紫杉醇的生物合成途径和参与催化的酶

通过了解紫杉醇生物合成途径和途径中的催化酶 ,特别是催化限速步骤的关键酶以及这些酶的编码基因 ,从而从分子水平对该途径实施人工操纵 ,将是发展先进的生物工艺大量生产紫杉醇的前提。文章介绍了紫杉醇生物合成途径和催化酶类的研究进展 ,并简略讨论了紫杉醇生物合成研究领域所面临的问题     

Engineering Salidroside Biosynthetic Pathway in Hairy Root Cultures of Rhodiola crenulata Based on Metabolic Characterization of Tyrosine Decarboxylase

Tyrosine decarboxylase initializes salidroside biosynthesis. Metabolic characterization of tyrosine decarboxylase gene from Rhodiola crenulata (RcTYDC) revealed that it played an important role in salidroside biosynthesis. Recombinant 53 kDa RcTYDC converted tyrosine into tyramine. RcTYDC gene expression was induced coordinately with the expression of RcUDPGT (the last gene involved in salidroside biosynthesis) in SA/MeJA treatment; the expression of RcTYDC and RcUDPGT was dramatically upregulated by SA, respectively 49 folds and 36 folds compared with control. MeJA also significantly increased the expression of RcTYDC and RcUDPGT in hairy root cultures. The tissue profile of RcTYDC and RcUDPGT was highly similar: highest expression levels found in stems, higher expression levels in leaves than in flowers and roots. The gene expressing levels were consistent with the salidroside accumulation levels. This strongly suggested that RcTYDC played an important role in salidroside biosynthesis in R. crenulata. Finally, RcTYDC was used to engineering salidroside biosynthetic pathway in R. crenulata hairy roots via metabolic engineering strategy of overexpression. All the transgenic lines showed much higher expression levels of RcTYDC than non-transgenic one. The transgenic lines produced tyramine, tyrosol and salidroside at higher levels, which were respectively 3.21–6.84, 1.50–2.19 and 1.27–3.47 folds compared with the corresponding compound in non-transgenic lines. In conclusion, RcTYDC overexpression promoted tyramine biosynthesis that facilitated more metabolic flux flowing toward the downstream pathway and as a result, the intermediate tyrosol was accumulated more that led to the increased production of the end-product salidroside.     

植物花青素生物合成相关基因的研究及应用

花青素是决定植物花色的主要色素,使大多数花呈现从红到蓝的系列变化,是花色研究和开发的重点,并具有重要的营养和药用作用。目前花青素生物合成途径已日益清楚,并已分离到大量的相关酶和基因,并获得了一批具有商业价值的转基因植物新品种。本文重点介绍了花青素合成途径中关键基因的研究成果,并概述了国内外花青素基因在植物基因工程中的应用研究进展情况,同时对花青素基因的研究应用前景和发展趋势作一展望。     

Polyamine Biosynthetic Enzymes in Chlorella: Characterization of Ornithine and Arginine Decarboxylase

In a Chlorella culture grown asynchronously under autotrophicconditions, two biosynthetic enzymes of putrescine—ornithinedecarboxylase (ODC) and arginine decarboxylase (ADC)—weredetected. Both enzymes require pyridoxal phosphate and dithiothreitolfor their activity but differ in their optimal pH, the ionicstrength of their buffer, temperature of inactivation, and Km.In addition, L-canaline was found to inhibit the activity ofODC but not that of ADC. During the logarithmic phase of growth,ODC activity increased sharply, then decreased before the onsetof the stationary phase. ADC activity changed only slightlyduring growth. ³The work was performed in partial fulfillment of the requirementsfor the Ph.D. Thesis of E.C. (Received September 25, 1982; Accepted June 1, 1983)     

假单胞菌生物合成L-半胱氨酸途径中脱巯基酶的研究

通过PCR方法扩增得到假单胞菌TS1138L-半胱氨酸脱巯基酶基因（cd）,将其克隆至pBlueseript SKII载体,测定了含有L-半胱氨酸脱巯基酶基因的1．2kbDNA片段序列,并与其它菌株的脱巯基酶基因进行了同源性比较;同时,将其克隆至表达载体pET-21a（＋）,IPTG诱导表达,表达产物经Ni-NTA柱亲合层析后,得到纯化的重组蛋白。利用脱巯基酶的活性染色方法对重组表达的L-半胱氨酸脱巯基酶进行了鉴定,并探讨了L-半胱氨酸脱巯基酶的酶学性质,以及在生物转化合成L-半胱氨酸途径中的关键作用。     

Characterization of the Enzyme CbiH60 Involved in Anaerobic Ring Contraction of the Cobalamin (Vitamin B12) Biosynthetic Pathway

The anaerobic pathway for the biosynthesis of cobalamin (vitamin B₁₂) has remained poorly characterized because of the sensitivity of the pathway intermediates to oxygen and the low activity of enzymes. One of the major bottlenecks in the anaerobic pathway is the ring contraction step, which has not been observed previously with a purified enzyme system. The Gram-positive aerobic bacterium Bacillus megaterium has a complete anaerobic pathway that contains an unusual ring contraction enzyme, CbiH₆₀, that harbors a C-terminal extension with sequence similarity to the nitrite/sulfite reductase family. To improve solubility, the enzyme was homologously produced in the host B. megaterium DSM319. CbiH₆₀ was characterized by electron paramagnetic resonance and shown to contain a [4Fe-4S] center. Assays with purified recombinant CbiH₆₀ demonstrate that the enzyme converts both cobalt-precorrin-3 and cobalt factor III into the ring-contracted product cobalt-precorrin-4 in high yields, with the latter transformation dependent upon DTT and an intact Fe-S center. Furthermore, the ring contraction process was shown not to involve a change in the oxidation state of the central cobalt ion of the macrocycle.     

Production and Characterization of a New Specific Antiserum Against the Taurine Putative Biosynthetic Enzyme Cysteine Sulfinate Decarboxylase

Abstract: We have shown previously that cysteine sulfinate decarboxylase (CSD), the putative biosynthetic enzyme of taurine in the brain, is identical to the liver enzyme according to biochemical, kinetic, and immunochemical criteria. In the present work, CSD was purified in its native form from rat liver. The purification was performed in eight steps, which included conventional chromatography (diethylaminoethyl cellulose, hydroxylapatite), followed by HPLC (hydrophobic, adsorption, and ion-exchange HPLC). The purification factor was 11,000, and the final yield was around 2%. The procedure led to the enrichment of a protein, the molecular mass of which was 51,000 daltons as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The final fraction was more than 90% homogeneous. By using this fraction as the antigen, an antiserum was raised in rabbit that (a) quantitatively immunoprecipitated CSD activity from liver and brain extract, and (b) immunolabeled one band (51,000 daltons) on immunoblots of partially purified fractions from liver. Enrichment of CSD specific activity and that of the protein immunolabeled by the antiserum for a given step, e.g., hydrophobic HPLC, were consistently parallel. The antiserum was used to carry out CSD immunocytochemistry in cerebellum. Numerous small cells were labeled in the Purkinje cell layer, the granular layer, and the white matter. In the molecular layer, Bergmann radial fibers were im munostained. The Purkinje and stellate cells were devoid of any labeling at the cell body and terminal levels. The antiserum appears to be specific for CSD and suitable for immunocytochemical visualization of CSD in the brain.     

Characterization of the CDP-2-Glycerol Biosynthetic Pathway in Streptococcus pneumoniae

Capsule polysaccharide (CPS) plays an important role in the virulence of Streptococcus pneumoniae and is usually used as the pneumococcal vaccine target. Glycerol-2-phosphate is found in the CPS of S. pneumoniae types 15A and 23F and is rarely found in the polysaccharides of other bacteria. The biosynthetic pathway of the nucleotide-activated form of glycerol-2-phosphate (NDP-2-glycerol) has never been identified. In this study, three genes (gtp1, gtp2, and gtp3) from S. pneumoniae 23F that have been proposed to be involved in the synthesis of NDP-2-glycerol were cloned and the enzyme products were expressed, purified, and assayed for their respective activities. Capillary electrophoresis was used to detect novel products from the enzyme-substrate reactions, and the structure of the product was elucidated using electrospray ionization mass spectrometry and nuclear magnetic resonance spectroscopy. Gtp1 was identified as a reductase that catalyzes the conversion of 1,3-dihydroxyacetone to glycerol, Gtp3 was identified as a glycerol-2-phosphotransferase that catalyzes the conversion of glycerol to glycerol-2-phosphate, and Gtp2 was identified as a cytidylyltransferase that transfers CTP to glycerol-2-phosphate to form CDP-2-glycerol as the final product. The kinetic parameters of Gtp1 and Gtp2 were characterized in depth, and the effects of temperature, pH, and cations on these two enzymes were analyzed. This is the first time that the biosynthetic pathway of CDP-2-glycerol has been identified biochemically; this pathway provides a method to enzymatically synthesize this compound.Capsule polysaccharide (CPS) of Gram-positive bacteria, external to the cell wall, provides resistance to phagocytosis. CPS in Streptococcus pneumoniae is the most important virulence factor and the target of pneumococcal vaccines (2). Ninety individual CPS serotypes have been recognized so far by immunological and chemical techniques (9). Each has a structurally distinct CPS, composed of repeating oligosaccharide units joined by glycosidic linkages. The components of the repeat units are transferred from nucleoside diphosphate (NDP) derivatives. Among the 54 identified CPS structures, several sugars and related compounds have been found. Seven NDP-monosaccharide precursors (glucopyranose, N-acetylglucosamine, galactopyranose, N-acetylgalactosamine, 2-acetamido-4-amino-2,4,6-trideoxy-d-galactopyranose, ribitol-phosphate, and phosphorylcholine) are available from housekeeping metabolic pathways, and the biosynthetic genes for 14 NDP-monosaccharide precursors were found in the pneumococcal cps loci. Among the 14 components, the pathways of five (NDP-d-mannitol, NDP-d-arabinitol, NDP-ribofuranose [Rib], CDP-glycerol [CDP-Gro], and NDP-2-glycerol) are putative and have not yet been identified (1, 4).Glycerol-2-phosphate is rarely present in bacteria and has been found in S. pneumoniae types 15A and 23F. The NDP-2-glycerol biosynthetic pathway has been proposed to include three enzymes: Gtp1, Gtp2, and Gtp3. Gtp3 has been proposed to be a glyceraldehyde-2-phosphotransferase and to be involved in the synthesis of glyceraldehyde-2-phosphate from glyceraldehyde. Gtp1, a putative dehydrogenase, has been proposed to be responsible for the conversion of glyceraldehyde-2-phosphate to glycerol-2-phosphate. The last step of the synthesis of CDP-2-glycerol is catalyzed by the putative glycerol-2-phosphate cytidyltransferase Gtp2 (14). The three genes, gtp1, gtp2, and gtp3, have also been found to be present in the cps loci of S. pneumoniae serotypes 15B, 15C, 15F, 23A, 23B, 28A, and 28F (4). However, the biosynthetic pathway for NDP-2-glycerol has never been identified by molecular and biochemical methods.In this study, we found that the enzymes were not reactive by the previously proposed CDP-2-glycerol biosynthetic pathway. Therefore, a new pathway was proposed, and the three enzymes, Gtp1, Gtp2, and Gtp3, were identified and confirmed biochemically as 1,3-dihydroxyacetone/glyceraldehyde reductase, glycerol-2-phosphate cytidylyltransferase, and glycerol-2-phosphotransferase, respectively. This is the first report on the characterization of the CDP-2-glycerol biosynthetic pathway.     

Characterization of the Biosynthetic Pathway of Cellulose from Glucose and Fructose in Acetobacter xylinum

For characterization of the biosynthetic pathway of cellulose in a cellulose-producing Acetobacter xylinum strain BPR2001, the activities of several enzymes were measured. The activity of phosphoglucose isomerase catalyzing the conversion of fructose-6- phosphate into glucose-6-phosphate was greatly increased by fructose in the medium. The UDP-glucose pyrophosphorylase activity catalyzing the synthesis of UDP-glucose was very high in strain BPR2001, consistent with the idea that this is the key enzyme in cellulose biosynthesis. Strain BPR2001 was found to have a fructose-specific phosphoenolpyruvate-dependent phosphotransferase system (PTS).     

The Structure of NtdA,a Sugar Aminotransferase Involved in the Kanosamine Biosynthetic Pathway in Bacillus subtilis,Reveals a New Subclass of Aminotransferases

NtdA from Bacillus subtilis is a sugar aminotransferase that catalyzes the pyridoxal phosphate-dependent equatorial transamination of 3-oxo-α-d-glucose 6-phosphate to form α-d-kanosamine 6-phosphate. The crystal structure of NtdA shows that NtdA shares the common aspartate aminotransferase fold (Type 1) with residues from both monomers forming the active site. The crystal structures of NtdA alone, co-crystallized with the product α-d-kanosamine 6-phosphate, and incubated with the amine donor glutamate reveal three key structures in the mechanistic pathway of NtdA. The structure of NtdA alone reveals the internal aldimine form of NtdA with the cofactor pyridoxal phosphate covalently attached to Lys-247. The addition of glutamate results in formation of pyridoxamine phosphate. Co-crystallization with kanosamine 6-phosphate results in the formation of the external aldimine. Only α-d-kanosamine 6-phosphate is observed in the active site of NtdA, not the β-anomer. A comparison of the structure and sequence of NtdA with other sugar aminotransferases enables us to propose that the VI_β family of aminotransferases should be divided into subfamilies based on the catalytic lysine motif.     

Genetic Characterization of the Carotenoid Biosynthetic Pathway in Methylobacterium extorquens AM1 and Isolation of a Colorless Mutant

Genomic searches were used to reconstruct the putative carotenoid biosynthesis pathway in the pink-pigmented facultative methylotroph Methylobacterium extorquens AM1. Four genes for putative phytoene desaturases were identified. A colorless mutant was obtained by transposon mutagenesis, and the insertion was shown to be in one of the putative phytoene desaturase genes. Mutations in the other three did not affect color. The tetracycline marker was removed from the original transposon mutant, resulting in a pigment-free strain with wild-type growth properties useful as a tool for future experiments.     

Identification, Cloning, and Characterization of a Lactococcus lactis Branched-Chain α-Keto Acid Decarboxylase Involved in Flavor Formation

The biochemical pathway for formation of branched-chain aldehydes, which are important flavor compounds derived from proteins in fermented dairy products, consists of a protease, peptidases, a transaminase, and a branched-chain α-keto acid decarboxylase (KdcA). The activity of the latter enzyme has been found only in a limited number of Lactococcus lactis strains. By using a random mutagenesis approach, the gene encoding KdcA in L. lactis B1157 was identified. The gene for this enzyme is highly homologous to the gene annotated ipd, which encodes a putative indole pyruvate decarboxylase, in L. lactis IL1403. Strain IL1403 does not produce KdcA, which could be explained by a 270-nucleotide deletion at the 3′ terminus of the ipd gene encoding a truncated nonfunctional decarboxylase. The kdcA gene was overexpressed in L. lactis for further characterization of the decarboxylase enzyme. Of all of the potential substrates tested, the highest activity was observed with branched-chain α-keto acids. Moreover, the enzyme activity was hardly affected by high salinity, and optimal activity was found at pH 6.3, indicating that the enzyme might be active under cheese ripening conditions.     

Carbon Balance of a Mannitol Fermentation and the Biosynthetic Pathway

The carbon balance was determined for a fermentation in which mannitol is produced from glucose by an Aspergillus species. The products found were: cells (17% of carbon input), CO(2) (26%), mannitol (35%), glycerol (10%), erythritol (2.5%), glycogen (1%), and unidentified compounds (8%). Thus, 92% of the carbon input was accounted for. Cell-free enzyme studies showed that mannitol was synthesized via the reduction of fructose-6-phosphate and not by the direct reduction of fructose. If the cell yield from glucose was assumed to be 50% and the theoretical conversion efficiency from glucose to polyols was 90%, as calculated from the energy balance, then 34% of the glucose carbon was used for growth and 53% was used for polyol formation.     

Detection,characterization, and phytotoxic activity of the nucleoside antibiotics,Blasticidin S and 5-Hydroxylmethyl-Blasticidin S

Screening for herbicidal compounds carried out on culture broths of Streptomyces strains isolated from soil resulted in the detection of potent phytotoxic activity. The active principles were isolated, and identified as the nucleoside antibiotics Blasticidin S (Bl-S) and 5-Hydroxymethyl-Blasticidin S (H-M-Bl-S). Bl-S was more active than H-M-Bl-S on seedling germination in petri dishes and in postemergence greenhouse tests. Moreover both antibiotics were more phytotoxic to dicotyledonous than to monocotyledonous plants. The increased sensitivity of dicots was confirmed in carrot and rice cell cultures. Both compounds also inhibited [¹⁴C]amino acid incorporation into proteins of rice and carrot cell cultures. Protein synthesis was more affected in carrot than rice.     

蓝粒小麦花青素生物合成途径中的二氢黄酮醇4-还原酶基因的分子克隆

蓝色色素在蓝粒小麦种子糊粉层中的生物合成途径的分子生物学机制至今仍不清楚.应用RT-PCR和RACE方法从蓝粒小麦正在发育的种子中克隆到一个编码二氢黄酮醇4-还原酶的基因(DFR).推测其为花青素生物合成途径中的一个关键基因,且与蓝粒小麦中蓝色色素形成密切相关;其开放阅读框编码一个包含354个氨基酸残基的多肽,与一些从其他植物中已克隆到的DFR有很高的同源性:大麦(94%)、水稻(83%)、玉米(84%).从长穗偃麦草(2n=70)、蓝粒小麦、浅蓝粒小麦自交产生的白粒后代小麦以及中国春的基因组中分别分离到一个全长DFR序列.经聚类分析表明DFR cDNA核甘酸序列与从中国春基因组中克隆的DFR具有100%的同源性,且与长穗偃麦草、蓝粒小麦、白粒小麦基因组中分离的DFR均有很高的同源性.4个DFR基因组DNA均含有3个内含子,且它们之间的差异主要在内含子区,表明该基因在进化上很保守.经Southern杂交分析,DFR在小麦中至少有3～5个拷贝,不同小麦材料间未见明显差异,但与长穗偃麦草有明显差异,属于一个DFR超基因家族.Northern分析表明该DFR在蓝粒和白粒种子的不同发育时期的表达存在明显差异,都在开花后大约18 d表达最强,在同一时期的蓝白种子中,DFR在蓝粒种子中的表达量高于白粒.DFR转录本在小麦和长穗偃麦草的幼叶中积累多,但在芽鞘中的表达显著低于幼叶中;在小麦的根和长穗偃麦草的发育种子中均未检测到DFR的表达.推测蓝粒小麦中可能存在调控DFR在蓝粒小麦中表达的调控基因,类似于玉米花青素合成途径中的调节基因.     

蓝粒小麦花青素生物合成途径中的二氢黄酮醇4-还原酶基因的分子克隆

蓝色色素在蓝粒小麦种子糊粉层中的生物合成途径的分子生物学机制至今仍不清楚。应用RT—PCR和RACE方法从蓝粒小麦正在发育的种子中克隆到一个编码二氢黄酮醇4-还原酶的基因(DFR)。推测其为花青素生物合成途径中的一个关键基因,且与蓝粒小麦中蓝色色素形成密切相关;其开放阅读框编码一个包含354个氨基酸残基的多肽,与一些从其他植物中已克隆到的DFR有很高的同源性：大麦(94％)、水稻(83％)、玉米(84％)。从长穗偃麦草(2n=70)、蓝粒小麦、浅蓝粒小麦自交产生的白粒后代小麦以及中国春的基因组中分别分离到一个全长DFR序列。经聚类分析表明DFR cDNA核甘酸序列与从中国春基因组中克隆的DFR具有100％的同源性,且与长穗偃麦草、蓝粒小麦、白粒小麦基因组中分离的DFR均有很高的同源性。4个DFR基因组DNA均含有3个内含子,且它们之间的差异主要在内含子区,表明该基因在进化上很保守。经Southern杂交分析,DFR小麦中至少有3-5个拷贝,不同小麦材料间未见明显差异,但与长穗偃麦草有明显差异,属于一个DFR超基因家族。Northern分析表明该DFR在蓝粒和白粒种子的不同发育时期的表达存在明显差异,都在开花后大约18d表达最强,在同一时期的蓝白种子中,DFR蓝粒种子中的表达量高于白粒。DFR转录本在小麦和长穗偃麦草的幼叶中积累多,但在芽鞘中的表达显著低于幼叶中;在小麦的根和长穗偃麦草的发育种子中均未检测到DFR的表达。推测蓝粒小麦中可能存在调控DFR在蓝粒小麦中表达的调控基因,类似于玉米花青素合成途径中的调节基因。     

A Branched Biosynthetic Pathway Is Involved in Production of Roquefortine and Related Compounds in Penicillium chrysogenum

Profiling and structural elucidation of secondary metabolites produced by the filamentous fungus Penicillium chrysogenum and derived deletion strains were used to identify the various metabolites and enzymatic steps belonging to the roquefortine/meleagrin pathway. Major abundant metabolites of this pathway were identified as histidyltryptophanyldiketopiperazine (HTD), dehydrohistidyltryptophanyldi-ketopiperazine (DHTD), roquefortine D, roquefortine C, glandicoline A, glandicoline B and meleagrin. Specific genes could be assigned to each enzymatic reaction step. The nonribosomal peptide synthetase RoqA accepts L-histidine and L-tryptophan as substrates leading to the production of the diketopiperazine HTD. DHTD, previously suggested to be a degradation product of roquefortine C, was found to be derived from HTD involving the cytochrome P450 oxidoreductase RoqR. The dimethylallyltryptophan synthetase RoqD prenylates both HTD and DHTD yielding directly the products roquefortine D and roquefortine C without the synthesis of a previously suggested intermediate and the involvement of RoqM. This leads to a branch in the otherwise linear pathway. Roquefortine C is subsequently converted into glandicoline B with glandicoline A as intermediates, involving two monooxygenases (RoqM and RoqO) which were mixed up in an earlier attempt to elucidate the biosynthetic pathway. Eventually, meleagrin is produced from glandicoline B involving a methyltransferase (RoqN). It is concluded that roquefortine C and meleagrin are derived from a branched biosynthetic pathway.     

PqqD Is a Novel Peptide Chaperone That Forms a Ternary Complex with the Radical S-Adenosylmethionine Protein PqqE in the Pyrroloquinoline Quinone Biosynthetic Pathway

Pyrroloquinoline quinone (PQQ) is a product of a ribosomally synthesized and post-translationally modified pathway consisting of five conserved genes, pqqA-E. PqqE is a radical S-adenosylmethionine (RS) protein with a C-terminal SPASM domain, and is proposed to catalyze the formation of a carbon-carbon bond between the glutamate and tyrosine side chains of the peptide substrate PqqA. PqqD is a 10-kDa protein with an unknown function, but is essential for PQQ production. Recently, in Klebsiella pneumoniae (Kp), PqqD and PqqE were shown to interact; however, the stoichiometry and K_D were not obtained. Here, we show that the PqqE and PqqD interaction transcends species, also occurring in Methylobacterium extorquens AM1 (Me). The stoichiometry of the MePqqD and MePqqE interaction is 1:1 and the K_D, determined by surface plasmon resonance spectroscopy (SPR), was found to be ∼12 μm. Moreover, using SPR and isothermal calorimetry techniques, we establish for the first time that MePqqD binds MePqqA tightly (K_D ∼200 nm). The formation of a ternary MePqqA-D-E complex was captured by native mass spectrometry and the K_D for the MePqqAD-MePqqE interaction was found to be ∼5 μm. Finally, using a bioinformatic analysis, we found that PqqD orthologues are associated with the RS-SPASM family of proteins (subtilosin, pyrroloquinoline quinone, anaerobic sulfatase maturating enzyme, and mycofactocin), all of which modify either peptides or proteins. In conclusion, we propose that PqqD is a novel peptide chaperone and that PqqD orthologues may play a similar role in peptide modification pathways that use an RS-SPASM protein.