微生物异源合成植物异喹啉生物碱的新进展

植物异喹啉生物碱(plant isoquinoline alkaloids,PIAs)包括吗啡、可待因、加兰他敏及小糵碱等药用活性产物和其他天然活性产物。从植物中提取异喹啉生物碱,受制于低含量、种植季节及提取方法。人们开始研究利用微生物异源合成和改造天然异喹啉生物碱,从而获得低成本的药用活性物质。异喹啉生物碱合成途径长,反应复杂,为实现微生物异源合成带来了诸多挑战。随着合成途径和酶的解析和鉴定,合成生物学技术为在微生物中合成异喹啉生物碱提供了可能。综述了PIAs合成途径解析的最新进展,以及微生物异源合成PIAs的代谢工程策略,讨论了目前存在的问题和未来的发展趋势。     

微生物法生产烷烃研究进展

烷烃在自然界中广泛存在。它不仅是化石能源的主要组成部分,而且在润滑剂、化妆品、水果保藏、植物防护等方面具有广泛的应用价值。本文概述了天然产烷烃的微生物及其烷烃的天然合成途径及其途径中关键酶催化的作用机理,并对近几年国内外运用代谢工程手段改造微生物使其细胞合成烷烃的研究进展作了介绍。微生物生产烷烃可以通过改造烷烃的天然合成菌株或通过在模式微生物中引入异源烷烃合成途径两种方法来强化。最后文章讨论了微生物法生产烷烃存在的不足和今后研究的方向与展望。     

大肠杆菌异源合成三萜化合物研究进展和前景分析

三萜化合物具有可观的药用价值和经济价值,但是目前的生产过程复杂、产量低,利用微生物异源合成三萜化合物已成为当前研究趋势,大肠杆菌作为常用萜类合成底盘细胞具有异源合成三萜化合物及其前体的天然优势和研究前景。对三萜化合物微生物异源合成研究进展进行了综述,从三萜化合物合成代谢途径、关键酶的特点及大肠杆菌三萜表达模块和底盘细胞适配三个方面对该途径进行了阐述和分析,针对实现大肠杆菌高效合成三萜类化合物所需要解决的基础问题进行讨论,为扩展大肠杆菌作为三萜化合物合成底盘细胞提供建议和前景分析。     

代谢工程改造酿酒酵母合成植物萜类D-柠檬烯的策略

植物萜类化合物是以异戊二烯为结构单位的一大类植物天然的次生代谢产物。D-柠檬烯属于单萜类化合物,由于它具有抑菌、增香、抗癌、止咳、平喘等多种功能,已被广泛应用于食品、香料、医疗等行业。目前D-柠檬烯的工业生产主要是从植物的果皮或者果肉中提取的,但提取方法存在着分离纯化复杂、产率低、能耗大等缺点。而本世纪初合成生物学技术的兴起,为微生物异源合成天然活性化合物带来了全新的理念与工具,打破了物种间的界限,使微生物异源合成D-柠檬烯成为现实。构建定向、高效的异源合成D-柠檬烯的微生物细胞工厂,实现微生物发酵法替换传统的植物提取法,具有重要的经济与社会效益。本文主要回顾了近几年利用代谢工程改造酿酒酵母异源合成萜类化合物取得的成就,阐述了以酿酒酵母作为底盘微生物,利用代谢工程和合成生物学的手段构建高产D-柠檬烯的合成策略。     

萜类合成酶定向进化的新思路

萜类化合物是天然产物中种类最多且主要存在于植物和微生物体内的一类化合物。随着越来越多具有应用价值的萜类化合物被挖掘,其应用前景引起了人们的关注,但由于含量低、提取成本高等缺点,因此制约了萜类化合物的广泛应用。合成生物学的兴起,为异源合成具有应用价值的萜类化合物提供了新思路,使构建定向、高效的微生物细胞工厂成为现实。萜类合成酶常作为萜类化合物异源合成代谢调控的靶酶,但天然的萜类合成酶存在催化效率低、底物专一性差、立体/区域选择性差、稳定性差等问题,严重影响萜类化合物的产量。萜类合成酶的定向进化可以有效地解决上述问题,为实现微生物细胞工厂异源、高效合成萜类化合物奠定基础。本文综述了近年来酶的定向进化技术的最新进展及应用,并提出了萜类合成酶定向进化的策略。     

香豆素类化合物功能及生物合成研究进展*

香豆素类化合物是自然界中一类重要的化合物,具有抗肿瘤、抗凝血、抗菌、杀虫等多种生物活性,应用领域广泛。目前大多数香豆素类化合物从植物中提取,受环境因素影响较大,得率低、成本高,不利于大规模生产,从而限制了其应用和推广。利用合成生物学的思路合成香豆素类化合物具有无污染、原料易得、成本低、过程可控等优势。对香豆素类化合物生物合成途径的研究,尤其是靶标天然产物生物合成表达元件、宿主和发酵条件的优化,以及合成途径中关键酶的挖掘,已经成为研究热点。综述香豆素类化合物及其衍生物的结构、功能和生物合成研究进展,为其生物合成路径中的基因挖掘及异源表达提供参考。     

基于莽草酸途径微生物合成芳香族化合物及其衍生物的研究进展

芳香族化合物是一类重要的天然产物,在自然界中广泛存在,应用于食品、医药、化工等多个领域,主要通过化学合成、植物提取等方式获得。近年来,随着石化资源减少、人类环保意识的加强,微生物合成芳香族化合物及其衍生物成为热点。莽草酸途径合成的芳香族化合物及其衍生物多种多样。现重点综述通过莽草酸途径合成的"达菲"药物前体莽草酸、大宗化学品己二酸前体顺,顺-粘康酸、芳香族氨基酸及其他高附加值芳香族氨基酸衍生物的微生物合成研究进展,为建立生产高附加值化合物的细胞工厂提供参考。     

工程微生物合成香草醛的进展与挑战

香草醛是广泛使用的一种天然香料和药物原料,目前主要通过化学合成和天然提取的方法获取。市场上的香草醛主要来源于化学合成,但有毒有害试剂的使用无法满足绿色化学的要求和可持续的发展理念。代谢工程和合成生物学技术的发展在微生物宿主中实现了构建香草醛合成途径,并以可再生资源为底物合成香草醛。因此,本文全面综述了已经发现的香草醛生物合成途径以及优化与调控途径合成效率的各种工程策略,提出了微生物异源合成香草醛面临的挑战,为香草醛的高效生物合成提供参考。     

谷氨酸棒状杆菌异源合成萜类化合物的研究进展

萜类化合物具有可观的商业价值,但生产过程复杂,产量低,利用微生物异源合成萜类化合物已成为热点。谷氨酸棒状杆菌内含合成萜类色素的途径,具有异源合成萜类化合物的天然优势和研究前景。首次对谷氨酸棒状杆菌合成萜类化合物进行了综述,从萜类合成途径、关键酶和全局调控机制三个方面进行了途经介绍。概述了谷氨酸棒状杆菌中单萜、倍半萜、四萜类化合物的异源合成,并对利用谷氨酸棒状杆菌高效合成萜类化合物所需解决的问题进行讨论,为谷氨酸棒状杆菌高效合成萜类化合物提供建议。     

计算预测天然产物生物合成

正植物天然产物(Plant natural products,PNPs)及其衍生物是重要的但未被充分开发的药物分子来源。为了利用这种未开发的潜力,在工程微生物中重组植物天然产物及其衍生物生物合成途径可以用于探索和生产新的PNP衍生物。2021年3月19日Nature Communications报道,斯坦福大学研究者开发了一个计算工作流,用于系统地筛选生物分子合成的衍生途径,该工作流在预测代谢工程中的反应、途径和酶上具有很高的价值。     

Measurement of unbound caffeic acid in rat blood by on-line microdialysis coupled with liquid chromatography and its application to pharmacokinetic study

To monitor the levels of caffeic acid in rat blood, an on-line microdialysis system coupled with liquid chromatography was developed. The microdialysis probe was inserted into the jugular vein/right atrium of male Sprague-Dawley rats. Caffeic acid (100 mg/kg, i.v.) was then administered via the femoral vein. Dialysates were automatically injected onto a liquid chromatographic system via an on-line injector. Samples were eluted with a mobile phase containing methanol–100 mM monosodium phosphoric acid (35:65, v/v, pH 2.5). The UV detector wavelength was set at 320 nm. The detection limit of caffeic acid was 20 ng/ml. The in vivo recoveries of the microdialysis probe for caffeic acid at 0.5 and 1 μg/ml were 48.34±2.68 and 47.64±3.43%, respectively (n=6). Intra- and inter-assay accuracy and precision of the analyses were ≤10% in the range of 0.05 to 10 μg/ml. Pharmacokinetics analysis of results obtained using such a microdialysis–chromatographic method indicated that unbound caffeic acid in the rat fitted best to a biexponential decay model.     

Development and validation of a liquid chromatographic-tandem mass spectrometric method for the determination of caffeic acid phenethyl ester in rat plasma and urine

Caffeic acid phenethyl ester (CAPE) is one of the most bioactive compounds of propolis, a resinous substance collected and elaborated by honeybees. A new liquid chromatography-electrospray ionisation tandem mass spectrometric method was developed and validated for its determination in rat plasma and urine, using taxifolin as internal standard. After sample preparation by liquid/liquid extraction with ethyl acetate, chromatographic separations were carried out with an ODS-RP column using a binary mobile phase gradient of acetonitrile in water. Detection was performed using a turboionspray source operated in negative ion mode and by multiple reaction monitoring. The method was validated, showing good selectivity, sensitivity (LOD = 1 ng/ml), linearity (5-1000 ng/ml; r > or = 0.9968), intra- and inter-batch precision and accuracy (< or =14.5%), and recoveries (94-106%) in both plasma and urine. Stability assays have shown that CAPE is rapidly hydrolysed by plasmatic esterases, which are however inhibited by sodium fluoride. The method was applied to the determination of CAPE levels in rat plasma and urine after oral administration, showing that CAPE is rapidly absorbed and excreted in urine both as unmodified molecule and as glucuronide conjugate.     

Production of caffeic,chlorogenic and rosmarinic acids in plants and suspension cultures of Glechoma hederacea

Glechoma hederacea L. (Lamiaceae) is a perennial plant, which is distributed widely in Europe, Asia and America. Important anti-oxidant compounds are caffeic acid esters like rosmarinic acid (RA) and chlorogenic acid (CA). Phenylalanine ammonia-lyase (PAL) and rosmarinic acid synthase (RAS, 4-coumaroyl-CoA:hydroxyphenyllactic acid hydroxycinnamoyltransferase) contribute to the formation of RA. Our aim in this study was to follow the accumulation of RA, CA and caffeic acid in a suspension culture of G. hederacea. Growth, medium and secondary metabolism parameters were determined during a culture period of 14 days. The maximal PAL activity was observed on day 5 and the maximal RAS activity on day 8. The RA content was exceedingly high and reached 25.9% of the dry mass on day 7. Caffeic acid and CA contents remained rather low. Furthermore, the presence of RA, CA and caffeic acid and the expression patterns of RAS and hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyltransferase (HST), an important enzyme of monolignol formation, in leaves, flowers, stems and roots of naturally grown G. hederacea were assessed. The expression of RAS and HST genes was detectable in all organs except roots. Flowers accumulated 12.5% RA in their dry mass, leaves, stems and roots about 1%. CA was highest in leaves (2.0%), while it was at 1.6% in flowers, 1.3% in stems and almost undetectable in roots. The caffeic acid content remained at or below 0.4% of the dry weight in all organs.     

Caffeic acid derivatives: A new type of influenza neuraminidase inhibitors

Recently, many natural products, especially some plant-derived polyphenols have been found to exert antiviral effects against influenza virus and show inhibitory activities on neuraminidases (NAs). In our research, we took caffeic acid which contained two phenolic hydroxyl groups as the basic fragment to build a small compound library with various structures. The enzyme inhibition result indicated that some compounds exhibited moderate activities against NA and compound 15d was the best with IC₅₀ = 7.2 μM and 8.5 μM against N2 and N1 NAs, respectively. The 3,4-dihydroxyphenyl group from caffeic acid was important for the activity according to the docking analysis. Besides, compound 15d was found to be a non-competitive inhibitor with K_i = 11.5 ± 0.25 μM by the kinetic study and also presented anti-influenza virus activity in chicken embryo fibroblast cells. It seemed promising to discover more potent NA inhibitors from caffeic acid derivatives to cope with influenza virus.     

Heterologous production of caffeic acid from tyrosine in Escherichia coli

Caffeic acid is a plant secondary metabolite and its biological synthesis has attracted increased attention due to its beneficial effects on human health. In this study, Escherichia coli was engineered for the production of caffeic acid using tyrosine as the initial precursor of the pathway. The pathway design included tyrosine ammonia lyase (TAL) from Rhodotorula glutinis to convert tyrosine to p-coumaric acid and 4-coumarate 3-hydroxylase (C3H) from Saccharothrix espanaensis or cytochrome P450 CYP199A2 from Rhodopseudomonas palustris to convert p-coumaric acid to caffeic acid. The genes were codon-optimized and different combinations of plasmids were used to improve the titer of caffeic acid. TAL was able to efficiently convert 3 mM of tyrosine to p-coumaric acid with the highest production obtained being 2.62 mM (472 mg/L). CYP199A2 exhibited higher catalytic activity towards p-coumaric acid than C3H. The highest caffeic acid production obtained using TAL and CYP199A2 and TAL and C3H was 1.56 mM (280 mg/L) and 1 mM (180 mg/L), respectively. This is the first study that shows caffeic acid production using CYP199A2 and tyrosine as the initial precursor. This study suggests the possibility of further producing more complex plant secondary metabolites like flavonoids and curcuminoids.     

Activity of caffeic acid derivatives against Candida albicans biofilm

The aim of this study was to evaluate the caffeic acid (1) and ester derivatives (2–10) against Candida albicans biofilm and to investigate whether these compounds are able to inhibit the biofilm formation or destroy pre-formed biofilm.Caffeic acid ester 7, cinnamic acid ester 8 and 3,4-dihydroxybenzoic acid ester 10 are more active than fluconazole, used as reference drug, both on biofilm in formation with MIC₅₀ values of 32, 32 and 16 μg/mL, respectively, and in the early stage of biofilm formation (4 h) with MIC₅₀ values of 64, 32 and 64 μg/mL, respectively. These esters result also more active than fluconazole on mature biofilm (24 h), especially 8 and 10 with MIC₅₀ values of 64 μg/mL.     

Characterization of a multifunctional methyltransferase from the orchid <Emphasis Type="Italic">Vanilla planifolia</Emphasis>

The final enzymatic step in the synthesis of the flavor compound vanillin (4-hydroxy-3-methoxybenzaldehyde) is believed to be methylation of 3,4-dihydroxybenzaldehyde. We have isolated and functionally characterized a cDNA that encodes a multifunctional methyltransferase from Vanilla planifolia tissue cultures that can catalyze the conversion of 3,4-dihydroxybenzaldehyde to vanillin, although 3,4-dihydroxybenzaldehyde is not the preferred substrate. The higher catalytic efficiency of the purified recombinant enzyme with the substrates caffeoyl aldehyde and 5-OH-coniferaldehyde, and its tissue distribution, suggest this methyltransferase may primarily function in lignin biosynthesis. However, since the enzyme characterized here does have 3,4-dihydroxybenzaldehyde-O-methyltransferase activity, it may be useful in engineering strategies for the synthesis of natural vanillin from alternate sources.Abbreviations COMT Caffeic acid O-methyltransferase - DOMT 3,4-Dihydroxybenzaldehyde-O-methyltransferase - OMTs O-Methyltransferases - SAM S-adenosyl-l-methionine     

Further discovery of caffeic acid derivatives as novel influenza neuraminidase inhibitors

Eight series of compounds, each series containing two to five compounds were prepared by structural modifications of a lead, which was previously discovered as a mild influenza neuraminidase (NA) inhibitor. On the basis of the biological result, a detailed structure–activity relationship (SAR) was derived and discussed. Several caffeic acid derivatives that acted as non-competitive inhibitors were close or superior to the lead and also presented good antiviral activities in cells. Besides, it was interesting to find that modifications of the lead with different strategies could result in selective inhibition against N1 or N2. The preliminary docking analysis indicated that the 150-cavity of the enzymes played an important role in the selective inhibition.     

Efficacy of caffeic acid in preventing nickel induced oxidative damage in liver of rats

Nickel (Ni), a major environmental pollutant, is known for its wide toxic manifestations. In the present study caffeic acid (CA), one of the most commonly occurring phenolic acids in fruits, grains and dietary supplements, was evaluated for its protective effect against the Ni induced oxidative damage in liver. In this investigation, Ni (20 mg/kg body weight) was administered intraperitoneally for 20 days to induce toxicity. CA was administered orally (15, 30 and 60 mg/kg body weight) for 20 days with intraperitoneal administration of Ni. Ni induced liver damage was clearly shown by the increased activities of serum hepatic enzymes namely aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), gamma glutamyl transferase (GGT) and lactate dehydrogenase (LDH) along with increased elevation of lipid peroxidation indices (thiobarbituric reactive acid substances (TBARS) and lipid hydroperoxides). The toxic effect of Ni was also indicated by significantly decreased levels of enzymatic (superoxide dismutase (SOD), catalase (CAT) glutathione peroxidase (GPx) and glutathione S-transferase (GST)) and non-enzymatic antioxidants (glutathione (GSH), vitamin C and vitamin E). CA administered at a dose of 60 mg/kg body weight significantly reversed the activities of hepatic marker enzymes to their near normal levels when compared with other two doses. In addition, CA significantly reduced lipid peroxidation and restored the levels of antioxidant defense in the liver. All these changes were supported by histological observations. The results indicate that CA may be beneficial in ameliorating the Ni induced oxidative damage in the liver of rats.     

Caffeic acid ethanolamide prevents cardiac dysfunction through sirtuin dependent cardiac bioenergetics preservation

Background

Cardiac oxidative stress, bioenergetics and catecholamine play major roles in heart failure progression. However, the relationships between these three dominant heart failure factors are not fully elucidated. Caffeic acid ethanolamide (CAEA), a synthesized derivative from caffeic acid that exerted antioxidative properties, was thus applied in this study to explore its effects on the pathogenesis of heart failure.

Results

In vitro studies in HL-1 cells exposed to isoproterenol showed an increase in cellular and mitochondria oxidative stress. Two-week isoproterenol injections into mice resulted in ventricular hypertrophy, myocardial fibrosis, elevated lipid peroxidation, cardiac adenosine triphosphate and left ventricular ejection fraction decline, suggesting oxidative stress and bioenergetics changes in catecholamine-induced heart failure. CAEA restored oxygen consumption rates and adenosine triphosphate contents. In addition, CAEA alleviated isoproterenol-induced cardiac remodeling, cardiac oxidative stress, cardiac bioenergetics and function insufficiency in mice. CAEA treatment recovered sirtuin 1 and sirtuin 3 activity, and attenuated the changes of proteins, including manganese superoxide dismutase and hypoxia-inducible factor 1-α, which are the most likely mechanisms responsible for the alleviation of isoproterenol-caused cardiac injury

Conclusion

CAEA prevents catecholamine-induced cardiac damage and is therefore a possible new therapeutic approach for preventing heart failure progression.