Identification of a key prenyltransferase involved in biosynthesis of the most abundant fungal meroterpenoids derived from 3,5-dimethylorsellinic acid

Destroying aromaticity: A novel prenyltransferase (Trt2) involved in fungal meroterpenoid biosynthesis was shown to catalyze an unusual aromatic addition reaction onto a fully substituted aromatic ring. The prenylated product serves as a key intermediate in the biosynthesis of the most abundant series of meroterpenoids in fungi.     

An Improved Technique for the Rapid Chemical Characterisation of Bacterial Terpene Cyclases

A derivative of the pET28c(+) expression vector was constructed. It contains a yeast replication system (2μ origin of replication) and a yeast selectable marker (URA3), and can be used for gene cloning in yeast by efficient homologous recombination, and for heterologous expression in E. coli. The vector was used for the expression and chemical characterisation of three bacterial terpene cyclases.     

Volatile Terpenes from Actinomycetes: A Biosynthetic Study Correlating Chemical Analyses to Genome Data

The volatile terpenes of 24 actinomycetes whose genomes have been sequenced (or are currently being sequenced) were collected by use of a closed‐loop stripping apparatus and identified by GC/MS. The analytical data were compared against a phylogenetic analysis of all 192 currently available sequences of bacterial terpene cyclases (excluding geosmin and 2‐methylisoborneol synthases). In addition to the several groups of terpenes with known biosynthetic origin, selinadienes were identified as a large group of biosynthetically related sesquiterpenes that are produced by several streptomycetes. The detection of a large number of previously unrecognised side products of known terpene cyclases proved to be particularly important for an in depth understanding of biosynthetic pathways to known terpenes in actinomycetes. Interpretation of the chemical analytical data in the context of the phylogenetic tree of bacterial terpene cyclases pointed to the function of three new enzymes: (E)‐β‐caryophyllene synthase, selina‐3,7(11)‐diene synthase and aristolochene synthase.     

Pheromone biosynthesis in lepidoptera

Pheromone components for many lepidopteran species are produced by the use of unique chain-shortening and 9, 10, and 11 desaturase systems. Correlations in the Tortricidae indicate that the pheromone components derived from 9 and 01 desaturases are found in the more primitive species (those possessing morphological plesiomorphies). The precise blend ofZ andE acetates in a number of species is regulated in the final reduction sequence from acyl intermediates. Preliminary research has been conducted on the characterization of the various desaturase enzymes used and on the important blend regulating sequence. Initial purification work on the 11 desaturase enzyme found in the cabbage looper moth is reported.     

Induction of pseudopterosin biosynthesis in the gorgonian Pseudopterogorgia elisabethae

A number of biological and physical stimuli have been evaluated as inducers of pseudopterosin biosynthesis in the sea whip Pseudopterogorgia elisabethae. Data indicate that the production of pseudopterosins can be significantly increased in response to high levels of predation by the mollusk Cyphoma gibbosum and in response to decreased levels of UV/visible radiation. High levels of feeding by Chaetodon capistratus and an artificial wounding of the sea whip did not result in increased pseudopterosin levels.     

Determination of the cryptic stereochemistry of the first PKS chain-extension step in ansamitocin biosynthesis by Actinosynnema pretiosum

The biosynthesis of the antitumor antibiotic, ansamitocin, involves the assembly of a linear octaketide on the ansamitocin (asm) polyketide synthase (PKS), which is then cyclized to proansamitocin and further modified to the final product. In the first chain-extension step on the asm PKS, a stereocenter is generated which is then obliterated in a subsequent double-bond migration. The cryptic configuration at this stereocenter was determined by first synthesizing the two enantiomers of the intermediate diketide as their N-acetylcysteamine (SNAC) thioesters. These were then used to demonstrate that only the R enantiomer complements a 3-amino-5-hydroxybenzoic acid (AHBA) deficient mutant of Actinosynnema pretiosum to restore ansamitocin formation. The low efficiency of complementation by the diketide, compared to AHBA, is due to inefficient loading onto the PKS and not the inhibition of the enzyme. A presumed next chain-extension intermediate-the triketide with an unrearranged double bond-was also synthesized as its SNAC ester, but did not complement the AHBA(-) mutant.     

Glyceryl-S-acyl carrier protein as an intermediate in the biosynthesis of tetronate antibiotics

The biosynthetic pathway to the unusual tetronate ring of certain polyketide natural products, including the antibiotics abyssomicin and tetronomycin (TMN) and the antitumour compound chlorothricin (CHL), is presently unknown. The gene clusters governing chlorothricin and tetronomycin biosynthesis both contain a gene encoding an atypical member of the FkbH family of enzymes, which has previously been shown to synthesise glyceryl-S-acyl carrier protein (ACP) as the first step in production of unusual extender units for modular polyketide biosynthesis. We show here that purified recombinant FkbH-like protein, Tmn16, from the TMN gene cluster catalyses the efficient transfer of a glyceryl moiety from D-1,3-bisphosphoglycerate (1,3-BPG) to either of the dedicated ACPs, Tmn7a and ChlD2, to form glyceryl-S-ACP, which directly implicates this compound as an intermediate in tetronate biosynthesis as well. Neither Tmn16 nor Tmn7a produced glyceryl-S-ACP when incubated, respectively, with analogous ACP and FkbH-like proteins from a known extender-unit pathway; this indicates a highly selective channelling of glycolytic metabolites into tetronate biosynthesis.     

有机介质中包衣菌体催化合成cis9,trans11- 共轭亚油酸

以三种脂肪醇正辛醇、月桂醇和肉豆蔻醇为原料，合成得到三种谷氨酸二烷基酯核糖醇（2C8GE、2C12GE和2C14GE），比较了不同谷氨酸二烷基酯核糖醇及其浓度和所用溶剂，以及缓冲液pH、包衣时间、温度和干燥方式对包衣Lactobacillus helveticus L7菌体酶活的影响。确定了制备包衣菌体的条件为：表面活性剂为谷氨酸二十二烷基酯核糖醇(2C12GE)，配制成质量百分比1.0%（w/w）的丙酮溶液；菌体用pH5.8的磷酸盐缓冲液重悬；在4℃下处理4 h经冷冻干燥而成。在以亚油酸为底物时包衣菌体中亚油酸异构酶的Km为35.7 mM，Vmax为5.6 mM/h。经包衣处理后，菌体中亚油酸异构酶和底物的亲和力有所降低。     

Membrane Permeant Analogs for Independent Cellular Introduction of the Terpene Precursors Isopentenyl- and Dimethylallyl-Pyrophosphate

Isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) are the central five-carbon precursors to all terpenes. Despite their significance, exogenous, independent delivery of IPP and DMAPP to cells is impossible as the negatively charged pyrophosphate makes these molecules membrane impermeant. Herein, we demonstrate a facile method to circumvent this challenge through esterification of the β-phosphate with two self-immolative esters (SIEs) that neutralize the negatively charged pyrophosphate to yield membrane-permeant analogs of IPP and DMAPP. Following cellular incorporation, general esterase activity initiates cleavage of the SIEs, resulting in traceless release of IPP and DMAPP for metabolic utilization. Addition of the synthesized IPP and DMAPP precursor analogs rescued cell growth of glioblastoma (U-87MG) cancer cells concurrently treated with the HMG-CoA reductase inhibitor pitavastatin, which otherwise abrogates cell growth via blocking production of IPP and DMAPP. This work demonstrates a new application of a prodrug strategy to incorporate a metabolic intermediate and promises to enable future interrogation of the distinct biological roles of IPP and DMAPP.