a novel esterase subfamily sharing an identified arm sequence (ArmEst) by gene-specific metagenomic PCR.

A gene-specific, metagenomic PCR method has led to the discovery of a novel esterase subfamily consisting of five homologous members. Sequence analysis of this esterase subfamily, named the ArmEst subfamily, revealed a unique conserved pattern with a significant variable interior sequence flanked by two symmetric and identical long arm sequences. The two homologous long arm sequences had 100 % sequence identity and symmetry at both ends between the five members of this esterase class, but only 17-58 % identity was shared for the internal sequence. The biochemical properties of two of the ArmEst esterases definitively demonstrated that they are true active esterases rather than pseudogenes. This is the first report presenting an esterase subfamily containing a unique arm sequence, indicating a rare homologous recombination occurring in the coding area of a functional gene to generate their functional diversity.

Versatile Strategy for the Construction of a Transcription Factor-Based Orthogonal Gene Expression Toolbox in Monascus spp.

Gene expression is needed to be conducted in an orthogonal manner and controllable independently from the host's native regulatory system. However, there is a shortage of gene expression regulatory toolboxes that function orthogonally from each other and toward the host. Herein, we developed a strategy based on the mutant library to generate orthogonal gene expression toolboxes. A transcription factor, MaR, located in the Monascus azaphilone biosynthetic gene cluster, was taken as a typical example. Nine DNA-binding residues of MaR were identified by molecular simulation and site-directed mutagenesis. We created five MaR multi-site saturation mutagenesis libraries consisting of 10743 MaR variants on the basis of five cognate promoters. A functional analysis revealed that all five tested promoters were orthogonally regulated by five different MaR variants, respectively. Furthermore, fine gene expression tunability and high signal sensitivity of this toolbox are demonstrated by introducing chemically inducible expression modules, designing synthetic promoter elements, and creating protein-protein interaction between MaRs. This study paves the way for a bottom-up approach to build orthogonal gene expression toolboxes.

The antitumor effect of the novel agent MCL/ACT001 in pancreatic ductal adenocarcinoma.

PURPOSE: Pancreatic ductal adenocarcinoma (PDAC) is a major challenge in cancer therapy, there are more than four hundred thousand deaths per year, and the 5-year survival rate is less than 10%. The incidence continues to rise. Treatment with classic drugs offers limited therapeutic benefits. The aim of this study was to investigate the mechanism and effect of the new agent ACT001, the active metabolite of Micheliolide (MCL), in vitro and in vivo against PDAC. METHODS: MTT assay, wound healing assay, and flow cytometry were used to assess the effects of MCL/ACT001 in vitro. DCFH-DA assay was used to assess ROS accumulation. Western blotting, immunohistochemical staining and TUNEL assay were also conducted to determine the mechanisms. PANC-1-Luc cells and bioluminescent reporter imaging were used to assess antitumor effect of ACT001 using a orthotopic xenograft model in vivo. RESULTS: MCL/ACT001 significantly inhibited cell growth in PDAC in a dose-dependent manner, induced cell apoptosis, cell migration and reactive oxygen species (ROS) accumulation in vitro. In vivo, ACT001 (400 mg/kg/day) inhibited PDAC tumor growth in orthotopic xenograft mice. We verified that EGFR and Akt were markedly overexpressed in PDAC cells and patient tumors. Mechanistic investigations revealed that MCL exerted its antitumor activity via regulation of the EGFR-Akt-Bim signaling pathway, thus inducing Bim expression both in vitro and in vivo. CONCLUSION: MCL/ACT001 is a highly promising agent in the treatment of PDAC patients.

Modular and Flexible Molecular Device for Simultaneous Cytosine and Adenine Base Editing at Random Genomic Loci in Filamentous Fungi.

Random base editing is regarded as a fundamental method for accelerating the genomic evolution in both scientific research and industrial applications. In this study, we designed a modular interaction-based dual base editor (MIDBE) that assembled a DNA helicase and various base editors through dockerin/cohesin-mediated protein-protein interactions, resulting in a self-assembled MIDBE complex capable of editing bases at any locus in the genome. The base editing type of MIDBE can be readily controlled by the induction of cytidine or/and adenine deaminase gene expression. MIDBE exhibited the highest editing efficiency 2.3 × 103 times greater than the native genomic mutation rate. To evaluate the potential of MIDBE in genomic evolution, we developed a removable plasmid-based MIDBE tool, which led to a remarkable 977.1% increase of lovastatin production in Monascus purpureus HJ11. MIDBE represents the first biological tool for generating and accumulating base mutations in Monascus chromosome and also offers a bottom-up strategy for designing the base editor.

Glutaredoxin-1 promotes lymphangioleiomyomatosis progression through inhibiting Bim-mediated apoptosis via COX2/PGE2/ERK pathway.

BACKGROUND: Lymphangioleiomyomatosis (LAM) is a female-predominant interstitial lung disease, characterized by progressive cyst formation and respiratory failure. Clinical treatment with the mTORC1 inhibitor rapamycin could relieve partially the respiratory symptoms, but not curative. It is urgent to illustrate the fundamental mechanisms of TSC2 deficiency to the development of LAM, especially mTORC1-independent mechanisms. Glutaredoxin-1 (Glrx), an essential glutathione (GSH)-dependent thiol-oxidoreductase, maintains redox homeostasis and participates in various processes via controlling protein GSH adducts. Redox signalling through protein GSH adducts in LAM remains largely elusive. Here, we demonstrate the underlying mechanism of Glrx in the pathogenesis of LAM. METHODS: 1. Abnormal Glrx expression in various kinds of human malignancies was identified by the GEPIA tumour database, and the expression of Glrx in LAM-derived cells was detected by real-time quantitative reverse transcription (RT-qPCR) and immunoblot. 2. Stable Glrx knockdown cell line was established to evaluate cellular impact. 3. Cell viability was determined by CCK8 assay. 4. Apoptotic cell number and intracellular reactive oxygen species (ROS) level were quantified by flow cytometry. 5. Cox2 expression and PGE2 production were detected to clarify the mechanism of Bim expression modulated by Glrx. 6. S-glutathionylated p65 was enriched and detected by immunoprecipitation and the direct regulation of Glrx on p65 was determined. 7. The xenograft animal model was established and photon flux was analyzed using IVIS Spectrum. RESULTS: In LAM, TSC2 negatively regulated abnormal Glrx expression and activation in a mTORC1-independent manner. Knockdown of Glrx increased the expression of Bim and the accumulation of ROS, together with elevated S-glutathionylated proteins, contributing to the induction of apoptotic cell death and inhibited cell proliferation. Knockdown of Glrx in TSC2-deficient LAM cells increased GSH adducts on nuclear factor-kappa B p65, which contributed to a decrease in the expression of Cox2 and the biosynthesis of PGE2. Inhibition of PGE2 metabolism attenuated phosphorylation of ERK, which led to the accumulation of Bim, due to the imbalance of its phosphorylation and proteasome degradation. In xenograft tumour models, knockdown of Glrx in TSC2-deficient LAM cells inhibited tumour growth and increased tumour cell apoptosis. CONCLUSIONS: Collectively, we provide a novel redox-dependent mechanism in the pathogenesis of LAM and propose that Glrx may be a beneficial strategy for the treatment of LAM or other TSC-related diseases.

Inhibition of Polo-like kinase 1 (PLK1) triggers cell apoptosis via ROS-caused mitochondrial dysfunction in colorectal carcinoma.

BACKGROUND: Colorectal cancer (CRC) is one of the most frequently diagnosed cancers. Polo-like kinase 1 (PLK1), a member of the serine/threonine kinase PLK family, is the most investigated and essential in the regulation of cell cycle progression, including chromosome segregation, centrosome maturation and cytokinesis. However, the nonmitotic role of PLK1 in CRC is poorly understood. In this study, we explored the tumorigenic effects of PLK1 and its potential as a therapeutic target in CRC. METHODS: GEPIA database and immunohistochemistry analysis were performed to evaluate the abnormal expression of PLK1 in CRC patients. MTT assay, colony formation and transwell assay were performed to assess cell viability, colony formation ability and migration ability after inhibiting PLK1 by RNAi or the small molecule inhibitor BI6727. Cell apoptosis, mitochondrial membrane potential (MMP) and ROS levels were evaluated by flow cytometry. Bioluminescence imaging was performed to evaluate the impact of PLK1 on CRC cell survival in a preclinical model. Finally, xenograft tumor model was established to study the effect of PLK1 inhibition on tumor growth. RESULTS: First, immunohistochemistry analysis revealed the significant accumulation of PLK1 in patient-derived CRC tissues compared with adjacent healthy tissues. Furthermore, PLK1 inhibition genetically or pharmacologically significantly reduced cell viability, migration and colony formation, and triggered apoptosis of CRC cells. Additionally, we found that PLK1 inhibition elevated cellular reactive oxygen species (ROS) accumulation and decreased the Bcl2/Bax ratio, which led to mitochondrial dysfunction and the release of Cytochrome c, a key process in initiating cell apoptosis. CONCLUSION: These data provide new insights into the pathogenesis of CRC and support the potential value of PLK1 as an appealing target for CRC treatment. Overall, the underlying mechanism of inhibiting PLK1-induced apoptosis indicates that the PLK1 inhibitor BI6727 may be a novel potential therapeutic strategy in the treatment of CRC.

Targeting glucose-6-phosphate dehydrogenase by 6-AN induces ROS-mediated autophagic cell death in breast cancer.

Dysregulation of G6PD involved in the pentose phosphate pathway (PPP) is known to promote tumorigenesis. The PPP plays a pivotal role in meeting the anabolic demands of cancer cells. However, the detailed underlying molecular mechanisms of targeting the G6PD-regulated PPP in breast cancer remain unclear. In this study, we aimed to elucidate the molecular pathways mediating the effects of G6PD on cancer progression. Clinical sample analysis found that the expression of G6PD in breast cancer patients was higher than that in normal controls, and patients with higher G6PD expression had poor survival. Gene knockdown or inhibition of G6PD by 6-AN in MCF-7 and MDA-MB-231 cells significantly decreased cell viability, migration, and colony formation ability. G6PD enzyme activity was inhibited by 6-AN treatment, which caused a transient upregulation of ROS. The elevated ROS was independent of cell apoptosis and thus associated with abnormal activated autophagy. Accumulated ROS levels induced autophagic cell death in breast cancer. Inhibition of G6PD suppresses tumour growth in preclinical models of breast cancer. Our results indicate that targeting the G6PD-regulated PPP could restrain tumours in vitro and in vivo, inhibiting G6PD caused cell death by over-activating autophagy, therefore leading to inhibited proliferation and tumour formation.

Retrieval of glycoside hydrolase family 9 cellulase genes from environmental DNA by metagenomic gene specific multi-primer PCR.

A new method, termed metagenomic gene specific multi-primer PCR (MGSM-PCR), is presented that uses multiple gene specific primers derived from an isolated gene from a constructed metagenomic library rather than degenerate primers designed based on a known enzyme family. The utility of MGSM-PCR was shown by applying it to search for homologues of the glycoside hydrolase family 9 cellulase in metagenomic DNA. The success of the multiplex PCR was verified by visualizing products on an agarose gel following gel electrophoresis. A total of 127 homologous genes were amplified with combinatorial multi-primer reactions from 34 soil DNA samples. Multiple alignments revealed extensive sequence diversity among these captured sequences with sequence identity varying from 26 to 99.7%. These results indicated that significantly diverse homologous genes were indeed readily accessible when using multiple metagenomic gene specific primers.

Programing a cyanide-free transformation of aldehydes to nitriles and one-pot synthesis of amides through tandem chemo-enzymatic cascades.

Nitriles are broadly applied to synthesize pharmaceuticals, agrochemicals, and materials because of their versatile transformation. Although various methods have been developed for introducing a nitrile group into organic molecules, most of them entail the use of highly toxic chemicals, transition metals, or harsh conditions. In this work, we reported a greener chemo-enzymatic cascade to synthesize alky and aryl nitriles from readily accessible aldehydes, that were further transformed into corresponding amides via an artificial enzyme cascade. A biphasic reaction system was designed to bridge chemical synthesis and enzymatic catalysis through simple phase separation. The biphasic system mainly perfectly avoided the inactivation of hydroxylamine on aldoxime dehydratase from Pseudomonas putida (OxdF1) and nitrile hydratase from Aurantimonas manganoxydans ATCC BAA-1229 (NHase1229). For the synthesis of various nitriles, moderate isolation yields of approximately 60% were obtained by the chemo-enzymatic cascade. Interestingly, two seemingly conflicting reactions of dehydration and hydration were sequentially proceeded to synthesize amides by the synergistic catalysis of OxdF1 and NHase1229 in E. coli cells. An isolation yield of approximately 62% was achieved for benzamide at the one-liter scale. In addition, the shuttle transport of substrates and products between two phases is convenient for the product separation and n-hexane recycling. Thus, the chemo-enzymatic cascade shows a potential application in the cyanide-free and large-scale synthesis of nitriles and amides.

Controllably crosslinked dual enzymes enabled by genetic-encoded non-standard amino acid for efficiently enantioselective hydrogenation.

In traditional method for preparing crosslinked enzymes aggregates using glutaraldehyde, random linkage is inevitable, which often destroys the enzyme active sites and severely decreases the activity. To address this issue, using genetic encode expanding, nonstandard amino acids (NSAAs) were inserted into enzyme proteins at the preselected sites for crosslinking. When aldehyde ketone reductase (AKR), alcohol dehydrogenase (ADH) and glucose dehydrogenase (GDH) were utilized as model enzymes, their mutants containing p-azido-L-phenylalanine were bio-orthogonally crosslinked with diyne to form crosslinked dual enzymes (CLDEs) acting as a cascade biological oxidation and reduction system. Then, the resultant self-purified CLDEs were characterized using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), scanning electron microscopy (SEM), and confocal laser scanning microscopy (CLSM), etc. In the asymmetric synthesis of (S)-1-(2,6-dichloro-3-fluorophenyl) ethanol using CLDEs, high product yield (76.08%), ee value (99.99%) and reuse stability were achieved. The yield and ee value were 12.05 times and 1.39 times higher than those using traditional crosslinked enzyme aggregates, respectively. Thus, controllable insertion NSAAs in number and location can engender reasonable linkage and metal-free self-purification for target enzyme proteins. This facile and sustainable method could be further expanded to other dual and multienzyme systems for cascade biocatalysis.