产番茄红素基因工程菌的研究进展

番茄红素是一种重要的类胡萝卜素,具有许多生物功能和生物活性,尤其在保护人类健康方面起着重要的作用。随着番茄红素生物合成途径的阐明及其相关基因的克隆,运用基因工程手段调控番茄红素的合成已经成为可能。本文首先综述了番茄红素生物合成途径及合成途径中相关基因的克隆,然后对近年来构建的番茄红素基因工程菌进行了全面的总结,包括：运用DNA重组技术使异源微生物大肠杆菌、酵母等生产番茄红素,以及通过过表达特定基因从而提高霉菌等产番茄红素的量,最后分析了改造过程中存在的主要问题,并展望了未来的研究方向。     

The nitrogen-fixing symbiotic bacterium Mesorhizobium loti has and expresses the gene encoding pyridoxine 4-oxidase involved in the degradation of vitamin B6

The gene product of mll6785 of a nitrogen-fixing symbiotic bacterium Mesorhizobium loti MAFF303099 was identified as pyridoxine 4-oxidase, the first enzyme in the vitamin B6-degradation pathway. The gene was cloned and ligated into pET-21a+. Escherichia coli BL21(DE3) was co-transformed with the constructed plasmid plus pKY206 containing groESL genes encoding chaperonins. The overexpressed protein was purified to homogeneity by the ammonium sulfate fractionation and three chromatography steps. The enzymatic properties of the purified protein, such as K(m) values for pyridoxine (213+/-19 microM) and oxygen (78+/-10 microM), were compared to those of pyridoxine 4-oxidase from two bacteria with known vitamin B6-degradation pathway. M. loti grown in a Rhizobium medium showed the enzyme activity. The results suggest that M. loti also contains the degradation pathway of vitamin B6.     

人B7—1和B7—2cDNA的克隆及鉴定

目的：为探索性构建全新型重组人B7－PE40绿脓杆菌外毒素融合蛋白以长期诱导免疫耐受,本研究从急性B淋巴细胞白血病细胞株Raji中隆N－末端分别缺失34和16个氨基酸的人B7－1和B7－2基因胞外区,并构建含此基因的重组质粒。方法：根据B7－1和B7－2基因序列设计合成可增B7－1和B7－2cDNA的特异性引物,用RT－PCR的方法从Raji细胞总RNA中扩增B7－1和B7－2cDNA,并克隆至pGEM－T载体中,经酶切鉴定后再进行序列分析。结果和结论：从Raji细胞中扩增出预期 的624和675bp的B7－1和B7－2cDNA,将其克隆至pGEM-T载体中,分别经EcoRI/HindⅢ和BamHI/SphI双酶切电泳和序列分析确证,为进一步构建人B7－PE40外毒素融合蛋白奠定了基础。     

Synthesis of vitamin D3 analogues with A-ring modifications to directly measure vitamin D levels in biological samples

C-3-substituted 25-hydroxyvitamin D₃ analogues were synthesized as tools to directly measure levels of vitamin D in biological samples. The strategy involves vinyloxycarbonylation of the 3β-hydroxy group and formation of a carbamate bond with a hydroxyl or amino group at the end of the alkyl chain. Biotinylated conjugates of synthesized derivatives were generated to be linked with vitamin D binding protein (DBP). The spacer group present in the alkyl chain is important in the binding of antibodies to the analogue–DBP complex. When compared to 25-hydroxyvitamin D₃-DBP, the binding of some antibodies to the analogue–DBP complex of the 25-hydroxyvitamin D₃ derivative 10 that posses an 8-aminoctyl alkyl chain is significantly reduced, but this analogue displaced [26,27-³H]-25-hydroxyvitamin D₃ from DBP. In contrast, the 8-hydroxyoctyl alkyl chain analogue 9 showed less displacement.     

1,25-Dihydroxyvitamin D3 suppresses inflammation-induced expression of plasminogen activator inhibitor-1 by blocking nuclear factor-κB activation

Plasminogen activator inhibitor (PAI)-1 is a major fibrinolytic inhibitor. High PAI-1 is associated with increased renal and cardiovascular disease risk. Previous studies demonstrated PAI-1 down-regulation by 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), but the molecular mechanism remains unknown. Here we show that exposure of mouse embryonic fibroblasts to TNFα or LPS led to a marked induction of PAI-1, which was blunted by 1,25(OH)₂D₃, NF-κB inhibitor or p65 siRNA, suggesting the involvement of NF-κB in 1,25(OH)₂D₃-induced repression. In mouse Pai-1 promoter a putative cis-κB element was identified at −299. EMSA and ChIP assays showed that TNF-α increased p50/p65 binding to this κB site, which was disrupted by 1,25(OH)₂D₃. Luciferase reporter assays showed that PAI-1 promoter activity was induced by TNFα or LPS, and the induction was blocked by 1,25(OH)₂D₃. Mutation of the κB site blunted TNFα, LPS or 1,25(OH)₂D₃ effects. 1,25(OH)₂D₃ blocked IκBα degradation and arrested p50/p65 nuclear translocation. In mice LPS stimulated PAI-1 expression in the heart and macrophages, and the stimulation was blunted by pre-treatment with a vitamin D analog. Together these data demonstrate that 1,25(OH)₂D₃ down-regulates PAI-1 by blocking NF-κB activation. Inhibition of PAI-1 production may contribute to the reno- and cardio-protective effects of vitamin D.     

紫杉醇生物合成途径及调控研究进展

本文综述了紫杉醇的生物合成途径、代谢调控及基因工程方面的研究进展,总结了代谢调控与基因工程方法提高红豆杉属植物细胞培养紫杉醇合成量的研究状况,并在探讨生物合成途径理论的基础上,对紫杉醇生物合成的限速步骤进行了阐述,指出解决侧链合成的根速步骤问题会显著提高紫杉醇的生物合成量。     

Red HE7B degradation using desulfonation by Pseudomonas desmolyticum NCIM 2112

Red HE7B (RHE7B, 100 mg l⁻¹), a sulfonated azo dye, was decolorized at static condition by Pseudomonas desmolyticum NCIM 2112 in 72 h with 71% reduction in chemical oxygen demand (COD). Extracellular lignin peroxidase (LiP) has played a crucial role in breakdown of the dye by asymmetric cleavage and reductases in the initial 24 h incubation to break azo bonds of some dye molecules. Dye also induced the activity of aminopyrine N-demethylase, one of the enzymes of mixed function oxidase system. Decolorization and degradation were analyzed by using UV–vis and high-pressure liquid chromatography (HPLC). The Fourier transform infrared spectroscopy (FTIR) analysis revealed that P. desmolyticum preferred C–N and SO bonds to break down the RHE7B. GC–MS identification of 8-amino-naphthalene-1,3,6,7-tetraol and 2-hydroxyl-6-oxalyl-benzoic acid as final metabolites supports the degradation of RHE7B by desulfonation before and after ring cleavage. Aerobic degradation of amines and reduced phytotoxicity increased the applicability of this microorganism for dye removal.

Scientific relevance of the paper

This is the first report on degradation of Red HE7B by oxidative enzymes and on further degradation by desulfonation before and after ring cleavage.     

Functional analysis of cholesterol biosynthesis by RNA interference

Inborn errors of cholesterol biosynthesis caused by dysfunctionality of single enzymes are known to cause severe malformation syndromes like X-linked chondrodysplasia punctata (CDPX2), CHILD syndrome or Smith–Lemli–Opitz-syndrome (SLOS). In this study we established the method of RNA interference (RNAi) for analyzing the molecular mechanisms underlying disrupted cholesterol biosynthesis. For different genes involved in the cholesterol biosynthesis pathway-NAD(P) dependent steroid dehydrogenase-like (NSDHL), 17-beta hydroxysteroid dehydrogenase type 7 (HSD17B7) and emopamil binding protein (EBP)-shRNA sequences were designed and tested for their effectiveness. For a better comparability of the experiments and to avoid different transfection efficiencies, examined shRNA sequences which reached a knock down of at least 80% were stably transfected in a HeLa cell line with a tetracycline-regulated expression (HeLa T-REx). These stable transfected cell lines represent novel tools for the analysis of cholesterol biosynthesis.     

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.     

异戊二烯生物合成研究进展

异戊二烯(isoprene),又名2-甲基-1、3-丁二烯,是最简单的类异戊二烯化合物,是橡胶的重要前体物质,在精细化工如香料、新型农药等方面应用广泛。异戊二烯主要依赖化石燃料合成,但生产成本较高、易污染环境,生物法合成异戊二烯具有巨大的潜在应用价值,本文综述了生物法合成异戊二烯的主要途径与研究进展。     

Formation of a heterooctameric complex between aspartate α-decarboxylase and its cognate activating factor, PanZ, is CoA-dependent

The existence of a fifth essential protein for pantothenate biosynthesis in some enteric bacteria has recently been reported by Stuecker et al. [10] and Nozaki et al. (in press) [9]. This protein, PanZ, catalyses the activation of the PanD zymogen to form ADC and is essential for prototrophic growth. In this paper, we characterise the interaction of PanZ with coenzyme A and a constitutively inactive mutant of PanD using a combination of isothermal titration calorimetry and mass spectrometry. These approaches reveal that the two proteins interact with nanomolar affinity in a CoA-dependent fashion to form a heterooctameric complex.     

ERK7的结构性活化与功能

ERK7是细胞外信号调节激酶家族中的新成员.尽管ERK7激酶的活化环上含有ERK家族成员共有的TEY基序,但是在活化上与其它ERK家族成员截然不同,ERK7无需典型活化ERK的细胞外刺激或JNK和p38激酶活化物而发生自磷酸化并足以使其在缺乏上游激酶时发生活化,且发现ERK7中非激酶结构域的C端区域调节其结构性活化、核定位、生长抑制.此外,N端的20个氨基酸作为ERK7降解的首要决定因素调节ERK7的表达.新近研究表明ERK7与乳腺肿瘤、神经元分化、胚胎发生密切相关.本文就ERK7的结构特性活化、调控及功能等作了综述.