Protein engineering of oxidoreductases utilizing nicotinamide-based coenzymes,with applications in synthetic biology

Two natural nicotinamide-based coenzymes(NAD and NADP)are indispensably required by the vast majority of oxidoreductases for catabolism and anabolism,respectively.Most NAD(P)-dependent oxidoreductases prefer one coenzyme as an electron acceptor or donor to the other depending on their different metabolic roles.This coenzyme preference associated with coenzyme imbalance presents some challenges for the construction of high-efficiency in vivo and in vitro synthetic biology pathways.Changing the coenzyme preference of NAD(P)-dependent oxidoreductases is an important area of protein engineering,which is closely related to product-oriented synthetic biology projects.This review focuses on the methodology of nicotinamide-based coenzyme engineering,with its application in improving product yields and decreasing production costs.Biomimetic nicotinamide-containing coenzymes have been proposed to replace natural coenzymes because they are more stable and less costly than natural coenzymes.Recent advances in the switching of coenzyme preference from natural to biomimetic coenzymes are also covered in this review.Engineering coenzyme preferences from natural to biomimetic coenzymes has become an important direction for coenzyme engineering,especially for in vitro synthetic pathways and in vivo bioorthogonal redox pathways.

Effect of coenzyme Q10 supplementation on post-vitrification mouse embryo development

Objective:To investigate the effects of coenzyme Q10(CoQ10)supplementation on post-vitrification embryo development and gross morphology.Methods:Balb/c mouse embryos were cultured in potassium simplex optimised medium(KSOM)with varying CoQ10 concentrations[0(control),20,40,and 60μM].The most effective CoQ10 concentration(40μM)was selected for subsequent post-vitrification morphology study.Embryos were randomly divided into four groups:Group A(non-vitrified without CoQ10),Group B(non-vitrified with CoQ10),Group C(vitrified without CoQ10),and Group D(vitrified with CoQ10),followed by vitrification at the 8-cell stage.Survival rates and development until the blastocyst stage were evaluated through morphological examinations using ASEBIR's system,distinguishing normal and abnormal embryos.Results:Supplementation of 40μM CoQ10 significantly increased blastocyst formation(95%)compared to the control group(92%),20μM(62%),and 60μM(56%)(P<0.001).Following vitrification,Group D exhibited a significant increase in blastocyst formation(92%)compared to Group C(82%)(P<0.05).Morphological assessments indicated superior embryo quality in Group B over Group D during the cleavage stage,morula,and blastocyst(P<0.05).Conclusions:CoQ10 supplementation exhibits promising potential to enhance preimplantation embryo development,increase blastocyst formation rates,and improve embryo quality post-vitrification.This offers a promising approach to mitigate oxidative stress on embryos,potentially improving overall assisted reproductive technology outcomes.

辅酶Q10及其杂质结构鉴定

The structure of coenzyme Q10 sample was identified by MS,IR and NMR.And the impurities were identified by HPLC-ESI MS.The unknown impurities were coenzyme Q10’s structure isomeride,Q9,Q11

碱皂化法提取麸皮中辅酶Q_(10)的工艺研究

以麸皮为原料,利用碱皂化法提取辅酶Q10,紫外分光光度法测定含量。试验结果表明:最佳碱皂化处理条件为:pH为11,料液比1∶12(g/mL),皂化温度为90℃,皂化时间为90min。

Hrd1转基因小鼠的构建与验证

背景 内质网应激在糖尿病视网膜病变（DR）中发挥着重要作用.羟甲基戊二酰辅酶A还原酶降解蛋白1（Hrd1,又名Syvn1）作为一种内质网相关蛋白降解（ERAD）途径中的核心蛋白,能够减少内质网应激. 目的 构建Hrd1高表达小鼠,为今后研究Hrd1在DR中的作用提供动物模型. 方法 构建Hrd1系统表达重组质粒,酶切、测序后显微注射3 p1到685枚C57BL/6小鼠受精卵中,移植到代孕母鼠,得到Hrd1转基因小鼠.利用鼠尾PCR检测法鉴定F0代小鼠的基因型.得到Hrd1基因检测阳性鼠后,使其分别与野生型鼠杂交,得到F1代Hrd1转基因小鼠,取鼠尾组织,用PCR检测法再次鉴定F1代小鼠基因型. 结果 成功构建了Hrd1重组载体,重组质粒pRP.ExBi-EF1 a-Syvn1-IRES-eGFP的全序列测定结果显示,cDNA序列均正确.接受了Hrd1-pcDNA显微注射的685枚受精卵中成活598枚.将存活的受精卵移植到代孕母鼠后共产子鼠41只,获F0代子鼠8只,生理活动均良好.PCR电泳显示339 bp处阳性条带,该基因型可以传递到子代F1.结论 成功构建了Hrd1转基因小鼠.

Electrocatalytic Oxidation of NADH Based on Self-assembled Colloidal Gold and Nafion Matrixes and Co Complex Mediator

A novel approach based on self-assembled colloidal gold and Nation matrixes and Co complex mediator to construct Co（bpy）3^3＋/nano-Au/Co（bpy）3^3＋/nafion/GC electrode, on which formed stable redox-active films. This electrode can decrease the overpotential about 330 mV for the oxidation of NADH with high stability, wide linear range and low detection limit.

Non-Conducting Poly(O-Aminophenol) Films in the Field of the Bioelectrochemistry

The practical use of non-conducting poly(o-aminophenol) (POAP) films in the field of the bioelectrochemistry is discussed in this paper. Particular emphasis is given to the effects of applied potential, solution pH and interferents on the response current of biosensors based on POAP.

超声波破碎辅酶Q_(10)工艺研究

通过单因素试验和正交试验对超声波提取辅酶Q10的工艺条件进行了优化,确定了类球红细菌辅酶Q10的最佳提取条件为:输出功率为55%、每次辐射时间为3s、工作总时间为6min、菌液浓度OD为23.4,条件优化后辅酶Q10单位细胞产量达到11.86mg/L。

辅酶Q10对青少年病毒性心肌炎的心肌保护作用研究

目的评价辅酶Q10对青少年病毒性心肌炎的心肌保护作用。方法收集病人98例,随机分组。疗后15天予心肌酶学、肌钙蛋白检查,疗后30天予综合疗效总有效率为评价指标。结果心肌酶学、肌钙蛋白分析及综合疗效总有效率,治疗前后两组均有明显改善,且观察组优于对照组,P<0.05,差异具有统计学意义。结论辅酶Q10对青少年病毒性心肌炎患者具有较好的心肌保护作用。

Regulation and deregulation of cholesterol homeostasis: The liver as a metabolic "power station"

Cholesterol plays several structural and metabolic roles that are vital for human biology. It spreads along the entire plasma membrane of the cell, modulating fluidity and concentrating in specialized sphingolipid-rich domains called rafts and caveolae. Cholesterol is also a substrate for steroid hormones. However, too much cholesterol can lead to pathological pictures such as atherosclerosis, which is a consequence of the accumu- lation of cholesterol into the cells of the artery wall. The liver is considered to be the metabolic power station of mammalians, where cholesterol homeostasis relies on an intricate network of cellular processes whose deregulations can lead to several life-threatening pathologies, such as familial and age-related hypercholesterolemia. Cholesterol homeostasis maintenance is carried out by: biosynthesis, via 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) activity; uptake, through low density lipoprotein receptors (LDLr); lipoprotein release in the blood; storage by esterification; and degradation and conversion into bile acids. Both HMGR and LDLr are transcribed as a function of cellular sterol amount by a family of transcription factors called sterol regulatory element binding proteins that are responsible for the maintenance of cholesterol homeostasis through an intricate mechanism of regulation. Cholesterol obtained by hepatic de novo synthesis can be esterified and incorporated into apolipoprotein B-100-containing very low density lipoproteins, which are then secreted into the bloodstream for transport to peripheral tissues. Moreover, dietary cholesterol is transferred from the intestine to the liver by high density lipoproteins (HDLs); all HDL particles are internalized in the liver, interacting with the hepatic scavenger receptor (SR-B1). Here we provide an updated overview of liver cholesterol metabolism regulation and deregulation and the causes of cholesterol metabolism-related diseases. Moreover, current pharmacological treatment and novel hypocho-lesterolemic strategies will also be introduced.

Weaning methods affect ruminal methanogenic archaea composition and diversity in Holstein calves

The objective of the present study was to examine the effect of different weaning methods on the ruminal methanogenic archaea composition and diversity in Holstein calves.Thirty-six newborn Holstein bull calves were assigned to 1 of 3 treatments:(1)conventional weaning(d 56)and fed a high proportion of solid feed(CWS);(2)conventional weaning(d 56)and fed a high proportion of liquid feed(CWL);(3)early weaning(d 42)and fed with a high proportion of solid feed(EWS).High-throughput sequencing of the methyl coenzyme M reductase(mcr A)gene,which encodes theα-subunit of methyl coenzyme M reductase-the enzyme that catalyzes the final step in methanogenesis was used to determine the composition and diversity of rumen methanogens.No significant difference(P>0.05)was observed for operational taxonomic units(OTUs)or richness indices,but diversity indices increased(P<0.05)for calves fed high dietary solids.Predominant families across the three treatments were Methanobacteriaceae,Thermoplasmataceae and Methanomassiliicoccaceae.Calves in the EWS treatment had a higher(P<0.05)relative abundance of Methanobrevibacter sp.strain AbM4 and Methanosphaera stadtmanae,while calves in the CWL treatment had a higher(P<0.05)abundance of Methanosphaera sp.strain SM9.A positive(P<0.05)relationship was identified between butyrate and Methanobrevibacter sp.strain AbM4.In conclusion,the composition and diversity of methanogens in the rumen of Holstein calves varied under the different weaning methods.This study identified a positive relationship between butyrate and Methanobrevibacter sp.strain AbM4,potentially reflecting correlations between ruminal fermentation variables and methanogenesis function.These in-depth analyses provide further understanding of weaning methods for intensified production systems.

Multiple Strategies for Metabolic Engineering of Escherichia coli for Efficient Production of Coenzyme Q_（10）

Escherichia coli BW25113 was metabolically engineered for CoQ10 production by replacing ispB with ddsA from Gluconobacter suboxydans.Effects of precursor balance and reduced nicotinamide-adenine dinucleotide phosphate （NADPH） availability on CoQ10 production in E.coli were investigated.The knockout of pykFA along with pck overexpression could maintain a balance between glyceraldehyde 3-phosphate and pyruvate,increasing CoQ10 production.Replacement of native NAD-dependent gapA with NADP-dependent gapC from Clostridium acetobutylicum,together with the overexpression of gapC,could increase NADPH availability and then enhanced CoQ10 production.Three effects,overexpressions of various genes in CoQ biosynthesis and central metabolism,different vectors and culture conditions on CoQ10 production in E.coli,were all investigated.The investigation of different vectors indicated that low copy number vector may be more beneficial for CoQ10 production in E.coli.The recombinant E.coli （△ispB：：ddsA,△pykFA and △gapA：：gapC）,harboring the two plasmids encoding pck,dxs,idi and ubiCA genes under the control of PT5 on pQE30,ispA,ddsA from Gluconobacter suboxydans and gapC from Clostridium acetobutylicum under the control of PBAD on pBAD33,could produce CoQ10 up to 3.24 mg·g-1 dry cell mass simply by changing medium from M9YG to SOB with phosphate salt and initial culture pH from 7.0 to 5.5.The yield is unprecedented and 1.33 times of the highest production so far in E.coli.

Variation of Coenzyme F_(420) Activity and Methane Yield in Landfill Simulation of Organic Waste

A simulated landfill anaerobic bioreactor was used to characterize the anaerobic biodegradation and biogas generation of organic waste which was mainly composed of residuals of vegetables and foods. We investigated the dynamics of the coenzyme F420 activity and determined correlations between biogas yields, methane yields, methane concentration and coenzyme F420 activity. The experiment was carded out under different conditions from control without any treatment, addition of Fe^3＋, microorganism inoculation to a combination of Fe3＋ addition and inoculation at a temperature of 36±2℃. The experiment was lasted 120 d and coenzyme F420 activity was analyzed using ultraviolet spectrophotornetry. Experimental results indicated that activity of the coenzyme F420 treated by Fe and microorganism inoculation increased substantially. The waste treated by inoculation had the greatest increase. When the waste was treated by Fe^3＋, inoculation and the combination of Fe^3＋ and inoculation, biogas yields increased by 46.9%, 132.6% and 153.1%, respectively; while the methane yields increased 4, 97 and 98 times. Methane concentration varied between 0 and 6% in the control reactor, from 0 to 14% for waste treated by the addition of Fe^3＋, from 0 to 59% for waste treated by inoculation and from 0 to 63% for waste treated by Fe^3＋ addition and inoculation. Correlations between coenzyme F420 activity and biogas production, methane production and methane concentration proved to be positively significant （p〈0.05）, except for the control. Consequently, coenzyme F420 activity could be used as an index for monitoring the activity of methanogens during anaerobic biodegradation of the organic fraction of municipal solid waste.

Advances in the Coenzyme Q10 Biosynthesis Pathway in Rhodobacter sphaeroides and the Enhancement of Coenzyme Q10 Production Based on Metabolic Engineering

Coenzyme Q10 is widely used in food,cosmetics and pharmaceuticals,possessing a broad market.Rhodobacter sphaeroides is enriched in natural coenzyme Q10 and is becoming an important microorganism for producing natural coenzyme Q10.The paper reviewed the biosynthesis pathways of coenzyme Q10 in R.sphaeroides and the advances in enhancement of coenzyme Q10 production in R.sphaeroides based on metabolic engineering.

Antioxidative stress-induced damage in cochlear explants

The imbalance of reactive oxygen species and antioxidants is considered to be an important factor in the cellular injury of the inner ear. At present, great attention has been placed on oxidative stress. However,little is known about fighting oxidative stress. In the current study, we evaluated antioxidant-induced cochlear damage by applying several different additional antioxidants. To determine whether excessive antioxidants can cause damage to cochlear cells, we treated cochlear explants with 50 m M M40403, a superoxide dismutase mimetic, 50 m M coenzyme Q-10, a vitamin-like antioxidant, or 50 m M d-methionine, an essential amino acid and the important antioxidant glutathione for 48 h. Control cochlear explants without the antioxidant treatment maintained their normal structures after incubation in the standard serum-free medium for 48 h, indicating the maintenance of the inherent oxidative and antioxidant balance in these cochlear explants. In contrast, M40403 and coenzyme Q-10-treated cochlear explants displayed significant hair cell damage together with slight damage to the auditory nerve fibers.Moreover, d-methiodine-treated explants exhibited severe damage to the surface structure of hair cells and the complete loss of the spiral ganglion neurons and their peripheral fibers. These results indicate that excessive antioxidants are detrimental to cochlear cells, suggesting that inappropriate dosages of antioxidant treatments can interrupt the balance of the inherent oxidative and antioxidant capacity in the cell.

Progression of intervention-focused research for Gulf War illness

The Persian Gulf War of 1990 to 1991 involved the deployment of nearly 700,000 American troops to the Middle East.Deployment-related exposures to toxic substances such as pesticides,nerve agents,pyridostigmine bromide(PB),smoke from burning oil wells,and petrochemicals may have contributed to medical illness in as many as 250,000 of those American troops.The cluster of chronic symptoms,now referred to as Gulf War Illness(GWI),has been studied by many researchers over the past two decades.Although over$500 million has been spent on GWI research,to date,no cures or condition-specific treatments have been discovered,and the exact pathophysiology remains elusive.Using the 2007 National Institute of Health(NIH)Roadmap for Medical Research model as a reference framework,we reviewed studies of interventions involving GWI patients to assess the progress of treatment-related GWI research.All GWI clinical trial studies reviewed involved investigations of existing interventions that have shown efficacy in other diseases with analogous symptoms.After reviewing the published and ongoing registered clinical trials for cognitivebehavioral therapy,exercise therapy,acupuncture,coenzyme Q10(CoQ10),mifepristone,and carnosine in GWI patients,we identified only four treatments(cognitive-behavioral therapy,exercise therapy,CoQ10,and mifepristone)that have progressed beyond a phase II trial.We conclude that progress in the scientific study of therapies for GWI has not followed the NIH Roadmap for Medical Research model.Establishment of a standard case definition,prioritized GWI research funding for the characterization of the pathophysiology of the condition,and rapid replication and adaptation of early phase,single site clinical trials could substantially advance research progress and treatment discovery for this condition.

Photocatalytic oxidation of primary and secondary benzyl alcohol catalyzed by two coenzyme NAD^+ models

Photocatalytic oxidation of primary and secondary benzyl alcohol to corresponding benzaldehyde or acetophenone using Acr＋Cl04- or PhAcr＋Cl04- as photocatalysts under visible light irradiation at room temperature.

Do statins reduce hepatitis C RNA titers during routine clinical use?

AIM: To compare hepatitis C virus (HCV) titers in patients with chronic hepatitis C with and without exposure to 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins).METHODS: Medical records were reviewed for 6463 patients with documented HCV infection at a single center between March 2004 and September 2006. Patients with confi rmed viremia and meeting inclusion criteria were assigned to one of three groups: Group A (n = 50), dyslipidemic patients with statin usage during HCV RNA polymerase chain reaction (PCR) determination; Group B (n = 49), dyslipidemic patients with prior or future statin usage but not at the time of HCV RNA PCR determination; and Group C (n = 102), patients without statin usage during the study period. The primary analysis explored the effect of statin therapy on HCV viremia. Secondary analyses assessed class effect, dose response, and effect of other lipid-lowering therapies on HCV viral titers.RESULTS: Median HCV RNA titers did not signif icantly differ among the three groups (Group A: 4 550 000 IU/mL, Group B: 2 850 000 IU/mL, Group C: 3 055 000 IU/mL).For those subjects with longitudinal assessment of HCV viremia prior to and while on statins, there were no signif icant differences between pre- and post-HCV viral titers. Additionally, no differences in HCV titers were observed at any dose level of the most prescribed statin, simvastatin. However, hypertriglyceridemia independently correlated with HCV titers, and niacin exposure was associated with signif icantly lower viral titers (P < 0.05).CONCLUSION: There was no apparent effect of statins on HCV viral replication in this analysis. Further investigation is warranted to explore the possible antiviral properties of triglyceride-lowering agents and their potential role as adjuncts to standard HCV therapy.

Study on the Reaction of Benzimidazolium Salt with Amine Compounds

The addition-hydrolysis reaction of benzimidazolium salt with some mono- and bifunctional amine nucleophiles is reported, and a novel method of biomimetic synthesis for formamides and heterocycle compounds is provided.

Roles of Long-chain Acyl Coenzyme A Synthetase in Absorption and Transport of Fatty Acid

Long-chain acyl coenzyme A synthetase(ACSL) is a member of the synthetase family encoded by a multigene family;it plays an important role in the absorption and transport of fatty acid.Here we review the roles of ACSL in the regulating absorption and transport of fatty acid,as well as the connection between ACSL and some metabolic diseases.