引入新型异戊二烯醇利用途径促进解脂耶氏酵母中β-胡萝卜素的合成*

目的:微生物体内异戊二烯类化合物的前体物异戊烯焦磷酸酯的天然合成路径受到严格的代谢调控,因此限制了异戊二烯类化合物的高效生物合成,而新型异戊二烯醇利用途径独立于生物体内源性代谢路径,通过在微生物中引入IUP能够进行异戊烯焦磷酸酯的大量合成,从而促进异戊二烯类化合物的大量合成。方法:在油脂酵母解脂耶氏酵母中引入IUP,强化异戊烯焦磷酸酯生物合成,促进β-胡萝卜素的高效积累。结果:通过生物信息学的方法预测IUP中两个关键蛋白酿酒酵母来源的胆碱激酶ScCK和拟南芥来源的异戊烯磷酸激酶AtIPK,均为酸性亲水性蛋白,无跨膜区和信号肽,二者都具有疏松不稳定的结构特征,显著富集于磷酸类物质的合成通路中。在解脂耶氏酵母中利用同源重组技术引入外源β-胡萝卜素合成关键基因carRP和carB,强化甲羟戊酸途径的关键基因thmgR和ggs1,使工程菌株中积累2.68 mg/L β-胡萝卜素。通过Cre-loxP系统回收基因组上的ura标签,再将IUP进一步整合到工程菌株染色体上。当培养基中含有20 mM异戊二烯醇作为底物、碳氮比为4/3且发酵96 h后,重组解脂耶氏酵母中β-胡萝卜素的产量提高到410.2 mg/L,较原始工程菌的产量提高了近200倍。结论:IUP能够促进解脂耶氏酵母中β-胡萝卜素的高效积累,为利用IUP开展β-胡萝卜素和其他异戊二烯类化合物的高效生物合成提供新思路。     

Plant carotene cis-trans isomerase CRTISO: a new member of the FAD(RED)-dependent flavoproteins catalyzing non-redox reactions

The carotene cis-trans isomerase CRTISO is a constituent of the carotene desaturation pathway as evolved in cyanobacteria and prevailing in plants, in which a tetra-cis-lycopene species, termed prolycopene, is formed. CRTISO, an evolutionary descendant of the bacterial carotene desaturase CRTI, catalyzes the cis-to-trans isomerization reactions leading to all-trans-lycopene, the substrate for the subsequent lycopene cyclization to form all-trans-α/β-carotene. CRTISO and CRTI share a dinucleotide binding motif at the N terminus. Here we report that this site is occupied by FAD in CRTISO. The reduced form of this cofactor catalyzes a reaction not involving net redox changes. Results obtained with C(1)- and C(5)-deaza-FAD suggest mechanistic similarities with type II isopentenyl diphosphate: dimethylallyl diphosphate isomerase (IDI-2). CRTISO, together with lycopene cyclase CRTY and IDI-2, thus represents the third enzyme in isoprenoid metabolism belonging to the class of non-redox enzymes depending on reduced flavin for activity. The regional specificity and the kinetics of the isomerization reaction were investigated in vitro using purified enzyme and biphasic liposome-based systems carrying specific cis-configured lycopene species as substrates. The reaction proceeded from cis to trans, recognizing half-sides of the symmetrical prolycopene and was accompanied by one trans-to-cis isomerization step specific for the C(5)-C(6) double bond. Rice lycopene β-cyclase (OsLCY-b), when additionally introduced into the biphasic in vitro system used, was found to be stereospecific for all-trans-lycopene and allowed the CRTISO reaction to proceed toward completion by modifying the thermodynamics of the overall reaction.     

Isoprenoid biosynthesis in bacteria: Two different pathways?

Abstract The biosynthesis of isopentenylpyrophosphate, a central intermediate of isoprenoid formation, was investigated in six different bacterial organisms. Cell-free extracts of Myxococcus fulvus, Staphylococcus carnosus, Lactobacillus plantarum and Halobacterium cutirubrum converted [¹⁴C]acetyl-CoA or [¹⁴C]hydroxymethylglutaryl-CoA to [¹⁴C]mevalonic acid. Furthermore, [¹⁴C]mevalonic acid, [¹⁴C]mevalonate-5-phosphate and [¹⁴C]mevalonate-5-pyrophosphate were metabolized to [¹⁴C]isopentenylpyrophosphate in bacteria. In contrast, no intermediates of this reaction sequence could be detected using cell-free extracts of Zymomonas mobilis and Escherichia coli . These results indicate that at least two different pathways for the biosynthesis of isopentenylpyrophosphate are present in bacteria.     

Effect of Lipid Particle Biogenesis on the Subcellular Distribution of Squalene in the Yeast Saccharomyces cerevisiae

Squalene belongs to the group of isoprenoids and is a precursor for the synthesis of sterols, steroids, and ubiquinons. In the yeast Saccharomyces cerevisiae, the amount of squalene can be increased by variation of growth conditions or by genetic manipulation. In this report, we show that a hem1Δ mutant accumulated a large amount of squalene, which was stored almost exclusively in cytoplasmic lipid particles/droplets. Interestingly, a strain bearing a hem1Δ deletion in a dga1Δlro1Δare1Δare2Δ quadruple mutant background (QMhem1Δ), which is devoid of the classical storage lipids, triacylglycerols and steryl esters, and lacks lipid particles, accumulated squalene at similar amounts as the hem1Δ mutant in a wild type background. In QMhem1Δ, however, increased amounts of squalene were found in cellular membranes, especially in microsomes. The fact that QMhem1Δ did not form lipid particles indicated that accumulation of squalene solely was not sufficient to initiate proliferation of lipid particles. Most importantly, these results also demonstrated that (i) squalene was not lipotoxic under the conditions tested, and (ii) organelle membranes in yeast can accommodate relatively large quantities of this non-polar lipid without compromising cellular functions. In summary, localization of squalene as described here can be regarded as an unconventional example of non-polar lipid storage in cellular membranes.     

Enzymes of the mevalonate pathway of isoprenoid biosynthesis

The mevalonate pathway accounts for conversion of acetyl-CoA to isopentenyl 5-diphosphate, the versatile precursor of polyisoprenoid metabolites and natural products. The pathway functions in most eukaryotes, archaea, and some eubacteria. Only recently has much of the functional and structural basis for this metabolism been reported. The biosynthetic acetoacetyl-CoA thiolase and HMG-CoA synthase reactions rely on key amino acids that are different but are situated in active sites that are similar throughout the family of initial condensation enzymes. Both bacterial and animal HMG-CoA reductases have been extensively studied and the contrasts between these proteins and their interactions with statin inhibitors defined. The conversion of mevalonic acid to isopentenyl 5-diphosphate involves three ATP-dependent phosphorylation reactions. While bacterial enzymes responsible for these three reactions share a common protein fold, animal enzymes differ in this respect as the recently reported structure of human phosphomevalonate kinase demonstrates. There are significant contrasts between observations on metabolite inhibition of mevalonate phosphorylation in bacteria and animals. The structural basis for these contrasts has also recently been reported. Alternatives to the phosphomevalonate kinase and mevalonate diphosphate decarboxylase reactions may exist in archaea. Thus, new details regarding isopentenyl diphosphate synthesis from acetyl-CoA continue to emerge.     

Oral Administration of Lycopene Reverses Cadmium-suppressed Body Weight Loss and Lipid Peroxidation in Rats

Cadmium (Cd) exposure has been recognized to result in a wide variety of cellular responses, including oxidative stress and body weight loss. The aim of the present study was to examine the effect of lycopene supplementation on the antioxidant defense system, lipid peroxidation (LPO) level, nitric oxide (NO), tumor necrosis factor alpha (TNF-α) production, and body weight in Cd-exposed rats. Animals were divided into four groups (n = 7): control, Cd-treated, Cd plus lycopene-treated, and lycopene-treated. Cadmium (as CdCl₂) was administrated orally for 20 days (6.6 mg kg⁻¹ day⁻¹), and lycopene (10 mg kg⁻¹ day⁻¹) was similarly administered. Lycopene administration significantly suppressed Cd-induced LPO in plasma and kidney homogenates. Lycopene also reversed Cd-decreased body weight compared to the control. Cadmium treatment had diverse effects on the antioxidant enzyme activities. Although antioxidant superoxide dismutase activity was unchanged, glutathione peroxidase activity was decreased, and catalase activity was elevated in kidney homogenates of Cd-administrated group. However, lycopene treatment reversed Cd-changed enzyme activities to the control level. Xanthine oxidase activity and TNF-α concentration were not altered by Cd administration, indicating that superoxide anion production and inflammation were not stimulated. Cadmium did not change NO levels in kidney homogenates but decreased those in plasma, and this effect was not prevented by lycopene supplementation. The result suggests that consumption of adequate levels of lycopene may be useful to prevent heavy-metal-induced LPO and body weight loss.     

Cytokinins in macroalgae

Thirty-one seaweeds were collected from the warmer KwaZulu-Natal coast and the cooler Cape waters (South Africa). Plant material was extracted with 70% ethanol supplemented with deuterium labelled standards of all known isoprenoid cytokinins. The samples were then centrifuged and purified by combined DEAE-Sephadex×octadecylsilica column and immunoaffinity chromatography and finally analysed for cytokinins by HPLC-linked mass spectrometry and a photodiode array detector. The cytokinin profiles were similar in all the macroalgae regardless of their taxonomy and growing locality. The main type of isoprenoid cytokinins present were zeatins with cis forms being more common than trans forms and isopentenyladenine (iP) derivatives. Only a few dihydrozeatin-type cytokinins were detected at very low levels in only nine species. Aromatic cytokinins were also present but at lower levels and were represented by benzyladenine (BA) and ortho- and meta-topolin derivatives. The topolins were present in greater diversity and concentrations than BA. For all the cytokinin types, the free bases, O-glucosides and nucleotides were the most common with no N-glucosides being detected and ribosides present at very low levels. The results suggest that different pathways for regulating cytokinin concentrations operate in macroalgae than in higher plants.     

生物固碳途径研究进展

生物固碳是地球碳循环过程的重要组成部分。自然界已经发现了六条天然生物固碳途径,但自然途径不仅能量利用效率低下,而且人工改造提升固碳效率难度大。随着合成生物学的发展,新的人工固碳途径不断涌现。相对于天然途径,人工固碳途径具有路线短、耗能少、原子经济性高等优点,有望在不久的将来能够替代天然固碳途径,实现固碳效率的大幅提高,是解决人类能源与环境问题的有效途径之一。主要总结了天然固碳途径和人工固碳途径的代谢原理和关键固碳酶的酶学特征,并对未来发展趋势进行展望。     

A Novel Epimerase That Converts GlcNAc-P-P-undecaprenol to GalNAc-P-P-undecaprenol in Escherichia coli O157

Escherichia coli strain O157 produces an O-antigen with the repeating tetrasaccharide unit α-d-PerNAc-α-l-Fuc-β-d-Glc-α-d-GalNAc, preassembled on undecaprenyl pyrophosphate (Und-P-P). These studies were conducted to determine whether the biosynthesis of the lipid-linked repeating tetrasaccharide was initiated by the formation of GalNAc-P-P-Und by WecA. When membrane fractions from E. coli strains K12, O157, and PR4019, a WecA-overexpressing strain, were incubated with UDP-[³H]GalNAc, neither the enzymatic synthesis of [³H]GlcNAc-P-P-Und nor [³H]GalNAc-P-P-Und was detected. However, when membrane fractions from strain O157 were incubated with UDP-[³H]GlcNAc, two enzymatically labeled products were observed with the chemical and chromatographic properties of [³H]GlcNAc-P-P-Und and [³H]GalNAc-P-P-Und, suggesting that strain O157 contained an epimerase capable of interconverting GlcNAc-P-P-Und and GalNAc-P-P-Und. The presence of a novel epimerase was demonstrated by showing that exogenous [³H]GlcNAc-P-P-Und was converted to [³H]GalNAc-P-P-Und when incubated with membranes from strain O157. When strain O157 was metabolically labeled with [³H]GlcNAc, both [³H]GlcNAc-P-P-Und and [³H]GalNAc-P-P-Und were detected. Transformation of E. coli strain 21546 with the Z3206 gene enabled these cells to synthesize GalNAc-P-P-Und in vivo and in vitro. The reversibility of the epimerase reaction was demonstrated by showing that [³H]GlcNAc-P-P-Und was reformed when membranes from strain O157 were incubated with exogenous [³H]GalNAc-P-P-Und. The inability of Z3206 to complement the loss of the gne gene in the expression of the Campylobacter jejuni N-glycosylation system in E. coli indicated that it does not function as a UDP-GlcNAc/UDP-GalNAc epimerase. Based on these results, GalNAc-P-P-Und is synthesized reversibly by a novel GlcNAc-P-P-Und epimerase after the formation of GlcNAc-P-P-Und by WecA in E. coli O157.     

A high-performance liquid chromatography method for the analysis of intermediates of the deoxyxylulose phosphate pathway

A sensitive and versatile ion pair radio high-performance liquid chromatography (HPLC) method for the investigation of the deoxyxylulose phosphate (DXP) pathway has been developed, allowing the simultaneous separation of phosphorylated, nonphosphorylated, and nucleotide moieties bearing intermediates. Moreover, this method addresses the problem of separating the isomers isopentenyl diphosphate (IDP) and dimethylallyl diphosphate (DMADP). Because the majority of the intermediates of this isoprenoid pathway lack a chromophore, the combination with an on-line radiodetector provides a highly sensitive tool for their detection. Chromoplasts isolated from Capsicum annuum and Narcissus pseudonarcissus served as model systems for the testing of the analytical procedures after the application of radiolabeled precursors. This HPLC system, which represents an improvement in analytical methods developed for the analysis of the mevalonic acid pathway, should be easily adaptable to other plant and bacterial systems and should permit further elucidation of the regulatory mechanisms that control the flow of intermediates through the DXP pathway and the coordination with related metabolic pathways. Moreover, the system can serve as an analytical tool in the screening for inhibitors of this pathway, allowing the development of new antibiotics as well as herbicides, because this pathway is absent in vertebrates.     

Supply of precursors for carotenoid biosynthesis in plants

Carotenoids are isoprenoids of industrial and nutritional interest produced by all photosynthetic organisms, including plants. Too often, the metabolic engineering of plant carotenogenesis has been obstructed by our limited knowledge on how the endogenous pathway interacts with other related metabolic pathways, particularly with those involved in the production of isoprenoid precursors. However, recent discoveries are providing new insights into this field. All isoprenoids derive from prenyl diphosphate precursors. In the case of carotenoids, these precursors are produced predominantly by the methylerythritol 4-phosphate (MEP) pathway in plants. This review focuses on the progress in our understanding of how manipulation of the MEP pathway impacts carotenoid biosynthesis and on the discoveries underlining the central importance of coordinating the supply of MEP-derived precursors with the biosynthesis of carotenoids and other derived isoprenoids.     

Lycopene and ellagic acid prevent testicular apoptosis induced by cisplatin

The aim of this study was to investigate the possible protective effects of lycopene (LC) and ellagic acid (EA) on cisplatin (CP)-induced testicular apoptosis in male rats. The control group was treated with placebo; LC, EA and CP groups were given alone LC, EA and CP, respectively; the CP + LC group was treated with a combination of CP and LC; and the CP + EA group was treated with a combination of CP and EA. Although CP significantly increased the number of Bax-positive (apoptotic) cells it had no effect on the number of Bcl-2-positive (antiapoptotic) cells compared with the control group. Administration of CP caused significant increase in lipid peroxidation and nonsignificant decrease in superoxide dismutase (SOD) activity along with some histopathological lesions in testicular tissue. However, combined treatments of LC or EA in addition to CP tended to prevent the CP-induced testicular apoptosis, histopathological lesions and lipid peroxidation.     

Effects of farnesol on the physical properties of DMPC membranes

Farnesol interacts with membranes in a wide variety of biological contexts, yet our understanding of how it affects lipid bilayers is not yet complete. This study investigates how the 15-carbon isoprenoid, farnesol, influences the phase behaviour, lateral organization, and mechanical stability of dimyristol phosphatidylcholine (DMPC) model membranes. Differential scanning calorimetry (DSC) of multilamellar DMPC-farnesol mixtures (up to 26 mol% farnesol) demonstrates how this isoprenoid lowers and broadens the gel-fluid phase transition. A gel-fluid coexistence region becomes progressively more dominant with increasing farnesol concentration and at concentrations of and greater than 10.8 mol%, an upper transition emerges at about 35 °C. Atomic force microscopy images of supported farnesol-DMPC bilayers containing 10 and 20 mol% farnesol provide structural evidence of gel-fluid coexistence around the main transition. Above this coexistence region, membranes exhibit homogeneous lateral organization but at temperatures below the main gel-fluid coexistence region, another form of phase coexistence is observed. The solid nature of the gel phase is confirmed using micropipette aspiration. The combined thermodynamic, structural, and mechanical data allow us to construct a phase diagram. Our results show that farnesol preferentially partitions into the fluid phase and induces phase coexistence in membranes below the main transition of the pure lipid.     

Effects of lipids on n-alkane attenuation in media supporting oil-utilizing microorganisms from the oily Arabian Gulf coasts

The Arabian Gulf is one of the most extensively oil-polluted areas of the world. The major objectives of this work were to study whether hydrocarbon-utilizing microorganisms indigenous to that area would readily accumulate added lipids, and whether this might affect their hydrocarbon consumption potential. Two prokaryotes, Arthrobacter nicotianae KCC B35 and the unidentified organisms KCC B6, as well as one eukaryote, Candida parapsilosis KCC Y1, were selected for this study. Biomass samples of the test organisms were incubated in an inorganic medium containing various concentrations of cholesterol, stearic acid, triolein or egg-phospholipids. The results revealed that all lipid classes were readily accumulated by the three test organisms. In addition, biomass samples were incubated for 6 h in an inorganic medium containing mixtures of individual lipid classes and either n-octadecane or n-docosane. The cells were removed and the residual alkanes in the medium were quantitatively recovered and analyzed by GLC. The results showed that out of the tested lipid classes only stearic acid exhibited a common stimulatory effect on the consumption of both n-alkanes by all test organisms. Other lipid classes were either inhibitory or had less pronounced effects than stearic acid.     

Structural Influence of Sugar and Aglycon Moieties on the Acetolysis of Some Glycosylamines

The acetolysis was applied to a novel cleavage of N-p-tolyl-β-d-glycosylamines in glucogalacto- and manno-configurations and their 2-acetamido-2-deoxy sugars. The results were compared with the acetolysis of furanosyl- and pyranosylnucleosides. N-Acetyl-p-toluidine was isolated as crystals in up to 92% yield in the acetolysis of N-P-tolyl-β-d-glycosylamines of neutral sugars and in 1.5~2.0% yields in that of 2-acetamido-2-deoxy sugars. The stability of glycosylamine linkages against the acetolysis was found in the following order: pyranosylnucleosides (the most stable) > N-P-tolyl-β-d-glycosylamine of 2-acetamido-2-deoxy sugars > furanosylnucleosides > N-P-tolyl-β-d-glycosylamines of neutral sugars (the most unstable).