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.

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.

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.

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.

Engineering a Carbonyl Reductase for Scalable Preparation of (S)-3-Cyclopentyl-3-hydroxypropanenitrile, the Key Building Block of Ruxolitinib.

(S)-3-Cyclopentyl-3-hydroxypropanenitrile is the key precursor for the synthesis of ruxolitinib. The bioreduction of 3-cyclopentyl-3-ketopropanenitrile (1 a) offers an attractive method to access this important compound. A carbonyl reductase (PhADH) from Paraburkholderia hospita catalyzed the reduction of 1 a giving the (S)-alcohol (1 b) with 85 % ee. Rational engineering of PhADH resulted in a double mutant H93C/A139L, which enhanced the enantioselectivity from 85 % to >98 %, as well as a 6.3-fold improvement in the specific activity. The bioreduction of 1 a was performed at 200 g/L (1.5 M) substrate concentration, leading to isolation of (S)-1 b in 91 % yield. Similarly, using this mutant enzyme, 3-cyclohexyl-3-ketopropanenitrile (2 a) and 3-phenyl-3-ketopropanenitrile (3 a) were reduced at high concentration affording the corresponding alcohols in >99 % ee, and 90 % and 92 % yield, respectively. The results showed that the variant H93C/A139L was a powerful biocatalyst for reduction of ß-substituted-ß-ketonitriles.

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.

Ultrasensitive Double-Channel Microfluidic Biosensor-Based Cathodic Photo-electrochemical Analysis via Signal Amplification of SOD-Au@PANI for Cardiac Troponin I Detection.

Interesting double-channel microfluidic chip integration with a sandwich-type cathodic photo-electrochemical (PEC) biosensor is utilized for ultrasensitive and efficient detection of cardiac troponin I (cTnI) based on a signal amplification strategy. The Pd nanoparticles loading on the I-doped bismuth oxybromide with oxygen vacancies (Pd/I:BiOBr-OVs) as a sensing platform can effectively enhance cathodic photocurrent response by improving the visible light absorption ability with I doping, facilitating the efficiency separation of photogenerated electron-hole pairs with OVs, and increasing the electron-transfer rate with Pd loading, where the photogenerated electron could be captured by dissolved O2 to boost generation of a superoxide anion radical (•O2-). To further enhance the PEC response, a novel superoxide dismutase loaded on gold@polyaniline (SOD-Au@PANI) as a signal amplification label is developed for incubating the detection antibody (dAb). It is particularly noteworthy that SOD can effectively catalyze dismutation of the •O2- to produce H2O2 and O2, and Au@PANI with a good reduction and catalytic property can catalyze the produced H2O2 into H2O and O2. Then, the produced O2 that has been dissolved or adsorbed can capture more photogenerated electrons, resulting in more electron-hole pairs to separate, so as to the cathodic photocurrent signal of this system which can be amplified more significantly. Therefore, a signal amplification cathodic PEC biosensor is prepared for sensitively detecting cTnI, in which a good linearity ranging from 0.1 pg/mL to 100 ng/mL with a low detection limit of 0.042 pg/mL is obtained. Furthermore, the proposed biosensor exhibits excellent sensitivity and high selectivity, which could be extended to detect other disease markers in biological analysis and early disease diagnosis.

Microfluidic Ratiometric Photoelectrochemical Biosensor Using a Magnetic Field on a Photochromic Composite Platform: A Proof-of-Concept Study for Magnetic-Photoelectrochemical Bioanalysis.

Integrating a microfluidic sensor with a ratiometric photoelectrochemical (PEC) strategy to build a bioanalysis device for actual sample testing is often limited to large-volume space-resolution equipment and wavelength-dependent or potential-dependent paired photoactive materials. This work reports a microfluidic ratiometric magnetic-photoelectrochemical (M-PEC) biosensor on the photochromic composite platform to solve the above problems. In particular, as a proof-of-concept study, the platform Bi2WO6-x/amorphous BiOCl nanosheets/Bi2S3 (p-BWO-s) mediated by photochromic color centers and the magnetic photoactive secondary antibody marker ZnFe2O4@Ag2O are integrated on the microfluidic biosensor. By enhancement of the photochromic color centers, p-BWO-s outputs a considerable photocurrent signal. Meanwhile, the photoactivity of the secondary antibody marker can be changed with a magnetic field; thus, different photocurrent signals can be obtained to realize ratiometric detection. The quenching photocurrent signal without the magnetic field and the difference photocurrent signal under the magnetic field are quantitatively related to the target concentration, which unfolds a novel general strategy for bioanalysis. Different from traditional ratiometric PEC biosensors, this work characterizes the first ratiometric PEC biosensor based on an external magnetic field. Generally speaking, combined with different biorecognition cases, this scheme with good expansibility brings a unique new perspective.

Self-Powered Cathodic Photoelectrochemical Aptasensor Comprising a Photocathode and a Photoanode in Microfluidic Analysis Systems.

An intriguing self-powered cathodic photoelectrochemical (PEC) microfluidic aptasensor with enhanced cathodic photocurrent response is proposed for sensitive detection of prostate-specific antigen (PSA). The self-powered system is constructed by a cadmium sulfide-sensitized zinc oxide nanorod array (CdS/ZnO NA) as a photoanode with an iodide-doped bismuth oxychloride flower-array (I0.2:BiOCI0.8) as a photocathode, which can generate the electrical output under visible light irradiation with no external power supply. In addition, the p-type semiconductor I0.2:BiOCI0.8 with a special internal electric field between the iodide ion layer and the [Bi2O2]2+ layer could increase the cathodic photocurrent response by facilitating the separation of electron/hole pairs under visible light excitation. It is worth noting that dissolved oxygen as an electron acceptor can be reduced by the photogenerated electron to form a superoxide radical (•O2-) in the self-powered cathodic PEC system. The further enhanced cathodic photocurrent response can be achieved by eliminating •O2- that reacts with the luminol anion radical (L•-) to produce chemiluminescence emission, which serves as an inner excitation light source. What is more exciting is that the integration of the photoanode and the photocathode into a microfluidic chip could realize automatic sample injection and detection. On this basis, the proposed aptasensor presents excellent reproducibility and high sensitivity for detecting PSA and exhibits a good linearity range (50 fg·mL-1 to 50 ng·mL-1) with a low detection limit (25.8 fg·mL-1), which opens up a new horizon of potential for sensitively detecting other kinds of disease markers.

Long-term trends in the incidence of endometriosis in China from 1990 to 2019: a joinpoint and age-period-cohort analysis.

BACKGROUND: Trends in the incidence of endometriosis in China remain unknown. The purpose of this study was to examine the trends in the incidence of endometriosis and the effects of age, period, and cohort on them. METHODS: Trends in endometriosis incidence were estimated using joinpoint regression. Age-period-cohort analysis was used to analyze the effects of age, period, and cohort on these trends. Endometriosis incidences in China (1990-2019) were retrieved from the Global Burden of Disease Study 2019. Annual percentage change and average annual percent change (AAPC) were analyzed by joinpoint regression, and relative risks were analyzed using an age-period-cohort model. RESULTS: Age-standardized incidence rates (ASIRs) declined between 1990 and 2019 in China, with an overall AAPC of -1.2% (95% CI: -1.20, -1.10). Compared to 1990, the ASIR in 2019 decreased by almost 30%. Moreover, the joinpoint regression analysis revealed that endometriosis ASIRs showed a downward trend across all age groups. A significant age-related effect was seen for endometriosis incidence among young women aged 15-24 years, which then decreased with advancing age. Consistently, the effect of the period on endometriosis incidence showed a declining trend, and the effect of birth cohort decreased by 0.53 (42.7%) from 1938-1942 to 1998-2002. CONCLUSIONS: Endometriosis ASIRs declined from 1990 to 2019. The effects of period and birth cohort on endometriosis incidence exhibited a declining trend across all age groups. The effect of age on endometriosis incidence showed an increasing trend before the age of 24, followed by a decreasing trend with subsequent advancing age.

Crystal Structures and Catalytic Mechanism of l-erythro-3,5-Diaminohexanoate Dehydrogenase and Rational Engineering for Asymmetric Synthesis of ß-Amino Acids.

Amino acid dehydrogenases (AADHs) have shown considerable potential as biocatalysts in the asymmetric synthesis of chiral amino acids. However, compared to the widely studied α-AADHs, limited knowledge is available about ß-AADHs that enable the synthesis of ß-amino acids. Herein, we report the crystal structures of a l-erythro-3,5-diaminohexanoate dehydrogenase and its variants, the only known member of ß-AADH family. Crystal structure analysis, site-directed mutagenesis studies and quantum chemical calculations revealed the differences in the substrate binding and catalytic mechanism from α-AADHs. A number of rationally engineered variants were then obtained with improved activity (by 110-800 times) toward various aliphatic ß-amino acids without an enantioselectivity trade-off. Two ß-amino acids were prepared by using the outstanding variants with excellent enantioselectivity (>99 % ee) and high isolated yields (86-87 %). These results provide important insights into the molecular mechanism of 3,5-DAHDH, and establish a solid foundation for further design of ß-AADHs for the asymmetric synthesis of ß-amino acids.

Inverting the Enantiopreference of Nitrilase-Catalyzed Desymmetric Hydrolysis of Prochiral Dinitriles by Reshaping the Binding Pocket with a Mirror-Image Strategy.

A mirror-image strategy, that is, symmetry analysis of the substrate-binding pocket, was applied to identify two key amino acid residues W170 and V198 that possibly modulate the enantiopreference of a nitrilase from Synechocystis sp. PCC6803 towards 3-isobutyl glutaronitrile (1 a). Exchange of these two residues resulted in the enantiopreference inversion (S, 90 % ee to R, 47 % ee). By further reshaping the substrate-binding pocket via routine site-saturation and combinatorial mutagenesis, variant E8 with higher activity and stereoselectivity (99 % ee, R) was obtained. The mutant enzyme was applied in the preparation of optically pure (R)-3-isobutyl-4-cyanobutanoic acid ((R)-2 a) and showed similar stereopreference inversion towards a series of 3-substituted glutaronitriles. This study may offer a general strategy to switch the stereopreference of other nitrilases and other enzymes toward the desymmetric reactions of prochiral substrates with two identical reactive functional groups.

Etching Triangular Silver Nanoparticles by Self-generated Hydrogen Peroxide to Initiate the Response of an Electrochemiluminescence Sensing Platform.

This work outlines a versatile and high-performance electrochemiluminescence (ECL) platform that uses complex luminescent molecules [Ru(II) complex] formed by carbohydrazide (CON4H6) and tris(4,4'-dicarboxylicacid-2,2'-bipyridyl)ruthenium(II) dichloride [Ru(dcbpy)32+] as emitters to facilitate the intramolecular ECL mechanism for reducing the response distance and interference, and they were kept immobilized on a porous bismuth vanadate nanoarray (BiVO4 NA) to improve the orderliness of electron transfer. In addition, the detection was made depending on the etching of triangular silver nanoparticles (T-Ag NPs) by self-generated hydrogen peroxide (H2O2) to initiate the recovery response of the originally quenched ECL due to ECL-RET between the Ru(II) complex (donor) and T-Ag NPs (receptor). Because of the antibacterial application of dopamine, its own redox ability could produce more H2O2 for etching receptor T-Ag NPs under near-infrared (NIR) stimulation. Notably, in this system, the specific binding of antigens and antibodies with the autogenesis process of H2O2 and the ECL detection procedure are independent. Therefore, the proposed system can avert the impact of complex biological samples effectively, and the ECL efficiency of the Ru(II) complex can be readily utilized. On this basis, a biosensor is explored for the primary diagnosis of squamous cell carcinoma by detecting the biomarker named after cytokeratin fragment 19 (CYFRA21-1), from which an excellent linearity from 0.1 pg/mL to 50 ng/mL is achieved with a detection limit of 0.058 pg/mL. All of these results confirmed that this strategy can be a promising candidate for fabricating an ECL-based biosensor.

Highly Diastereoselective Synthesis of 2,2-Disubstituted Cyclopentane-1,3-diols via Stepwise Ketone Reduction Enabling Concise Chirality Construction.

A series of 2,2-disubstituted trans,cis-cyclopentane-1,3-diols were synthesized in >99% dr through enzymatic reduction of enantiopure 2,2-disubstituted 3-hydroxycyclopentane-1-ones, which were prepared by highly stereoselective enzymatic reduction of the corresponding cyclodiketones. For 2-benzyl-2-methyl-3-oxocyclopentyl acetate, acetylation of the hydroxyl group significantly affected the reduction stereoselectivity, giving trans,cis-, trans,trans-, and cis,cis-2-benzyl-2-methyl-cyclopentane-1,3-diols in stereomerically pure form. This efficient and environmentally friendly method provides a practical approach to the synthesis of these chiral building blocks in single stereoisomeric form, demonstrating the power of biocatalysis in the concise chirality construction of complex chiral molecules.

Distinct Regioselectivity of Fungal P450 Enzymes for Steroidal Hydroxylation.

In this study, we identified two P450 enzymes (CYP5150AP3 and CYP5150AN1) from Thanatephorus cucumeris NBRC 6298 by combination of transcriptome sequencing and heterologous expression in Pichia pastoris The biotransformation of 11-deoxycortisol and testosterone by Pichia pastoris whole cells coexpressing the cyp5150ap3 and por genes demonstrated that the CYP5150AP3 enzyme possessed steroidal 7ß-hydroxylase activities toward these substrates, and the regioselectivity was dependent on the structures of steroidal compounds. CYP5150AN1 catalyzed the 2ß-hydroxylation of 11-deoxycortisol. It is interesting that they display different regioselectivity of hydroxylation from that of their isoenzyme, CYP5150AP2, which possesses 19- and 11ß-hydroxylase activities.IMPORTANCE The steroidal hydroxylases CYP5150AP3 and CYP5150AN1 together with the previously characterized CYP5150AP2 belong to the CYP5150A family of P450 enzymes with high amino acid sequence identity, but they showed completely different regioselectivities toward 11-deoxycortisol, suggesting the regioselectivity diversity of steroidal hydroxylases of CYP5150 family. They are also distinct from the known bacterial and fungal steroidal hydroxylases in substrate specificity and regioselectivity. Biocatalytic hydroxylation is one of the important transformations for the functionalization of steroid nucleus rings but remains a very challenging task in organic synthesis. These hydroxylases are useful additions to the toolbox of hydroxylase enzymes for the functionalization of steroids at various positions.

Growth-coupled evolution of phosphoketolase to improve L-glutamate production by Corynebacterium glutamicum.

The introduction of the key non-oxidative glycolytic (NOG) pathway enzyme, phosphoketolases (PKTs), into heterologous hosts can improve the yield of a variety of acetyl CoA-derived products of interest. However, the low specific activity of existing PKTs compared with that of 6-phosphofructokinase (PFK), the key EMP pathway enzyme, largely limits their potential applications. To improve PKT activity, previous attempts have focused on increasing intracellular PKT concentration via the use of strong promoters. Herein, we report the establishment of a growth-coupled evolution strategy for the enrichment and selection of PKT mutants with improved specific activity in Corynebacterium glutamicum hosts with defective PFK. Five mutants from 9 Bifidobacterium adolescentis-source PKT (BA-PKT) mutant libraries were obtained. Site-directed mutagenesis analysis revealed 11 mutant sites which contributed to improved BA-PKT specific activity. Further structural analysis revealed that the mutant sites were located far away from the enzyme active site, which makes them almost unpredictable using a rational design approach. Mutant site recombination led to the construction of a novel mutant, PKTT2A/I6T/H260Y, with Vmax 29.77 ± 1.58 U/mg and Kcat/Km 0.32 ± 0.01 s-1/mM, which corresponds to 73.27 ± 3.25% and 80.16 ± 3.38% improvements, respectively, compared with the wildtype (Vmax; 17.17 ± 0.59 U/mg, Kcat/Km; 0.17 ± 0.01 s-1/mM). Expression of PKTT2A/I6T/H260 in C. glutamicum Z188 resulted in 16.67 ± 2.24% and 18.19 ± 0.53% improvement in L-glutamate titer and yield, respectively, compared with the wildtype BA-PKT. Our findings provide an efficient approach for improving the activity of PKTs. Furthermore, the novel mutants could serve as useful tools in improving the yield of L-glutamate and other acetyl CoA-associated products.

A Fungal P450 Enzyme from Thanatephorus cucumeris with Steroid Hydroxylation Capabilities.

In this study, we identified a P450 enzyme (STH10) and an oxidoreductase (POR) from Thanatephorus cucumeris NBRC 6298 by a combination of transcriptome sequencing and heterologous expression in Pichia pastoris The biotransformation of 11-deoxycortisol was performed by using Pichia pastoris whole cells coexpressing sth10 and por, and the product analysis indicated that the STH10 enzyme possessed steroidal 19- and 11ß-hydroxylase activities. This is a novel fungal P450 enzyme with 19-hydroxylase activity, which is different from the known steroidal aromatase cytochrome P450 19 (CYP19) and CYP11B families of enzymes.IMPORTANCE Hydroxylation is one of the most important reactions in steroid functionalization; in particular, C-19 hydroxylation produces a key intermediate for the synthesis of 19-nor-steroid drugs without a C-19 angular methyl group in three chemoenzymatic steps, in contrast to the current industrial process, which uses 10 chemical reactions. However, hydroxylation of the C-19 angular methyl group remains a very challenging task due to the high level of steric resistance to the C-19 methyl group between the A and B rings. The present report describes a novel fungal P450 enzyme with 19-hydroxylase activity. This opens a new venue for searching effective biocatalysts for the useful process of steroidal C-19 hydroxylation, although further studies for better understanding of the structural basis of the regioselectivity and substrate specificity of this fungal steroidal 19-hydroxylase are warranted to facilitate the engineering of this enzyme for industrial applications.

Characterization of new recombinant 3-ketosteroid-Δ1-dehydrogenases for the biotransformation of steroids.

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, we cloned 12 putative KstD-encoding (kstd) genes from both fungal and Gram-positive microorganisms and attempted to overproduce the recombinant proteins in E. coli BL21(DE3). Five successful recombinant enzymes catalyzed the Δ1-desaturation of a variety of steroidal compounds such as 4-androstene-3,17-dione (AD), 9α-hydroxy-4-androstene-3,17-dione (9-OH-AD), hydrocortisone, cortisone, and cortexolone. However, the substrate specificity and catalytic efficiency of the enzymes differ depending on their sources. The purified KstD from Mycobacterium smegmatis mc2155 (MsKstD1) displayed high catalytic efficiency toward hydrocortisone, progesterone, and 9-OH-AD, where it had the highest affinity (K m 36.9 ± 4.6 µM) toward 9-OH-AD. On the other hand, the KstD from Rhodococcus erythropolis WY 1406 (ReKstD) exhibited high catalytic efficiency toward androst-4,9(11)-diene-3,17-dione (Diene), 21-acetoxy-pregna-4,9(11),16-triene-3,20-dione (Triene), and cortexolone, where in all three cases the K m values (12.3 to 17.8 µM) were 2.5-4-fold lower than that toward hydrocortisone (46.3 µM). For both enzymes, AD was a good substrate although ReKstD had a 3-fold higher affinity than MsKstD1. Reaction conditions were optimized for the biotransformation of AD or hydrocortisone in terms of pH, temperature, and effects of hydrogen peroxide, solvent, and electron acceptor. For the biotransformation of hydrocortisone with 20 g/L wet resting E. coli cells harboring MsKstD1 enzyme, the yield of prednisolone was about 90% within 3 h at the substrate concentration of 6 g/L, demonstrating the application potential of the newly cloned KstDs.

Effect of smokeless tobacco products on human oral bacteria growth and viability.

To evaluate the toxicity of smokeless tobacco products (STPs) on oral bacteria, seven smokeless tobacco aqueous extracts (STAEs) from major brands of STPs and three tobacco-specific N-nitrosamines (TSNAs) were used in a growth and viability test against 38 oral bacterial species or subspecies. All seven STAEs showed concentration-dependent effects on the growth and viability of tested oral bacteria under anaerobic culture conditions, although there were strain-to-strain variations. In the presence of 1 mg/ml STAEs, the growth of 4 strains decreased over 0.32-2.14 log10 fold, while 14 strains demonstrated enhanced growth of 0.3-1.76 log10 fold, and the growth of 21 strains was not significantly affected. In the presence of 10 mg/ml STAEs, the growth of 17 strains was inhibited 0.3-2.11 log10 fold, 18 strains showed enhanced growth of 0.3-0.97 log10 fold, and 4 strains were not significantly affected. In the presence of 50 mg/ml STAEs, the growth of 32 strains was inhibited 0.3-2.96 log10 fold, 8 strains showed enhanced growth of 0.3-1.0 log10 fold, and 2 strains were not significantly affected. All seven STAEs could promote the growth of 4 bacterial strains, including Eubacterium nodatum, Peptostreptococcus micros, Streptococcus anginosus, and Streptococcus constellatus. Exposure to STAEs modulated the viability of some bacterial strains, with 21.1-66.5% decrease for 4 strains at 1 mg/ml, 20.3-85.7% decrease for 10 strains at 10 mg/ml, 20.0-93.3% decrease for 27 strains at 50 mg/ml, and no significant effect for 11 strains at up to 50 mg/ml. STAEs from snuffs inhibited more tested bacterial strains than those from snus indicating that the snuffs may be more toxic to the oral bacteria than snus. For TSNAs, cell growth and viability of 34 tested strains were not significantly affected at up to 100 µg/ml; while the growth of P. micros was enhanced 0.31-0.54 log10 fold; the growth of Veillonella parvula was repressed 0.33-0.36 log10 fold; and the cell viabilities of 2 strains decreased 56.6-69.9%. The results demonstrate that STAEs affected the growth of some types of oral bacteria, which may affect the healthy ecological balance of oral bacteria in humans. On the other hand, TSNAs did not significantly affect the growth of the oral bacteria.

Structural analysis reveals the substrate-binding mechanism for the expanded substrate specificity of mutant meso-diaminopimelate dehydrogenase.

A meso-diaminopimelate dehydrogenase (DAPDH) from Clostridium tetani E88 (CtDAPDH) was found to have low activity toward the D-amino acids other than its native substrate. Site-directed mutagenesis similar to that carried out on the residues mutated by Vedha-Peters et al. resulted in a mutant enzyme with highly improved catalytic ability for the synthesis of D-amino acids. The crystal structures of the CtDAPDH mutant in apo form and in complex with meso-diaminopimelate (meso-DAP), D-leucine (D-leu), and 4-methyl-2-oxopentanoic acid (MOPA) were solved. meso-DAP was found in an area outside the catalytic cavity; this suggested a possible two-step substrate-binding mechanism for meso-DAP. D-leu and MOPA each bound both to Leu154 and to Gly155 in the open form of CtDAPDH, and structural analysis revealed the molecular basis for the expanded substrate specificity of the mutant meso-diaminopimelate dehydrogenases.