Identification and structure-based engineering of a dipeptidase CpPepD from Clostridium perfringens for the synthesis of l-carnosine.

l-Carnosine (l-Car), an endogenous dipeptide presents in muscle and brain tissues of various vertebrates, has a wide range of application values. The enzymatic preparation of l-Car is a promising synthetic method because it avoids the protection and deprotection steps. In the present study, a dipeptidase gene (CpPepD) from Clostridium perfringens with high l-Car synthetic activity was cloned and characterized. In an effort to improve the performance of this enzyme, we carried out site saturation mutagenesis using CpPepD as the template. By the o-phthalaldehyde (OPA)-derived high throughput screening method, mutant A171S was obtained with 2.2-fold enhanced synthetic activity. The enzymatic properties of CpPepD and mutant A171S were investigated. Under the optimized conditions, 63.94 mM (14.46 g L-1) or 67.02 mM (15.16 g L-1) l-Car was produced at the substrate concentrations of 6 M ß-Ala and 0.2 M l-His using wild-type or mutant A171S enzyme, respectively. Although the mutation enhanced the enzyme activity, the reaction equilibrium was barely affected.

Engineered Imine Reductase for Asymmetric Synthesis of Dextromethorphan Key Intermediate.

(S)-1-(4-Methoxybenzyl)-1,2,3,4,5,6,7,8-octahydroisoquinoline ((S)-1-(4-methoxybenzyl)-OHIQ) is the key intermediate of the nonopioid antitussive dextromethorphan. In this study, (S)-IR61-V69Y/P123A/W179G/F182I/L212V (M4) was identified with a 766-fold improvement in catalytic efficiency compared with wide-type IR61 through enzyme engineering. M4 could completely convert 200 mM of 1-(4-methoxybenzyl)-3,4,5,6,7,8-hexahydroisoquinoline into (S)-1-(4-methoxybenzyl)-OHIQ in 77% isolated yield, with >99% enantiomeric excess and a high space-time yield of 542 g L-1 day-1, demonstrating a great potential for the synthesis of dextromethorphan intermediate in industrial applications.

Chemo-Enzymatic Strategy for the Efficient Synthesis of Steroidal Drugs with 10α-Methyl Group and a Side Chain at C17-Position from Biorenewable Phytosterols.

Steroidal pharmaceuticals with a 10α-methyl group or without the methyl group at C10-position are important medicines, but their synthesis is quite challenging, due to that the natural steroidal starting materials usually have a 10ß-methyl group which is difficult to be inverted to 10α-methyl group. In this study, 3-((1R,3aS,4S,7aR)-1-((S)-1-hydroxypropan-2-yl)-7a-methyl-5-oxooctahydro-1H-inden-4-yl) propanoic acid (HIP-IPA, 2e) was demonstrated as a valuable intermediate for the synthesis of this kind of active pharmaceutical ingredients (APIs) with a side chain at C17-position. Knockout of a ß-hydroxyacyl-CoA dehydrogenase gene and introduction of a sterol aldolase gene into the genetically modified strains of Mycobacterium fortuitum (ATCC 6841) resulted in strains N13Δhsd4AΩthl and N33Δhsd4AΩthl, respectively. Both strains transformed phytosterols into 2e. Compound 2e was produced in 62% isolated yield (25 g) using strain N13Δhsd4AΩthl, and further converted to (3S,3aS,9aS,9bS)-3-acetyl-3a,6-dimethyl-1,2,3,3a,4,5,8,9,9a,9b-decahydro-7H-cyclopenta[a]naphthalen-7-one, which is the key intermediate for the synthesis of dydrogesterone. This study not only overcomes a challenging synthetic problem by enabling an efficient synthesis of dydrogesterone-like steroidal APIs from phytosterols, the well-recognized cheap and readily available biobased raw materials, but also provides insights for redesigning the metabolic pathway of phytosterols to produce other new compounds of relevance to the steroidal pharmaceutical industry.

Reshaping the Substrate Binding Pocket of ß-Amino Acid Dehydrogenase for the Synthesis of Aromatic ß-Amino Acids.

By reshaping the substrate-binding pocket of ß-amino acid dehydrogenase (ß-AADH), some variants were obtained with up to 2560-fold enhanced activity toward the model substrates (S)-ß-homophenylalanine and (R)-ß-phenylalanine. A few aromatic ß-amino acids were prepared with >99% ee and high isolated yields via either kinetic resolution of racemates or reductive amination of the corresponding ß-keto acids. This work expands the catalytic capability of ß-AADHs and highlights their practical application in the synthesis of pharmaceutically relevant ß-amino acids.

Photoredox/Enzymatic Catalysis Enabling Redox-Neutral Decarboxylative Asymmetric C-C Coupling for Asymmetric Synthesis of Chiral 1,2-Amino Alcohols.

Photocatalysis offers tremendous opportunities for enzymes to access new functions. Herein, we described a redox-neutral photocatalysis/enzymatic catalysis system for the asymmetric synthesis of chiral 1,2-amino alcohols via decarboxylative radical C-C coupling of N-arylglycines and aldehydes by combining an organic photocatalyst, eosin Y, and carbonyl reductase RasADH. Notably, this protocol avoids using any sacrificial reductants. A possible reaction mechanism proposed is that the transformation proceeds through sequential photoinduced decarboxylative radical addition to an aldehyde and a photoenzymatic deracemization pathway. This redox-neutral photoredox/enzymatic strategy is promising not only for effective synthesis of a series of chiral amino alcohols in a green and sustainable manner but also for the design of other novel C-C radical coupling transformations for the synthesis of bioactive molecules.

Semirational Engineering of a Thermostable Carbonyl Reductase for the Precision Synthesis of (2R,3R)-2-Methyl-2-benzyl-3-hydroxycyclopentanone and Its Analogues.

2,2-Disubstituted-3-hydroxycyclopentanones are important chiral intermediates for natural products and pharmaceuticals. Through semirational engineering of a thermostable carbonyl reductase CBCR from Cupriavidus sp. BIS7, a mutant L91C/F93I was obtained. Mutant L91C/F93I showed 4- to 36-fold enhanced activities toward 2-methyl-2-benzyl-1,3-cyclopentanedione and its analogues, affording the (2R,3R)-stereoisomers with >99% ee and >99% de. Enzyme-substrate docking studies were performed to reveal the molecular basis for the activity and stereoselectivity improvements.

Engineering of the DNA replication and repair machinery to develop binary mutators for rapid genome evolution of Corynebacterium glutamicum.

Corynebacterium glutamicum is an important industrial workhorse for production of amino acids and chemicals. Although recently developed genome editing technologies have advanced the rational genetic engineering of C. glutamicum, continuous genome evolution based on genetic mutators is still unavailable. To address this issue, the DNA replication and repair machinery of C. glutamicum was targeted in this study. DnaQ, the homolog of ϵ subunit of DNA polymerase III responsible for proofreading in Escherichia coli, was proven irrelevant to DNA replication fidelity in C. glutamicum. However, the histidinol phosphatase (PHP) domain of DnaE1, the α subunit of DNA polymerase III, was characterized as the key proofreading element and certain variants with PHP mutations allowed elevated spontaneous mutagenesis. Repression of the NucS-mediated post-replicative mismatch repair pathway or overexpression of newly screened NucS variants also impaired the DNA replication fidelity. Simultaneous interference with the DNA replication and repair machinery generated a binary genetic mutator capable of increasing the mutation rate by up to 2352-fold. The mutators facilitated rapid evolutionary engineering of C. glutamicum to acquire stress tolerance and protein overproduction phenotypes. This study provides efficient tools for evolutionary engineering of C. glutamicum and could inspire the development of mutagenesis strategy for other microbial hosts.

Directed evolution of a neutrophilic and mesophilic methanol dehydrogenase based on high-throughput and accurate measurement of formaldehyde.

Methanol is a promising one-carbon feedstock for biomanufacturing, which can be sustainably produced from carbon dioxide and natural gas. However, the efficiency of methanol bioconversion is limited by the poor catalytic properties of nicotinamide adenine dinucleotide (NAD+)-dependent methanol dehydrogenase (Mdh) that oxidizes methanol to formaldehyde. Herein, the neutrophilic and mesophilic NAD+-dependent Mdh from Bacillus stearothermophilus DSM 2334 (MdhBs) was subjected to directed evolution for enhancing the catalytic activity. The combination of formaldehyde biosensor and Nash assay allowed high-throughput and accurate measurement of formaldehyde and facilitated efficient selection of desired variants. MdhBs variants with up to 6.5-fold higher Kcat/KM value for methanol were screened from random mutation libraries. The T153 residue that is spatially proximal to the substrate binding pocket has significant influence on enzyme activity. The beneficial T153P mutation changes the interaction network of this residue and breaks the α-helix important for substrate binding into two short α-helices. Reconstructing the interaction network of T153 with surrounding residues may represent a promising strategy to further improve MdhBs, and this study provides an efficient strategy for directed evolution of Mdh.

Correlation of systemic immune-inflammatory response index with clinical data in patients with malignant ovarian tumor.

OBJECTIVE: To determine the correlation of the systemic immune-inflammatory response index (SIRI) with clinical data in patients with malignant ovarian tumor. METHODS: The clinical data of 118 patients with ovarian cancer (OC) treated in Ningbo Women's and Children's Hospital from February 2016 to January 2018 were retrospectively studied. Patients were divided into high and low SIRI expression groups according to the receiver operator curve (ROC) optimal cut-off (Cut-off) value, and the association of SIRI with the patient's clinical data was analyzed. Cox regression was adopted for the analysis of prognostic factors impacting the patients' 5-year survival. The associations of SIRI with tumor markers were also analyzed. A risk prediction model was constructed based on the Cox regression coefficient. RESULTS: The group of patients who died showed notably higher neutrophil (NEUT) and SIRI levels than the surviving group, and also showed a significantly lower lymphocyte (LYM) level than the surviving group (P < 0.001). The areas under the ROC curves (AUC) for CA125, NEUT, LYM, and SIRI for predicting death from OC were 0.779, 0.754, 0.776, and 0.848, respectively. In addition, the AUC of each index was ranked CA125 > SIRI > LYM > NEUT. The high-expression group had a higher proportion of patients with stage III-IV and lymph node metastasis (LNM) than the low-expression group (P < 0.05). SIRI showed a positive correlation with serum carbohydrate antigen 125 (CA125), CA153, and HE4 (all P < 0.05), but had no correlation with CA199, AFP, or CEA (all P > 0.05). According to multivariate Cox regression analysis, age, FIGO stage, SIRI, and therapeutic regimen were independent prognostic factors for the 5-year survival of OC patients (all P < 0.05). The risk score was significantly higher in the death group than that in the surviving group (P < 0.001), and the AUC of this risk score for predicting 5-year survival was 0.876. CONCLUSION: Patients with an increased SIRI level account for a large proportion of OC patients with a high FIGO stage and LNM. The 5-year survival rate of patients with a high SIRI level is unfavorable, suggesting the use of SIRI as an observation index for the prognosis of OC.

Enantiodivergent Synthesis of 1-Heteroaryl Tetrahydroisoquinolines Catalyzed by Imine Reductases.

Two enantiocomplementary imine reductases (IREDs) with high enantioselectivity were identified with catalytic activity toward the reduction of 1-heteroaryl dihydroisoquinolines through a screening of wild-type IREDs and enzyme engineering. Furthermore, (R)-IR141-L172M/Y267F and (S)-IR40 were applied to access a series of different 1-heteroaryl tetrahydroisoquinolines with high to excellent ee values (82 to >99%) and isolated yields (80 to 94%), thereby providing an effective method to construct this class of pharmaceutically important alkaloids, such as the intermediate of kinase inhibitor TAK-981.

Systems metabolic engineering of Escherichia coli for hyper-production of 5­aminolevulinic acid.

BACKGROUND: 5-Aminolevulinic acid (5-ALA) is a promising biostimulant, feed nutrient, and photodynamic drug with wide applications in modern agriculture and therapy. Although microbial production of 5-ALA has been improved realized by using metabolic engineering strategies during the past few years, there is still a gap between the present production level and the requirement of industrialization. RESULTS: In this study, pathway, protein, and cellular engineering strategies were systematically employed to construct an industrially competitive 5-ALA producing Escherichia coli. Pathways involved in precursor supply and product degradation were regulated by gene overexpression and synthetic sRNA-based repression to channel metabolic flux to 5-ALA biosynthesis. 5-ALA synthase was rationally engineered to release the inhibition of heme and improve the catalytic activity. 5-ALA transport and antioxidant defense systems were targeted to enhance cellular tolerance to intra- and extra-cellular 5-ALA. The final engineered strain produced 30.7 g/L of 5-ALA in bioreactors with a productivity of 1.02 g/L/h and a yield of 0.532 mol/mol glucose, represent a new record of 5-ALA bioproduction. CONCLUSIONS: An industrially competitive 5-ALA producing E. coli strain was constructed with the metabolic engineering strategies at multiple layers (protein, pathway, and cellular engineering), and the strategies here can be useful for developing industrial-strength strains for biomanufacturing.

Photoelectrochemical Immunosensor Based on a 1D Fe2O3/3D Cd-ZnIn2.2Sy Heterostructure as a Sensing Platform for Ultrasensitive Detection of Neuron-Specific Enolase.

Lung cancer is a high-mortality cancer related to the concentration of neuron-specific enolase (NSE). In this work, a sandwich-type photoelectrochemical (PEC) immunosensor was constructed for ultrasensitive detection of NSE, which is based on iron trioxide/indium zinc cadmium sulfide (Fe2O3/Cd-ZnIn2.2Sy) as a sensing platform and Ag-modified polyaniline (Ag@PANI) as a signal amplification label. The 1D Fe2O3 porous nanorods with a large specific surface area were synthesized by calcination of Fe-MIL-88A and etching of NaOH. To improve the photocurrent response, the 3D architecture Cd-ZnIn2.2Sy was combined with the 1D Fe2O3 porous nanorods to form a 1D Fe2O3/3D Cd-ZnIn2.2Sy heterostructure. Specifically, the Fe2O3/Cd-ZnIn2.2Sy heterostructure with a good energy level matching (the two can form a stepped energy level matching, which accelerates the transfer rate of electrons) can improve the separation efficiency of electron-hole pairs (e-/h+) under visible light irradiation, which enhances the photocurrent response. Ag@PANI has a strong electron transport capability and can be used as a secondary antibody marker for the signal amplification of the immunosensor. The sensor exhibits a good linear detection range of 100 fg/mL to 100 ng/mL with a low detection limit of 33.5 fg/mL. Moreover, the constructed sandwich-type PEC immunosensor shows good performance and possesses excellent specificity, selectivity, and stability over a period of 4 weeks for NSE detection. With these excellent properties, the immunosensor can be extended to analyze and diagnose other disease biomarkers.

Tunable 0D/2D/2D Nanocomposite Based on Green Zn-Doped CuInS2 Quantum Dots and MoS2/rGO as Photoelectrodes for Solar Hydrogen Production.

Charge separation, transmission, and light absorption properties are critical to determining the performance of photoelectrochemical (PEC) devices. An important strategy to control such properties is based on using heterostructured materials. Herein, a tunable zero-dimensional (0D)/two-dimensional (2D) heterostructure is designed based on quantum dots (QDs) and 2D nanosheets (NSs). Specifically, eco-friendly Zn-doped CuInS2 QDs prepared by hot injection were anchored on hierarchical (2D/2D) MoS2/rGO (MG) NSs through a facile sonication-assisted method to develop a 0D/2D/2D heterojunction-based photoelectrode for solar hydrogen production. The interfacial structure and band alignment between the proposed 0D QDs and 2D/2D MG NSs were engineered by modulating the Zn molar ratio during the QD synthesis. As proof of concept, the optimized 0D/2D/2D photoanode exhibits almost five times higher PEC activity than MG/CuInS2 and MoS2/Zn-CuInS2 NSs due to the enhanced light absorption, efficient charge separation, and transmission. Zn doping and the presence of graphene are essential in enhancing performance in the proposed heterostructure, reducing recombination of charge carriers, and improving sunlight absorption. This work shows how optimal band alignment control and carbon addition can facilitate charge transfer, enabling the development of highly efficient PEC devices based on 0D/2D/2D heterostructure nanocomposites.

Engineering of the Translesion DNA Synthesis Pathway Enables Controllable C-to-G and C-to-A Base Editing in Corynebacterium glutamicum.

Expanding the base conversion type is expected to largely broaden the application of base editing, whereas it requires decipherment of the machinery controlling the editing outcome. Here, we discovered that the DNA polymerase V-mediated translesion DNA synthesis (TLS) pathway controlled the C-to-A editing by a glycosylase base editor (GBE) in Escherichia coli. However, C-to-G conversion was surprisingly found to be the main product of the GBE in Corynebacterium glutamicum and subsequent gene inactivation identified the decisive TLS enzymes. Introduction of the E. coli TLS pathway into a TLS-deficient C. glutamicum mutant completely changed the GBE outcome from C-to-G to C-to-A. Combining the canonical C-to-T editor, a pioneering C-to-N base editing toolbox was established in C. glutamicum. The expanded base conversion capability produces greater genetic diversity and promotes the application of base editing in gene inactivation and protein evolution. This study demonstrates the possibility of engineering TLS systems to develop advanced genome editing tools.

A Direct Z-Scheme AgBr/CuBi2O4 Photocathode for Ultrasensitive Detection of Ciprofloxacin and Ofloxacin by Controlling the Release of Luminol in Self-Powered Microfluidic Photoelectrochemical Aptasensors.

An innovative self-powered microfluidic photoelectrochemical (PEC) aptasensor was developed that uses photoactive AgBr/CuBi2O4 (ACO) composites as the photocathode matrix for ultrasensitive detection of ciprofloxacin (CIP) and ofloxacin (OFL). The formation of direct Z-scheme heterojunctions in ACO composites greatly aided electron/hole pair separation. Meanwhile, ZnIn2S4-decorated CdS nanorod arrays (CZIS) as the photoanode were used instead of a platinum counter electrode to provide electrons. The "signal-off" CIP detection was accomplished through the steric hindrance effect in the photoanode due to the combination of aptamer(CIP) and CIP. To increase the cathodic photocurrent intensity for OFL determination, controlled release of luminol was first used. Luminol molecules were successfully embedded in the porous structure of silicon dioxide nanospheres (PSiO2) by the electrostatic adsorption between PSiO2 and aptamer(OFL). The luminol released by specific recognition between OFL and aptamer(OFL) could not only react with •O2- but also produce chemiluminescence emission, resulting in the "signal-on" state. Because of the signal "on-off-on", the proposed aptasensor exhibited wide linear ranges for CIP (0.001-100 ng/mL) and OFL (0.0005-100 ng/mL) detection. Furthermore, the low detection limits of CIP (0.06 pg/mL) and OFL (0.022 pg/mL) could achieve the ultrasensitive analysis.

CuO Nanozymes as Multifunctional Signal Labels for Efficiently Quenching the Photocurrent of ZnO/Au/AgSbS2 Hybrids and Initiating a Strong Fluorescent Signal in a Dual-Mode Microfluidic Sensing Platform.

A novel dual-mode microfluidic sensing platform based on CuO nanozymes as a photoelectrochemical (PEC)-fluorescent (FL) multifunctional signal label was developed for ultrasensitive neuron specific enolase (NSE) detection. Herein, ZnO/Au/AgSbS2 hybrids, possessing excellent PEC properties, were first exploited as a sensing matrix to provide a stable photocurrent. The controlled synthesis of photoactive ZnO nanoflowers (NFs) was successfully conducted using a microfluidic reactor in the scale of seconds. Furthermore, the photocurrent of ZnO NFs decorated by Au and AgSbS2 nanoparticles significantly improved, owing to the local surface plasma resonance effect of Au and matching band structure between ZnO and AgSbS2. A strategy of catalytic oxidation ascorbic acid (AA) by CuO nanozymes was proposed to quench the PEC signals and initiate FL signals. CuO nanoparticles growing on conductive carbon spheres (CuO@CSs) as secondary antibodies' labels could efficiently catalyze the oxidation of AA to achieve a PEC "signal-off" state. Then, the produced dehydroascorbic acid reacting with o-phenylenediamine opportunely generated a strong FL signal. Importantly, wide linear ranges of 0.0001-150 ng/mL for the PEC technique and 0.001-150 ng/mL for the FL method with a low detection limit of 0.028 and 0.25 pg/mL, respectively, could guarantee the sensitive detection of NSE.

A New 3-Ketosteroid-Δ1-Dehydrogenase with High Activity and Broad Substrate Scope for Efficient Transformation of Hydrocortisone at High Substrate Concentration.

3-Ketosteroid-Δ1-dehydrogenases (KstDs [EC 1.3.99.4]) catalyze the Δ1-dehydrogenation of steroids and are a class of important enzymes for steroid biotransformations. In this study, nine putative kstD genes from different origins were selected and overexpressed in Escherichia coli BL21(DE3). These recombinant enzymes catalyzed the Δ1-desaturation of a variety of steroidal compounds. Among them, the KstD from Propionibacterium sp. (PrKstD) displayed the highest specific activity and broad substrate spectrum. The detailed catalytic characterization of PrKstD showed that it can convert a wide range of 3-ketosteroid compounds with diverse substituents, ranging from substituents at the C9, C10, C11 and C17 position through substrates without C4-C5 double bond, to previously inactive C6-substituted ones such as 11ß,17-dihydroxy-6α-methyl-pregn-4-ene-3,20-dione. Reaction conditions were optimized for the biotransformation of hydrocortisone in terms of pH, temperature, co-solvent and electron acceptor. By using 50 g/L wet resting E. coli cells harboring PrKstD enzyme, the conversion of hydrocortisone was about 92.5% within 6 h at the substrate concentration of 80 g/L, much higher than the previously reported results, demonstrating the application potential of this new KstD.

Asymmetric Synthesis of N-Substituted 1,2-Amino Alcohols from Simple Aldehydes and Amines by One-Pot Sequential Enzymatic Hydroxymethylation and Asymmetric Reductive Amination.

The chiral N-substituted 1,2-amino alcohol motif is found in many natural and synthetic bioactive compounds. In this study, enzymatic asymmetric reductive amination of α-hydroxymethyl ketones with enantiocomplementary imine reductases (IREDs) enabled the synthesis of chiral N-substituted 1,2-amino alcohols with excellent ee values (91-99 %) in moderate to high yields (41-84 %). Furthermore, a one-pot, two-step enzymatic process involving benzaldehyde lyase-catalyzed hydroxymethylation of aldehydes and subsequent asymmetric reductive amination was developed, offering an environmentally friendly and economical way to produce N-substituted 1,2-amino alcohols from readily available simple aldehydes and amines. This methodology was then applied to rapidly access a key synthetic intermediate of anti-malaria and cytotoxic tetrahydroquinoline alkaloids.

CRISPR-assisted rational flux-tuning and arrayed CRISPRi screening of an L-proline exporter for L-proline hyperproduction.

Development of hyperproducing strains is important for biomanufacturing of biochemicals and biofuels but requires extensive efforts to engineer cellular metabolism and discover functional components. Herein, we optimize and use the CRISPR-assisted editing and CRISPRi screening methods to convert a wild-type Corynebacterium glutamicum to a hyperproducer of L-proline, an amino acid with medicine, feed, and food applications. To facilitate L-proline production, feedback-deregulated variants of key biosynthetic enzyme γ-glutamyl kinase are screened using CRISPR-assisted single-stranded DNA recombineering. To increase the carbon flux towards L-proline biosynthesis, flux-control genes predicted by in silico analysis are fine-tuned using tailored promoter libraries. Finally, an arrayed CRISPRi library targeting all 397 transporters is constructed to discover an L-proline exporter Cgl2622. The final plasmid-, antibiotic-, and inducer-free strain produces L-proline at the level of 142.4 g/L, 2.90 g/L/h, and 0.31 g/g. The CRISPR-assisted strain development strategy can be used for engineering industrial-strength strains for efficient biomanufacturing.