Oligomeric interactions maintain active-site structure in a noncooperative enzyme family.

The evolutionary benefit accounting for widespread conservation of oligomeric structures in proteins lacking evidence of intersubunit cooperativity remains unclear. Here, crystal and cryo-EM structures, and enzymological data, demonstrate that a conserved tetramer interface maintains the active-site structure in one such class of proteins, the short-chain dehydrogenase/reductase (SDR) superfamily. Phylogenetic comparisons support a significantly longer polypeptide being required to maintain an equivalent active-site structure in the context of a single subunit. Oligomerization therefore enhances evolutionary fitness by reducing the metabolic cost of enzyme biosynthesis. The large surface area of the structure-stabilizing oligomeric interface yields a synergistic gain in fitness by increasing tolerance to activity-enhancing yet destabilizing mutations. We demonstrate that two paralogous SDR superfamily enzymes with different specificities can form mixed heterotetramers that combine their individual enzymological properties. This suggests that oligomerization can also diversify the functions generated by a given metabolic investment, enhancing the fitness advantage provided by this architectural strategy.

Association between Troponin Elevation and Decreased Myocardial Blood Flow Reserve in Patients without Obstructive Coronary Artery Disease.

INTRODUCTION: To study the prognostic factors of patients with chest pain and without obstructive coronary artery disease is of great significance for the management of such patients. We assessed whether a high-sensitivity troponin I (hs-TnI) is associated with prognosis in patients with chest pain and without obstructive coronary artery disease. METHODS: From 2011 to 2017, 489 consecutively hospitalized patients with chest pain and without significant coronary artery stenosis (<50%) were tested for hs-TnI and underwent stress myocardial contrast echocardiography (MCE). Myocardial blood flow reserve (MBFR) was measured by stress MCE. Patients were followed (median, 41 months) for composite endpoints, including cardiovascular death and non-fatal myocardial infarction. Cox proportional hazards models were performed to determine associations between hs-TnI and the composite endpoints. RESULTS: Among 489 patients with chest pain and without significant coronary artery stenosis, 257 patients (52.6%) had elevated hs-TnI. Compared to patients with normal hs-TnI, patients with elevated hs-TnI were older (p = 0.013) and had a higher prevalence of atrial fibrillation (p = 0.003), higher left ventricular mass index (p = 0.002) and E/e' septal (p < 0.001), and a lower MBFR (p < 0.001). After adjustment, there was still a significant association between hs-TnI and MBFR (odds ratio = 1.145; 95% confidence interval [CI], 1.079-1.214; p < 0.001). Compared with patients with normal hs-TnI, patients with elevated hs-TnI had a greater cumulative event rate (log-rank p = 0.002). Males (hazard ratio [HR], 4.770; 95% CI, 1.175-19.363; p = 0.029) and reduced MBFR (HR, 2.496; 95% CI, 1.446-4.311; p = 0.001) were risk factors associated with composite endpoints in patients with elevated hs-TnI. CONCLUSIONS: In patients with chest pain and without obstructive coronary artery disease, elevated hs-TnI is associated with decreased myocardial perfusion by contrast echocardiography as well as a higher incidence of cardiovascular events.

Structure-based rational design of hydroxysteroid dehydrogenases for improving and diversifying steroid synthesis.

A group of steroidogenic enzymes, hydroxysteroid dehydrogenases are involved in steroid metabolism which is very important in the cell: signaling, growth, reproduction, and energy homeostasis. The enzymes show an inherent function in the interconversion of ketosteroids and hydroxysteroids in a position- and stereospecific manner on the steroid nucleus and side-chains. However, the biocatalysis of steroids reaction is a vital and demanding, yet challenging, task to produce the desired enantiopure products with non-natural substrates or non-natural cofactors, and/or in non-physiological conditions. This has driven the use of protein design strategies to improve their inherent biosynthetic efficiency or activate their silent catalytic ability. In this review, the innate features and catalytic characteristics of enzymes based on sequence-structure-function relationships of steroidogenic enzymes are reviewed. Combining structure information and catalytic mechanisms, progress in protein redesign to stimulate potential function, for example, substrate specificity, cofactor dependence, and catalytic stability are discussed.

Metabolite profiling analysis of hepatitis B virus-induced liver cirrhosis patients with minimal hepatic encephalopathy using gas chromatography-time-of-flight mass spectrometry and ultra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry.

This study used gas chromatography-time-of-flight mass spectrometry (GC-TOFMS) and ultra-performance liquid chromatography-quadrupole TOFMS (UPLC-QTOFMS) metabonomic analytical techniques in combination with bioinformatics and pattern recognition analysis methods to analyze the serum metabolite profiling of hepatitis B virus (HBV)-induced liver cirrhosis patients with minimal hepatic encephalopathy (MHE), to find the specific biomarkers of MHE, to reveal the pathogenesis of MHE, and to determine a promising approach for early diagnosis of MHE. Serum samples of 100 normal controls (NC group), 29 HBV-induced liver cirrhosis patients with MHE (MHE group), and 24 HBV-induced liver cirrhosis patients without MHE [comprising 12 cases of compensated cirrhosis (CS group) and 12 cases of decompensated cirrhosis (DS group)] were collected and employed into GC-TOFMS and UPLC-QTOFMS platforms for serum metabolite detection; the outcome data were then analyzed using principal component analysis and orthogonal partial least squares-discriminant analysis (OPLS-DA). There were no significant differential metabolites between the NC group and the CS group. A series of key differential metabolites were detected. According to the variable influence in projection values and P-values, 60 small-molecule metabolites were considered to be dysregulated in the MHE group (compared to the NC group); 27 of these 60 dysregulated differential metabolites were considered to be the potential biomarkers (see Table 4, marked in bold); 66 small-molecule metabolites were considered to be dysregulated in the DS group (compared to the NC group); 34 of these 66 dysregulated differential metabolites were considered to be the potential biomarkers (see Table 5, marked in bold). According to the fold-change values, 9 of these 27 metabolites, namely valine, oxalic acid, erythro-sphingosine, 4,7,10,13,16,19-docosahexaenoic acid, isoleucine, allo-isoleucine, thyroxine, rac-octanoyl carnitine, and tocopherol (vitamin E), were downregulated in the MHE group (compared to the NC group); the other 18, namely adenine, glycochenodeoxycholic acid, fucose, allothreonine, glycohyocholic acid, glycoursodeoxycholic acid, tyrosine, taurocheno-deoxycholate, phenylalanine, 2-hydroxy-3-methyl-butanoic acid, hydroxyacetic acid, taurocholate, sorbitol, rhamnose, tauroursodeoxycholate, tolbutamide, pyroglutamic acid, and malic acid, were upregulated; 6 of these 34 metabolites were downregulated in the DS group (compared to the NC group), and the other 28 were upregulated, as shown in Table 5. (a) GC-TOFMS and UPLC-QTOFMS metabonomic analytical platforms can detect a range of metabolites in the serum; this might be of great help to study the pathogenesis of MHE and may provide a new approach for the early diagnosis of MHE. (b) Metabonomics analysis in combination with pattern recognition analysis might have great potential to distinguish the HBV-induced liver cirrhosis patients who have MHE from the normal healthy population and HBV-induced liver cirrhosis patients without MHE.

Use of elicitors to enhance or activate the antibiotic production in streptomyces.

Streptomyces is the largest and most significant genus of Actinobacteria, comprising 961 species. These Gram-positive bacteria produce many versatile and important bioactive compounds; of these, antibiotics, specifically the enhancement or activation of their production, have received extensive research attention. Recently, various biotic and abiotic elicitors have been reported to modify the antibiotic metabolism of Streptomyces, which promotes the production of new antibiotics and bioactive metabolites for improvement in the yields of endogenous products. However, some elicitors that obviously contribute to secondary metabolite production have not yet received sufficient attention. In this study, we have reviewed the functions and mechanisms of chemicals, novel microbial metabolic elicitors, microbial interactions, enzymes, enzyme inhibitors, environmental factors, and novel combination methods regarding antibiotic production in Streptomyces. This review has aimed to identify potentially valuable elicitors for stimulating the production of latent antibiotics or enhancing the synthesis of subsistent antibiotics in Streptomyces. Future applications and challenges in the discovery of new antibiotics and enhancement of existing antibiotic production using elicitors are discussed.

Phase 2B randomized controlled trial of NP001 in amyotrophic lateral sclerosis: Pre-specified and post hoc analyses.

INTRODUCTION/AIMS: ALS is a heterogeneous disease that may be complicated or in part driven by inflammation. NP001, a regulator of macrophage activation, was associated with slowing disease progression in those with higher levels of the plasma inflammatory marker C-reactive protein (CRP) in phase 2A studies in ALS. Here, we evaluate the effects of NP001 in a phase 2B trial, and perform a post hoc analysis with combined data from the preceding phase 2A trial. METHODS: The phase 2B trial enrolled 138 participants within 3 y of symptom onset and with plasma hs-CRP values >1.13 mg/L. They were randomized 1:1 to receive either placebo or NP001 for 6 mo. Change from baseline ALSFRS-R scores was the primary efficacy endpoint. Secondary endpoints included vital capacity (VC) change from baseline and percentage of participants showing no decline of ALSFRS-R score over 6 mo (non-progressor). RESULTS: The phase 2B study did not show significant differences between placebo and active treatment with respect to change in ALSFRS-R scores, or VC. The drug was safe and well tolerated. A post hoc analysis identified a 40- to 65-y-old subset in which NP001-treated patients demonstrated slower declines in ALSFRS-R score by 36% and VC loss by 51% compared with placebo. A greater number of non-progressors were NP001-treated compared with placebo (p = .004). DISCUSSION: Although the phase 2B trial failed to meet its primary endpoints, post hoc analyses identified a subgroup whose decline in ALSFRS-R and VC scores were significantly slower than placebo. Further studies will be required to validate these findings.

Postmortem study of organ-specific toxicity in glioblastoma patients treated with a combination of temozolomide, irinotecan and bevacizumab.

PURPOSE: Systemic chemotherapy including monotherapy with temozolomide (TMZ) or bevacizumab (BEV); two-drug combinations, such as irinotecan (IRI) and BEV, TMZ and BEV and a three-drug combination with TMZ, IRI and BEV (TIB) have been used in treating patients with progressive high-grade gliomas including glioblastoma (GBM). Most patients tolerated these regimens well with known side effects of hypertension, proteinuria, and reversible clinical myelosuppression (CM). However, organ- or system- specific toxicities from chemotherapy agents have never been examined by postmortem study. This is the largest cohort used to address this issue in glioma patients. METHODS: Postmortem tissues (from all major systems and organs) were prospectively collected and examined by standard institution autopsy and neuropathological procedures from 76 subjects, including gliomas (N = 68, 44/M, and 24/F) and brain metastases (N = 8, 5/M, and 3/F) between 2009 and 2019. Standard hematoxylin and eosin (H&E) were performed on all major organs including brain specimens. Electronic microscopic (EM) study was carried out on 14 selected subject's kidney samples per standard EM protocol. Medical records were reviewed with adverse events (AEs) analyzed and graded according to the Common Terminology Criteria for Adverse Events (CTCAE), version 4.03. A swimmer plot was utilized to visualize the timelines of patient history by treatment group. The binary logistic regression models were performed to explore any associations between treatment strategies and incident myelosuppression. RESULTS: Twenty-four glioma subjects were treated with TIB [median: 5.5 (range: 1-25) cycles] at tumor recurrence. Exposure to IRI significantly increased the frequency of CM (p = 0.05). No unexpected adverse events clinically, or permanent end-organ damage during postmortem examination was identified in glioma subjects who had received standard or prolonged duration of BEV, TMZ or TIB regimen-based chemotherapies except rare events of bone marrow suppression. The most common causes of death (COD) were tumor progression (63.2%, N = 43) followed by aspiration pneumonia (48.5%, N = 33) in glioma subjects. No COD was attributed to acute toxicity from TIB. The study also demonstrated that postmortem kidney specimen is unsuitable for studying renal ultrastructural pathological changes due to autolysis. CONCLUSION: There is no organ or system toxicity by postmortem examinations among glioma subjects who received BEV, TMZ or TIB regimen-based chemotherapies regardless of durations except for occasional bone marrow suppression and reversible myelosuppression clinically. IRI, but not the extended use of TMZ, significantly increased CM in recurrent glioma patients. COD most commonly resulted from glioma tumor progression with infiltration to brain stem and aspiration pneumonia.

Efficient D-allulose synthesis under acidic conditions by auto-inducing expression of the tandem D-allulose 3-epimerase genes in Bacillus subtilis.

BACKGROUND: D-allulose, a hexulose monosaccharide with low calorie content and high sweetness, is commonly used as a functional sugar in food and nutrition. However, enzyme preparation of D-allulose from D-frutose was severely hindered by the non-enzymatic browning under alkaline and high-temperature, and the unnecessary by-products further increased the difficulties in separation and extraction for industrial applications. Here, to address the above issue during the production process, a tandem D-allulose 3-epimerase (DPEases) isomerase synergistic expression strategy and an auto-inducible promoter engineering were levered in Bacillus subtilis 168 (Bs168) for efficient synthesis of D-allulose under the acidic conditions without browning. RESULTS: First, based on the dicistron expression system, two DPEases with complementary functional characteristics from Dorea sp. CAG:317 (DSdpe) and Clostridium cellulolyticum H10 (RCdpe) were expressed in tandem under the promoter HpaII in one cell. A better potential strain Bs168/pMA5-DSdpe-RCdpe increases enzyme activity to 18.9 U/mL at acidic conditions (pH 6.5), much higher than 17.2 and 16.7 U/mL of Bs168/pMA5-DSdpe and Bs168/pMA5-RCdpe, respectively. Subsequently, six recombinant strains based on four constitutive promoters were constructed in variable expression cassettes for improving the expression level of protein. Among those engineered strains, Bs168/pMA5-PspoVG-DSdpe-PsrfA-RCdpe exhibited the highest enzyme activity with 480.1 U/mL on fed-batch fermentation process in a 5 L fermenter at pH 6.5, about 2.1-times higher than the 228.5 U/mL of flask fermentation. Finally, the maximum yield of D-allulose reached as high as 163.5 g/L at the fructose concentration (50% w/v) by whole-cell biocatalyst. CONCLUSION: In this work, the engineered recombinant strain Bs168/pMA5-PspoVG-DSdpe-PsrfA-RCdpe was demonstrated as an effective microbial cell factory for the high-efficient synthesis of D-allulose without browning under acidic conditions. Based on the perspectives from this research, this strategy presented here also made it possible to meet the requirements of the industrial hyper-production of other rare sugars under more acidic conditions in theory.

Ile258Met mutation of Brucella melitensis 7α-hydroxysteroid dehydrogenase significantly enhances catalytic efficiency, cofactor affinity, and thermostability.

NAD(H)-dependent 7α-hydroxysteroid dehydrogenase catalyzes the oxidation of chenodeoxycholic acid to 7-oxolithocholic acid. Here, we designed mutations of Ile258 adjacent to the catalytic pocket of Brucella melitensis 7α-hydroxysteroid dehydrogenase. The I258M variant gave a 4.7-fold higher kcat, but 4.5-fold lower KM, compared with the wild type, resulting in a 21.8-fold higher kcat/KM value for chenodeoxycholic acid oxidation. It presented a 2.0-fold lower KM value with NAD+, suggesting stronger binding to the cofactor. I258M produced 7-oxolithocholic acid in the highest yield of 92.3% in 2 h, whereas the wild-type gave 88.4% in 12 h. The I258M mutation increased the half-life from 20.8 to 31.1 h at 30 °C. Molecular dynamics simulations indicated increased interactions and a modified tunnel improved the catalytic efficiency, and enhanced rigidity at three regions around the ligand-binding pocket increased the enzyme thermostability. This is the first report about significantly improved catalytic efficiency, cofactor affinity, and enzyme thermostability through single site-mutation of Brucella melitensis 7α-hydroxysteroid dehydrogenase. KEY POINTS: • Sequence and structure analysis guided the site mutation design. • Thermostability, catalytic efficiency and 7-oxo-LCA production were determined. • MD simulation was performed to indicate the improvement by I258M mutation.

Efficient production of (S)-1-phenyl-1,2-ethanediol using xylan as co-substrate by a coupled multi-enzyme Escherichia coli system.

BACKGROUND: (S)-1-phenyl-1,2-ethanediol is an important chiral intermediate in the synthesis of liquid crystals and chiral biphosphines. (S)-carbonyl reductase II from Candida parapsilosis catalyzes the conversion of 2-hydroxyacetophenone to (S)-1-phenyl-1,2-ethanediol with NADPH as a cofactor. Glucose dehydrogenase with a Ala258Phe mutation is able to catalyze the oxidation of xylose with concomitant reduction of NADP+ to NADPH, while endo-ß-1,4-xylanase 2 catalyzes the conversion of xylan to xylose. In the present work, the Ala258Phe glucose dehydrogenase mutant and endo-ß-1,4-xylanase 2 were introduced into the (S)-carbonyl reductase II-mediated chiral pathway to strengthen cofactor regeneration by using xylan as a naturally abundant co-substrate. RESULTS: We constructed several coupled multi-enzyme systems by introducing (S)-carbonyl reductase II, the A258F glucose dehydrogenase mutant and endo-ß-1,4-xylanase 2 into Escherichia coli. Different strains were produced by altering the location of the encoding genes on the plasmid. Only recombinant E. coli/pET-G-S-2 expressed all three enzymes, and this strain produced (S)-1-phenyl-1,2-ethanediol from 2-hydroxyacetophenone as a substrate and xylan as a co-substrate. The optical purity was 100% and the yield was 98.3% (6 g/L 2-HAP) under optimal conditions of 35 °C, pH 6.5 and a 2:1 substrate-co-substrate ratio. The introduction of A258F glucose dehydrogenase and endo-ß-1,4-xylanase 2 into the (S)-carbonyl reductase II-mediated chiral pathway caused a 54.6% increase in yield, and simultaneously reduced the reaction time from 48 to 28 h. CONCLUSIONS: This study demonstrates efficient chiral synthesis using a pentose as a co-substrate to enhance cofactor regeneration. This provides a new approach for enantiomeric catalysis through the inclusion of naturally abundant materials.

The prevalence, risk factors, and clinical characteristics of insulin resistance in Chinese patients with schizophrenia.

BACKGROUND: Studies have shown that patients with schizophrenia are at a high risk of developing insulin resistance (IR). We investigated the prevalence of IR and its clinical correlates in hospitalized Chinese patients with schizophrenia. METHODS: A total of 193 patients with schizophrenia (113 males and 80 females) were recruited for the study. We collected their demographic and clinical data, including data on their plasma glucose and lipid levels. All patients were rated using the Repeatable Battery for the Assessment of Neuropsychological Status (RBANS) to assess cognitive function, while Positive and Negative Syndrome Scale (PANSS) was used to assess psychopathology. The cut-off value for the homeostasis model assessment of insulin resistance (HOMA-IR) was set at 1.7. RESULTS: The prevalence of IR was 37.82% (73/193). The IR patients had significantly higher waist-to-hip ratio and body mass index (BMI), and higher fasting plasma glucose (FPG), triglyceride (TG), and low density lipoprotein (LDL) levels compared to non-IR patients (all p<.05). Binary logistic regression analysis showed that smoking, BMI, and TG and LDL levels are significant predictors of IR. In addition, correlation analysis showed that IR was significantly correlated with the waist-to-hip ratio, BMI, and LDL level (Bonferroni corrected p<.05). The multivariable linear regression analysis indicated that the BMI and FPG are associated with the IR index. There was no significant difference in IR index between patients who were taking different antipsychotics. CONCLUSION: We found a high prevalence of IR and its risk factors in Chinese patients with schizophrenia. Active weight control to reduce the BMI and waist circumference and reducing the number of cigarettes consumed, may be essential to decrease the incidence of IR in patients with schizophrenia.

Efficient chiral synthesis by Saccharomyces cerevisiae spore encapsulation of Candida parapsilosis Glu228Ser/(S)-carbonyl reductase II and Bacillus sp. YX-1 glucose dehydrogenase in organic solvents.

BACKGROUND: Saccharomyces cerevisiae AN120 osw2∆ spores were used as a host with good resistance to unfavorable environment. This work was undertaken to develop a new yeast spore-encapsulation of Candida parapsilosis Glu228Ser/(S)-carbonyl reductase II and Bacillus sp. YX-1 glucose dehydrogenase for efficient chiral synthesis in organic solvents. RESULTS: The spore microencapsulation of E228S/SCR II and GDH in S. cerevisiae AN120 osw2∆ catalyzed (R)-phenylethanol in a good yield with an excellent enantioselectivity (up to 99%) within 4 h. It presented good resistance and catalytic functions under extreme temperature and pH conditions. The encapsulation produced several chiral products with over 70% yield and over 99% enantioselectivity in ethyl acetate after being recycled for 4-6 times. It increased substrate concentration over threefold and reduced the reaction time two to threefolds compared to the recombinant Escherichia coli containing E228S and glucose dehydrogenase. CONCLUSIONS: This work first described sustainable enantioselective synthesis without exogenous cofactors in organic solvents using yeast spore-microencapsulation of coupled alcohol dehydrogenases.

Investigation of miR-490-3p Expression in Hepatocellular Carcinoma Based on Reverse Transcription-Polymerase Chain Reaction (RT-qPCR) and a Meta-Analysis of 749 Cases.

BACKGROUND miR-490-3p could play vital roles in multiple cancers. However, the role of miR-490-3p in hepatocellular carcinoma (HCC) remains uncertain. In this study, we sought to explore the underlying role of miR-490-3p in HCC. MATERIAL AND METHODS In this study, we explored the clinical role of miR-490-3p in HCC via quantitative reverse transcription-polymerase chain reaction (RT-qPCR) and The Cancer Genome Atlas (TCGA) database. Then, a meta-analysis was performed to evaluate the expression trend and diagnostic value of miR-490-3p in HCC. Furthermore, 12 miRNA prediction algorithms were applied to predict the potential target genes of miR-490-3p. The differentially expressed genes in HCC in the Gene Expression Profiling Interactive Analysis (GEPIA) database were also selected. Additionally, bioinformatics analyses were utilized to investigate the possible functions and pathways of the target genes. RESULTS miR-490-3p was clearly down-regulated in HCC based on RT-qPCR (P=0.002). Consistent with the results of RT-qPCR, miR-490 was more highly expressed in normal liver tissue than in HCC (P<0.001). Additionally, the meta-analysis confirmed the results from RT-qPCR and TCGA. Furthermore, based on the prediction algorithms and GEPIA, a total of 113 genes were selected. According to the bioinformatics analyses, we found that the most remarkably enriched functional terms included protein transport, poly(A) RNA binding, and intracellular organelle part. Additionally, the miR-490-3p target genes were significantly related to the pathways in cancer. CONCLUSIONS We found that miR-490-3p is down-regulated in HCC and is related to genes that have potential tumoral functions. However, the exact mechanism should be confirmed by functional experiments.

The high expression of Aspergillus pseudoglaucus protease in Escherichia coli for hydrolysis of soy protein and milk protein.

The hydrolysates of soy protein and milk protein are nutritional and functional food ingredients. Aspergillus pseudoglaucus aspergillopepsin I (App) is an acidic protease, including signal peptide, propeptide, and catalytic domain. Here, we cloned the catalytic domain App with or without propeptide in Escherichia coli. The results showed that the App without propeptide was not expressed or did not exhibit activity and App with propeptide (proApp) was highly expressed with a specific activity of 903 U/mg. Moreover, the denaturation temperature of proApp was 4.1 °C higher than App's. The proApp showed 104 U/mg and 252 U/mg hydrolysis activities towards soy protein and milk protein under acidic conditions. By RP-HPLC analysis, the peptides obtained from the hydrolysates of soy protein and milk protein were hydrophilic peptides. This work first demonstrates efficient proteolysis of soy protein and milk protein through the functional expression of full-length proApp, which will likely have valuable industrial applications.

Subchronic toxicity of herbal compound "Jiedu Huayu" granules in rats.

BACKGROUND: "Jiedu Huayu" (JDHY) granules are traditional Chinese herbal compounds that have been used to treat severe liver injury for many years. The purpose of the current study is to evaluate the safety of JDHY granules. METHODS: Subchronic toxicity was tested in male and female rats that were orally administered three different doses (80, 100, and 130 g/kg/d) of JDHY for 13 weeks. Clinical signs, bodyweight, food consumption, hematological and biochemical parameters, organ coefficients, and histological changes were observed during the study. RESULTS: There were no significant changes in toxicity observed in either sex at any dose of JDHY granules treatment. CONCLUSIONS: These results suggest that repeated oral administration of JDHY granules at dosage levels of ≤130 g/kg/d can be considered safe.

In situ expression of (R)-carbonyl reductase rebalancing an asymmetric pathway improves stereoconversion efficiency of racemic mixture to (S)-phenyl-1,2-ethanediol in Candida parapsilosis CCTCC M203011.

BACKGROUND: Candida parapsilosis (R)-carbonyl reductase (RCR) and (S)-carbonyl reductase (SCR) are involved in the stereoconversion of racemic (R,S)-1-phenyl-1,2-ethanediol (PED) to its (S)-isomer. RCR catalyzes (R)-PED to 2-hydroxyacetophenone (2-HAP), and SCR catalyzes 2-HAP to (S)-PED. However, the stereoconversion efficiency of racemic mixture to (S)-PED is not high because of an activity imbalance between RCR and SCR, with RCR performing at a lower rate than SCR. To realize the efficient preparation of racemic mixture to (S)-PED, an in situ expression of RCR and a two-stage control strategy were introduced to rebalance the RCR- and SCR-mediated pathways. RESULTS: An in situ expression plasmid pCP was designed and RCR was successfully expressed in C. parapsilosis. With respect to wild-type, recombinant C. parapsilosis/pCP-RCR exhibited over four-fold higher activity for catalyzing racemic (R,S)-PED to 2-HAP, while maintained the activity for catalyzing 2-HAP to (S)-PED. The ratio of k cat /K M for SCR catalyzing (R)-PED and RCR catalyzing 2-HAP was about 1.0, showing the good balance between the functions of SCR and RCR. Based on pH and temperature preferences of RCR and SCR, a two-stage control strategy was devised, where pH and temperature were initially set at 5.0 and 30 °C for RCR rapidly catalyzing racemic PED to 2-HAP, and then adjusted to 4.5 and 35 °C for SCR transforming 2-HAP to (S)-PED. Using these strategies, the recombinant C. parapsilosis/pCP-RCR catalyzed racemic PED to its (S)-isomer with an optical purity of 98.8 % and a yield of 48.4 %. Most notably, the biotransformation duration was reduced from 48 to 13 h. CONCLUSIONS: We established an in situ expression system for RCR in C. parapsilosis to rebalance the functions between RCR and SCR. Then we designed a two-stage control strategy based on pH and temperature preferences of RCR and SCR, better rebalancing RCR and SCR-mediated chiral biosynthesis pathways. This work demonstrates a method to improve chiral biosyntheses via in situ expression of rate-limiting enzyme and a multi-stage control strategy to rebalance asymmetric pathways.

Highly selective anti-Prelog synthesis of optically active aryl alcohols by recombinant Escherichia coli expressing stereospecific alcohol dehydrogenase.

Biocatalytic asymmetric synthesis has been widely used for preparation of optically active chiral alcohols as the important intermediates and precursors of active pharmaceutical ingredients. However, the available whole-cell system involving anti-Prelog specific alcohol dehydrogenase is yet limited. A recombinant Escherichia coli system expressing anti-Prelog stereospecific alcohol dehydrogenase from Candida parapsilosis was established as a whole-cell system for catalyzing asymmetric reduction of aryl ketones to anti-Prelog configured alcohols. Using 2-hydroxyacetophenone as the substrate, reaction factors including pH, cell status, and substrate concentration had obvious impacts on the outcome of whole-cell biocatalysis, and xylose was found to be an available auxiliary substrate for intracellular cofactor regeneration, by which (S)-1-phenyl-1,2-ethanediol was achieved with an optical purity of 97%e.e. and yield of 89% under the substrate concentration of 5 g/L. Additionally, the feasibility of the recombinant cells toward different aryl ketones was investigated, and most of the corresponding chiral alcohol products were obtained with an optical purity over 95%e.e. Therefore, the whole-cell system involving recombinant stereospecific alcohol dehydrogenase was constructed as an efficient biocatalyst for highly enantioselective anti-Prelog synthesis of optically active aryl alcohols and would be promising in the pharmaceutical industry.

[Efficient biosynthesis of (S)-1-phenyl-1,2-ethanediol catalyzed by (S)-carbonyl reductase â ¡ and glucose dehydrogenase].

Objective: To realize efficient biosynthesis of 2-hydroxyacetophenone to (S)-1-phenyl-1,2-ethanediol, we designed a co-expression system containing Candida parapsilosis CCTCC M203011 (S)-carbonyl reductase Ⅱ (SCRⅡ) and Bacillus sp. YX-1 glucose dehydrogenase (GDH) in Escherichia coli BL21(DE3), based on the optimal ratio between the specific activities of the two enzymes. Methods: The enzymes SCRⅡ and GDH were purified from their corresponding recombinant E. coli strains. When the purified SCRⅡ and GDH were used for the reduction of 2-hydroxyacetophenone to (S)-1-phenyl-1,2-ethanediol, the optimal ratio between their specific activities, the optimal temperature and pH were determined. Based on above results, a co-expression system E. coli BL21(DE3)/S-SD-AS-G harboring SCRⅡ and GDH was constructed. Results: SCRⅡ and GDH exhibited specific activities of 1.3 U/mg and 13.5 U/mg. When the total enzyme activity was 1 U, the optimal ratio of their activities is between 1:1 and 5:1, and the optimal temperature and pH are 30℃ and 7.0, respectively. So we designed a co-expression system E. coli BL21/S-SD-AS-G, in which the ratio of the SCRⅡ and GDH genes is 1:1. The specific activities of SCRⅡ and GDH are 0.76 U/mg and 0.73 U/mg in the cell-free extracts of E. coli BL21(DE3)/S-SD-AS-G, respectively. The ratio between SCRⅡ and GDH activity is 1:1. Under the optimal conditions, the system showed excellent performance to produce (S)-1-phenyl-1,2-ethanediol with an optical purity and a yield both over 99% during the reduction of 2-hydroxyacetophenone. With respect to the recombinant E. coli BL21(DE3)/pET-SCRⅡ, the co-expression system obviously improved the yield of (S)-1-phenyl-1,2-ethanediol and reduced biotransformation time from 24 h to 13 h. Conclusion: This work provides the research foundation on the construction of a co-expression system containing a target chiral catalyst and a cofactor-regeneration enzyme for efficient chiral biosynthesis based on the optimal ratio of SCRⅡ and GDH activities.

[Expression and functions of (S)-carbonyl reductase II in Saccharomyces cerevisiaes spores].

OBJECTIVE: To realize efficient biotransformation of (S)-1-phenyl-1,2-ethanediol by recombinant (S)-carbonyl reductase II, we expressed (S)- carbonyl reductase II from Candida parapsilosis CCTCC M203011 and embedded it in the spores of Saccharomyces cerevisiae AN120. METHODS: (S)-carbonyl reductase II gene was cloned from C. parapsilosis genome and expressed in S. cerevisiae AN120 by PCR amplification. When cultured with potassium acetate as the sole carbon source, the yeast spores were produced, and embedded the recombinant (S)-carbonyl reductase II. Using 10% W/V spores as biocatalysts, 6 g/L 2-hydroxyacetophenone as substrate, the biotransformation was carried out and the optical purity and yield of products were analyzed by HPLC. During the biotransformation of 2-hydroxyacetophenone to (S)-1-phenyl-1,2-ethanediol, the optimal temperature and pH, stability and reusability of the recombinant spores were determined. RESULTS: The recombinant yeast spores showed excellent performance to give (S)-1-phenyl-1,2- ethanediol with a high optical purity of 99.3% and a high yield of 99.0% at the optimal temperature (40 °C) and pH (6.0). Compared with the recombinant Escherichia coli, the spores improved the yield of (S)-1-phenyl-1,2-ethanediol from 89.7% to 99.0%, and shortened the biotransformation duration from 48 h to 4 h. After being reused for 10 times, the recombinant spores biotransformed (S)-1-phenyl-1,2-ethanediol with a stable optical purity of about 99% and a yield over 85%. CONCLUSION: The heterologous expression of oxidoreductases was first realized in yeast spores, which laid a solid foundation for efficient preparation of chiral compounds.

The rebalanced pathway significantly enhances acetoin production by disruption of acetoin reductase gene and moderate-expression of a new water-forming NADH oxidase in Bacillus subtilis.

Bacillus subtilis produces acetoin as a major extracellular product. However, the by-products of 2,3-butanediol, lactic acid and ethanol were accompanied in the NADH-dependent pathways. In this work, metabolic engineering strategies were proposed to redistribute the carbon flux to acetoin by manipulation the NADH levels. We first knocked out the acetoin reductase gene bdhA to block the main flux from acetoin to 2,3-butanediol. Then, among four putative candidates, we successfully screened an active water-forming NADH oxidase, YODC. Moderate-expression of YODC in the bdhA disrupted B. subtilis weakened the NADH-linked pathways to by-product pools of acetoin. Through these strategies, acetoin production was improved to 56.7g/l with an increase of 35.3%, while the production of 2,3-butanediol, lactic acid and ethanol were decreased by 92.3%, 70.1% and 75.0%, respectively, simultaneously the fermentation duration was decreased 1.7-fold. Acetoin productivity by B. subtilis was improved to 0.639g/(lh).