不同碳源对须糖多孢菌生长发育及丁烯基多杀菌素生物合成的影响

本文研究了不同碳源对须糖多孢菌生长以及丁烯基多杀菌素生物合成的影响,通过寻找优势碳源优化发酵培养基配方,促进须糖多孢菌丁烯基多杀菌素的生物合成。试验共设11个处理,1个对照,通过单因素试验比较不同处理组菌体OD600值和丁烯基多杀菌素产量,筛选获得最优碳源及其发酵培养基配方。结果表明,除可溶性淀粉和木糖外,须糖多孢菌在9种碳源中都能进行生长,对不同构型碳源显示较好的利用率。在以半乳糖、葡萄糖、果糖和甘露糖作为碳源时具有较好的生长速率,而以甘露糖为碳源时能显著促进丁烯基多杀菌素的合成。选择甘露糖最佳添加浓度为5 g/L,须糖多孢菌最高菌体浓度和丁烯基多杀菌素产量分别是初始配方条件的1. 32倍和1. 78倍,显著提高了丁烯基多杀菌素的产量。上述结果为培养基碳源对丁烯基多杀菌素生物合成影响机制的研究及丁烯基多杀菌素大规模工业化发酵生产提供了科学依据和新的技术途径。     

蛋白磷酸酶抑制剂变构霉素的生物合成及其抑制位点研究

变构霉素具有很强的蛋白磷酸酶抑制作用和良好的抗菌活性, 尤其是对油菜菌核病的抑制作用。本文综述了近年来有关变构霉素生物合成和蛋白磷酸酶抑制位点方面的最新研究进展。     

Streptomyces sp.NO1W98中杀黑星菌素的分离和生物合成基因簇的鉴定

[目的] 分离Streptomyces sp.NO1W98中的杀黑星菌素并鉴定其生物合成基因簇。[方法] 利用有机溶剂萃取法对Streptomyces sp.NO1W98放大规模发酵产物进行提取;以正向、反向色谱柱层析进行化合物的分离纯化;借助波谱学手段进行单体化合物的结构鉴定;采用Illumina Hiseq技术进行基因组序列测定,对得到的序列进行生物信息学分析、注释并定位杀黑星菌素的生物合成基因簇vtd,利用基于PCR-targeting的遗传操作系统构建vtd内相关基因的阻断突变株,同时利用pSET152AKE进行基因回补,并分析与野生菌株的发酵产物差异。[结果] 从NO1W98发酵产物提取物中初步分离鉴定了2个大环内酯类化合物杀黑星菌素A（1）和B（2）;NO1W98的基因组大小约为11.6 Mb,蕴涵49个次级代谢产物生物合成基因簇,其中scaffold 3上的Region 3.3可能负责杀黑星菌素的生物合成;基因阻断和回补实验初步鉴定了杀黑星菌素的生物合成基因簇,包含6个骨架基因、5个转运基因、2个调控基因以及9个后修饰基因。[结论] 杀黑星菌素的分离、结构鉴定和基因簇的鉴定以及生物合成途径的推导为其遗传改造和工程菌株的构建奠定了分子基础。     

真菌β—内酰胺次级代谢调控的分子机制

真菌中青霉素和头胞菌素生物合成起始于共同前体,异青霉素N是这两合成途径的分叉中间体,经各自合成途径分别转化为青霉素和头孢菌素。在转录及转录后水平上,β－内酰胺生物合成基因表达受多种顺式作用元件与反式作用因子以及营养源等多方面的调控。     

fcl基因对须糖多孢菌丁烯基多杀菌素生物合成及生长发育的影响

fcl基因编码的GDP-岩藻糖合成酶(GDP fucose synthetase,GFS),能催化由GDP-D-甘露糖合成GDP-L-岩藻糖过程中的两步差向异构酶和还原酶反应;还参与氨基糖和核糖的生物合成,是调控生物体糖代谢、核苷酸代谢的关键酶之一。通过前期基因组测序表明须糖多孢菌Saccharopolyspora pogona中存在fcl基因。利用基因工程技术构建了fcl基因的过表达菌株S. pogona-fcl和敲除菌株S. pogona-Δfcl。结果表明该基因对菌株生长发育、蛋白表达及其转录水平、杀虫活性、丁烯基多杀菌素的生物合成均存在影响。经HPLC分析显示,S. pogona-Δfcl的丁烯基多杀菌素产量增加为野生型菌株的130%,S.pogona-fcl的丁烯基多杀菌素产量降低了25%。生测结果显示,与野生型菌株相比S.pogona-Δfcl对棉铃虫的杀虫活性明显增强,而S.pogona-fcl的杀虫活性降低。利用扫描电镜观察发现,S. pogona-Δfcl菌丝体表面出现褶皱,呈现短棒状,S. pogona-fcl菌丝形态与野生型菌株一致。以上结果表明,fcl基因的敲除影响菌丝体的生长发育,能促进丁烯基多杀菌素的生物合成和增强杀虫活性,该基因的过表达抑制了丁烯基多杀菌素的生物合成和降低了杀虫活性。SDS-PAGE结果表明,三株菌株在96 h时蛋白表达差异最为明显。对差异蛋白通过实时荧光定量聚合酶链式反应结果显示,三菌株蛋白的转录水平存在显著表达差异。通过研究结果构建了网络代谢调控图,分析fcl基因对须糖多孢菌生长发育及丁烯基多杀菌素生物合成代谢调控网络途径的影响,初步构建了fcl基因调控的代谢途径,为揭示丁烯基多杀菌素生物合成的调控机制及相关后续研究提供了实验依据。     

竹黄遗传多样性与竹红菌素合成和抗癌作用的研究进展

竹黄在我国是一种重要的药用真菌,主要活性代谢产物为竹红菌素等苝醌类光敏色素。前人关于竹黄的研究主要集中在活性成分分析、临床应用和发酵培养等方面。竹黄遗传多样性的存在是不同居群、不同菌株之间竹红菌素生物合成代谢差异的原因,并且随着竹红菌素及其衍生物对肿瘤细胞的抑制机制被不断揭示,对于竹黄的遗传多样性、竹红菌素的合成代谢与抗癌作用机理等研究正持续深入。应用不同的分子标记发现,竹黄遗传多样性既可能来源于居群内,也可能来源于居群间;而通过化学修饰或物理修饰都使竹红菌素在肿瘤的光动力治疗(Photodynamic therapy,PDT)上展现出诱人的前景。同时,影响竹红菌素产生的聚酮合酶基因等新的研究发现最终将会有助于掌握竹黄中竹红菌素的生物合成途径。综述了竹黄的遗传多样性、竹红菌素及其衍生物对癌细胞的作用和竹红菌素生物合成的研究进展,旨在为竹黄的资源保育和竹红菌素的深入研发提供参考。     

杀念菌素/FR-008生物合成途径中转运基因fscTI与fscTII的功能

摘要:【目的】分析杀念菌素/FR-008 生物合成途径中转运基因fscTI和fscTII的功能。【方法】构建转运基因fscTI和fscTII的敲除质粒pJTU4137,并通过接合转移和同源重组双交换的方法得到转运基因缺失突变株。转运基因fscTI和fscTII也被克隆到高拷贝质粒pJTU1278上用于在链霉菌FR-008(Streptomyces sp．FR-008)的衍生菌株ZYJ-6中进行转运蛋白的过量表达。【结果】获得了转运蛋白缺失的双交换突变株LX10,发酵结果显示该突变株不再产生杀念菌素及其衍生物;过量表达转运蛋白的基因工程菌株LX11,其杀念菌素的产量约是对照菌株的1.5倍。【结论】体内遗传实验进一步证实FR-008生物合成途径中的fscTI和fscTII是ATP依赖的ABC转运基因,fscTI与fscTII的过量表达增加了杀念菌素的产量,为利用此方法提高其它多烯类抗生素的产量提供了例证。     

添加生物素和浅蓝菌素对裂殖壶菌发酵产DHA的影响

为提高发酵产量,根据裂殖壶菌生物合成二十二碳六烯酸(DHA)的途径,考察添加代谢途径关键酶的辅酶及酶的抑制剂对发酵裂殖壶菌的影响.结果表明:添加生物素可促进油脂积累,添加浅蓝菌素有利于DHA及不饱和脂肪酸含量的提高.添加生物素0.3 mg/L时,DHA占细胞干质量分数达11.26％,相对于对照提高了13％;当添加浅蓝菌素0.1mg/L时,DHA占细胞干质量分数可达12.2％.     

杀念菌素/FR-008生物合成途径中转运基因fscTⅠ与fscTⅡ的功能

[目的]分析杀念菌素/FR-008生物合成途径中转运基因fscTⅠ和ficTⅡ的功能.[方法]构建转运基因fscTⅠ和fscTⅡ的敲除质粒pJTU4137,并通过接合转移和同源重组双交换的方法得到转运基因缺失突变株.转运基因fscTⅠ和ficTⅡ也被克隆到高拷贝质粒pJTU 1278上用于在链霉菌FR-008(Streptomyces sp.FR-008)的衍生菌株ZYJ-6中进行转运蛋白的过量表达.[结果]获得了转运蛋白缺失的双交换突变株LX10,发酵结果显示该突变株不再产生杀念菌素及其衍生物 ;过量表达转运蛋白的基因工程菌株LX11,其杀念菌素的产量约是对照菌株的1.5倍.[结论]体内遗传实验进一步证实FR-008生物合成途径中的fscTⅠ和fscTⅡ是ATP依赖的ABC转运基因,fscTⅠ与fscTⅡ的过量表达增加了杀念菌素的产量,为利用此方法提高其它多烯类抗生素的产量提供了例证.     

黄脂菌素生物合成基因簇中调控基因xanR3的功能

【目的】研究黄脂菌素产生菌灰黄链霉菌中编码ArsR家族转录调控蛋白(Arsenical resistance regulator)的xanR3基因的功能。【方法】利用大肠杆菌和链霉菌双亲本接合转移的方法,构建xanR3基因缺失突变株及回补突变株。利用cDNA在相邻同方向的基因间隔区进行PCR确定黄脂菌素生物合成基因簇中的转录单元。利用荧光定量RT-PCR方法进行突变株中黄脂菌素生物合成基因簇转录水平的检测。【结果】对得到的xanR3基因缺失突变株及回补突变株进行发酵,发现xanR3基因缺失突变株产黄脂菌素能力下降,回补菌株中黄脂菌素产量相比缺失突变株有一定程度的恢复,但仍未达到野生型水平。经鉴定,黄脂菌素生物合成基因簇中共有18个共转录单元,其中4个共转录单元在?xanR3突变株中转录水平明显下降。【结论】ArsR家族转录调控基因xanR3是黄脂菌素生物合成的正调控基因。     

The trans-tubular network of the hepatocyte Golgi apparatus is part of the secretory pathway

We have recently demonstrated the presence of sialyltransferase and sialic acid in a trans-tubular network (TTN) continuous with trans Golgi apparatus cisternae of rat liver hepatocytes. Based on these findings, we concluded that this structure, which also exhibited thiamine pyrophosphatase and acid phosphatase activity, is an integral part of the Golgi apparatus and functions in sialylation. In the present study, by comparing the distribution of a major hepatocyte secretory product with that of sialyltransferase, we sought to determine whether the TTN is also part of the secretory pathway. Examination of adjacent serial thin sections labeled for albumin showed its presence throughout the TTN and simultaneously provided new details about the structural complexity of the TTN. Double-immunolabeling with protein A-gold allowed the direct demonstration of albumin throughout the sialyltransferase containing TTN. Additional double staining protocols (combination of preembedding enzyme cytochemistry with postembedding immunolabeling) revealed the presence of albumin in both the thiamine pyrophosphatase and acid phosphatase positive regions of the TTN. These data show that albumin, a nonglycosylated secretory protein, reaches the TTN where terminal glycosylation of glycoproteins occurs. Therefore, it appears that the TTN of rat hepatocytes which functions in terminal glycosylation is also part of the constitutive secretory pathway.     

Titin as a potential novel therapeutic target in colorectal cancer

Colorectal cancer (CRC) is identified as a primary cause of death around the world. The current chemotherapies are not cost-effective. Therefore, finding novel potential therapeutic target is urgent. Titin (TTN) is a muscle protein that is critical in hypertrophic cardiomyopathy. However, its role in CRC is not well understood. The study focused on exploring the possible role of TTN in CRC carcinogenesis. TTN mRNA and protein expression levels presented an obvious downregulation in CRC tissue samples, relative to normal control (p < 0.05). TTN expression significantly correlated with the clinical stage (normal vs. Stage 1, p < 0.05; normal vs. Stage 4, p < 0.05), node metastasis (normal vs. N1, p < 0.05; N1 vs. N2, p < 0.05), histological type (normal vs. adenocarcinoma, p < 0.05), race (Caucasian vs. Asian, p < 0.05; African-American vs. Asian, p < 0.05) and TP53 mutation (normal vs. TP53 mutation, p < 0.05), considering The Cancer Genome Atlas database. However, for patients who had higher TTN expression, the overall survival was remarkably shorter than patients who had low TTN expression. Furthermore, TTN was lowly expressed in four CRC cell lines. TTN overexpression facilitated CRC cells in terms of the proliferation, metastasis and invasion. Based on gene set enrichment analysis, the ERB pathway might be responsible for TTN-related CRC. Besides, TTN was involved in the response to azacitidine. Overall, TTN might serve as a potential novel therapeutic target for treating and overcoming chemotherapy resistance in CRC.     

Diversity of TITAN functions in Arabidopsis seed development.

The titan mutants of Arabidopsis exhibit striking defects in seed development. The defining feature is the presence of abnormal endosperm with giant polyploid nuclei. Several TTN genes encode structural maintenance of chromosome proteins (condensins and cohesins) required for chromosome function at mitosis. Another TTN gene product (TTN5) is related to the ARL2 class of GTP-binding proteins. Here, we identify four additional TTN genes and present a general model for the titan phenotype. TTN1 was cloned after two tagged alleles were identified through a large-scale screen of T-DNA insertion lines. The predicted gene product is related to tubulin-folding cofactor D, which interacts with ARL2 in fission yeast (Schizosaccharomyces pombe) and humans to regulate tubulin dynamics. We propose that TTN5 and TTN1 function in a similar manner to regulate microtubule function in seed development. The titan phenotype can therefore result from disruption of chromosome dynamics (ttn3, ttn7, and ttn8) or microtubule function (ttn1 and ttn5). Three other genes have been identified that affect endosperm nuclear morphology. TTN4 and TTN9 appear to encode plant-specific proteins of unknown function. TTN6 is related to the isopeptidase T class of deubiquitinating enzymes that recycle polyubiquitin chains following protein degradation. Disruption of this gene may reduce the stability of the structural maintenance of chromosome complex. Further analysis of the TITAN network should help to elucidate the regulation of microtubule function and chromosome dynamics in seed development.     

The triakontatetraneuropeptide (TTN) stimulates thymidine incorporation in rat astrocytes through peripheral-type benzodiazepine receptors

Astrocytes and astrocytoma cells actively express the diazepam-binding inhibitor (DBI) gene, suggesting that DBI-processing products may regulate glial cell activity. In the present study, we have investigated the possible effect of one of the DBI-derived peptides, the triakontatetraneuropeptide (TTN), on [(3)H]thymidine incorporation in cultured rat astrocytes. Reversed-phase HPLC analysis of incubation media indicated that TTN is the major form of DBI-derived peptides released by cultured astrocytes. At very low concentrations (10(-14)-10(-11) M), TTN induced a dose-dependent increase in [(3)H]thymidine incorporation, whereas at higher concentrations (10(-10)-10(-5) M) the effect of TTN gradually declined. In the same range of concentrations, the specific peripheral-type benzodiazepine receptor (PBR) agonist Ro 5-4864 mimicked the bell-shaped stimulatory effect of TTN on [(3)H]thymidine incorporation. The PBR antagonist PK11195 (10(-6) M) suppressed the stimulatory action of both TTN and Ro 5-4864 on [(3)H]thymidine incorporation, whereas the central-type benzodiazepine receptor antagonist flumazenil (10(-6) M) had no effect. The present study demonstrates that the endozepine TTN stimulates DNA synthesis in rat glial cells through activation of PBRs. These data strongly suggest that TTN exerts an autocrine/paracrine stimulatory effect on glial cell proliferation.     

Improved operational stability of chloroperoxidase through use of antioxidants

Chloroperoxidase (CPO) from Caldariomyces fumago is a potentially very useful enzyme due to its ability to catalyze a large variety of stereoselective oxidation reactions, but poor operational stability is a main limitation for commercial use. In the present study, the possibility of increasing the operational stability by use of antioxidants was investigated using the oxidation of indole as model reaction. Caffeic acid was the antioxidant showing the strongest positive effects, reaching a total turnover number (TTN) of 135,000 at pH 4 and 4 mM hydrogen peroxide, compared to 28,700 in the absence of antioxidant. Portion-wise addition of hydrogen peroxide in the presence of caffeic acid caused a further increase in TTN to 171,000. An alternative way to reach high TTN was to use tert-butyl hydroperoxide as oxidant instead of hydrogen peroxide: a TTN of 600,000 was achieved although the reaction was quite slow. In this case, antioxidants did not have any positive effect. Possible mechanisms for the observed inactivation of CPO are discussed.     

Advances in the Plant Isoprenoid Biosynthesis Pathway and Its Metabolic Engineering

Although the cytosolic isoprenoid biosynthetic pathway, mavolonate pathway, in plants has been known for many years, a new plastidial 1-deoxyxylulose-5-phosphate (DXP) pathway was identified in the past few years and its related intermediates, enzymes, and genes have been characterized quite recently. With a deep insight into the biosynthetic pathway of isoprenoids, investigations into the metabolic engineering of isoprenoid biosynthesis have started to prosper. In the present article, recent advances in the discoveries and regulatory roles of new genes and enzymes in the plastidial isoprenoid biosynthesis pathway are reviewed and examples of the metabolic engineering of cytosolic and plastidial isoprenoids biosynthesis are discussed.     

Interleukin-8 production induced by the endozepine triakontatetraneuropeptide in human neutrophils: role of calcium and pharmacological investigation of signal transduction pathways

The endozepine triakontatetraneuropeptide (TTN) induces intracellular calcium ([Ca(2+)](i)) changes and is chemotactic for human neutrophils (PMNs). Because interleukin-8 (IL-8) production is Ca(2+) dependent and can be induced by chemotactic stimuli, we have investigated the ability of TTN to induce IL-8 production in PMNs, as well as the signal transduction mechanisms involved. Our results show that TTN increases IL-8 release and IL-8 mRNA expression in a concentration- and time-dependent fashion, and these effects are prevented by the Ca(2+) chelator BAPTA-AM. TTN-induced [Ca(2+)](i) changes and IL-8 mRNA expression are sensitive to pertussis toxin, to the phospholipase C (PLC) inhibitor U73122 (but not to its inactive analogue U73343) and to the protein kinase C (PKC) inhibitor calphostin C. It is therefore suggested that TTN-induced IL-8 production in human PMNs results from a G protein-operated, PLC-activated [Ca(2+)](i) rise, and PKC contributes to this effect. These findings further support the possible role of TTN in the modulation of the inflammatory processes.     

Impact of oxidative stress on ascorbate biosynthesis in Chlamydomonas via regulation of the VTC2 gene encoding a GDP-L-galactose phosphorylase

The L-galactose (Smirnoff-Wheeler) pathway represents the major route to L-ascorbic acid (vitamin C) biosynthesis in higher plants. Arabidopsis thaliana VTC2 and its paralogue VTC5 function as GDP-L-galactose phosphorylases converting GDP-L-galactose to L-galactose-1-P, thus catalyzing the first committed step in the biosynthesis of L-ascorbate. Here we report that the L-galactose pathway of ascorbate biosynthesis described in higher plants is conserved in green algae. The Chlamydomonas reinhardtii genome encodes all the enzymes required for vitamin C biosynthesis via the L-galactose pathway. We have characterized recombinant C. reinhardtii VTC2 as an active GDP-L-galactose phosphorylase. C. reinhardtii cells exposed to oxidative stress show increased VTC2 mRNA and L-ascorbate levels. Genes encoding enzymatic components of the ascorbate-glutathione system (e.g. ascorbate peroxidase, manganese superoxide dismutase, and dehydroascorbate reductase) are also up-regulated in response to increased oxidative stress. These results indicate that C. reinhardtii VTC2, like its plant homologs, is a highly regulated enzyme in ascorbate biosynthesis in green algae and that, together with the ascorbate recycling system, the L-galactose pathway represents the major route for providing protective levels of ascorbate in oxidatively stressed algal cells.     

Characterization of the Tautomycin Biosynthetic Gene Cluster from Streptomyces spiroverticillatus Unveiling New Insights into Dialkylmaleic Anhydride and Polyketide Biosynthesis

Tautomycin (TTM) is a highly potent and specific protein phosphatase inhibitor isolated from Streptomyces spiroverticillatus. The biological activity of TTM makes it an important lead for drug discovery, whereas its spiroketal-containing polyketide chain and rare dialkylmaleic anhydride moiety draw attention to novel biosynthetic chemistries responsible for its production. To elucidate the biosynthetic machinery associated with these novel molecular features, the ttm biosynthetic gene cluster from S. spiroverticillatus was isolated and characterized, and its involvement in TTM biosynthesis was confirmed by gene inactivation and complementation experiments. The ttm cluster was localized to a 86-kb DNA region, consisting of 20 open reading frames that encode three modular type I polyketide synthases (TtmHIJ), one type II thioesterase (TtmT), five proteins for methoxymalonyl-S-acyl carrier protein biosynthesis (Ttm-ABCDE), eight proteins for dialkylmaleic anhydride biosynthesis and regulation (TtmKLMNOPRS), as well as two additional regulatory proteins (TtmF and TtmQ) and one tailoring enzyme (TtmG). A model for TTM biosynthesis is proposed based on functional assignments from sequence analysis, which agrees well with previous feeding experiments, and has been further supported by in vivo gene inactivation experiments. These findings set the stage to fully investigate TTM biosynthesis and to biosynthetically engineer new TTM analogs.Tautomycin (TTM)² is a polyketide natural product first isolated in 1987 from Streptomyces spiroverticillatus (1). The structure and stereochemistry of TTM were established on the basis of chemical degradation and spectroscopic evidence (2-4). TTM contains several features not common to polyketide natural products, including a spiroketal group, a methoxymalonate-derived unit, and an acyl chain bearing a dialkylmaleic anhydride moiety. Structurally related to TTM is tautomycetin (TTN), which was first isolated in 1989 from Streptomyces griseochromogenes following the discovery of TTM (5, 6). The structure of TTN was deduced by chemical degradation and spectroscopic analysis (6), and its stereochemistry was established by comparison of spectral data with those of TTN degradation products and synthetic fragments (7). Both TTM and TTN exist as tautomeric mixtures composed of two interconverting anhydride and diacid forms in approximately a 5:4 ratio under neutral conditions (Fig. 1A) (1, 2).Open in a separate windowFIGURE 1.A, structures of TTM and TTN in anhydride or diacid forms, and biosynthetic origin of the dialkylmaleic anhydride by feeding experiments using ¹³C-labeled acetate and propionate. The methoxymalonate-derived unit in TTM is highlighted by the dotted oval. R, polyketide moiety of TTM or TTN. B, selected natural product inhibitors of PP-1 and PP-2A featuring a spiroketal or dialkylmaleric anhydride moiety. C, selected natural products containing a dialkylmaleic anhydride moiety.Early studies of TTM revealed its ability to induce morphological changes in leukemia cells (8). However, it was later realized that TTM is a potent and specific inhibitor of protein phosphatases (PPs) PP-1 and PP-2A (9). PP-1 and PP-2A are two of the four major serine/threonine protein phosphatases that regulate diverse cellular events such as cell division, gene expression, muscle contraction, glycogen metabolism, and neuronal signaling in eukaryotic cells (10-12). Many natural product PP-1 and PP-2A inhibitors are known, including okadaic acid (13), calyculin-A (14), phoslactomycin, spirastrellolide, and cantharidin (15) (Fig. 1B), as well as TTM (16, 17), and TTN (18). They have served as useful tools to study PP-involved intracellular events in vivo and as novel leads for drug discovery (10-12). Among these PP inhibitors, TTM and TTN are unique because of their PP-1 selectivity. Despite their structural similarities, TTM exhibits potent specific inhibition of PP-1 and PP-2A with IC₅₀ values of 22-32 nm and only a slight preference for PP-1 (18). Conversely, TTN shows nearly a 40-fold higher binding affinity to PP-1 (IC₅₀ = 1.6 nm) than to PP-2A (IC₅₀ = 62 nm) (18). Because the major structural differences between TTM and TTN reside in the region distal to the dialkylmaleic anhydride moiety (Fig. 1A), it has been proposed that differences in these moieties might be responsible for the PP-1 selectivity (17-19). Finally, TTN also has an impressive immuno-suppressive activity (20, 21), which is apparently devoid for TTM. Clearly, the structural differences between these two polyketides translate into large, exploitable differences in bio-activities, yet an understanding of the biosynthetic origins of these differences remains elusive.The spiroketal and dialkylmaleic anhydride features of TTM are uncommon for polyketide natural products, as is the methoxymalonate-derived unit (Fig. 1A). Few studies have been carried out for spiroketal biosynthesis, yet it is reasonably common among the phosphatase inhibitors such as calyculin A, okadaic acid, and a few others (Fig. 1B). Less common, but still found in the phosphatase inhibitor cantharidin, as well as TTM and TTN, is the dialkylmaleic anhydride moiety (Fig. 1B); this unit appears in a number of other natural products (Fig. 1C), although the biosynthetic steps leading to this reactive moiety (a protected version of a dicarboxylate) have not been rigorously investigated. Feeding experiments with ¹³C-labeled precursors indicated that the anhydride of TTM and TTN is assembled from a propionate and an as yet undefined C-5 unit (Fig. 1A), which would require novel chemistry for polyketide biosynthesis (22). TTM differentiates itself from all known PP-1 and PP-2A inhibitors by virtue of its unique combination of both the dialkymaleic anhydride and spiroketal functionalities.Multiple total syntheses of TTM and a small number of analogs have been reported, confirming the predicted structure and absolute stereochemistry and facilitating structure-activity relationship studies on PP inhibition and apoptosis induction (19, 23-25). These studies revealed that: (i) the C22-C26 carbon chain and the dialkylmaleic anhydride are the minimum requirements for TTM bioactivity; (ii) the C18-C21 carbon chain and 22-hydroxy group are important for PP inhibition; (iii) the spiroketal moiety determines the affinity to specific protein phosphatases; (iv) the active form is most likely the dicarboxylate; and (v) 3′-epi-TTM exhibits 1,000-fold less activity than TTM. However, taken as a whole, none of the analogs had an improved potency or selectivity for PP-1 inhibition than the natural TTM (19, 22-25). As a result, a more specific inhibitor of PP-1 is urgently awaited to differentiate the physiological roles of PP-1 and PP-2A in vivo and to explore PPs as therapeutic targets for drug discovery.We have undertaken the cloning and characterization of the TTM biosynthetic gene cluster from S. spiroverticillatus as the first step toward engineering TTM biosynthesis for novel analogs (26). We report here: (i) cloning and sequencing of the complete ttm gene cluster, (ii) determination of the ttm gene cluster boundaries, (iii) bioinformatics analysis of the ttm cluster and a proposal for TTM biosynthesis, and (iv) genetic characterization of the TTM pathway to support the proposed pathway. Of particular interest has been the identification of genes possibly related to dialkylmaleic anhydride biosynthesis, the unveiling of the ttm polyketide synthase (PKS) genes predicted to select and incorporate four different starter and extender units for TTM production, and the apparent lack of candidate genes associated with spiroketal formation. These findings now set the stage to engineer TTM analogs for novel PP-1- and PP-2A-specific inhibitors by applying combinatorial biosynthetic methods to the TTM biosynthetic machinery.