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Biosynthesis of the highly biologically active long-chain polyunsaturated fatty acids, arachidonic (ARA), eicosapentaenoic (EPA), and docosahexaenoic (DHA) acids, in vertebrates requires the introduction of up to three double bonds catalyzed by fatty acyl desaturases (Fad). Synthesis of ARA is achieved by A6 desaturation of 18:2n- 6 to produce 18:3n- 6 that is elongated to 20:3n- 6 followed by A5 desaturation. Synthesis of EPA from 18:3n- 3 requires the same enzymes and pathway as for ARA, but DHA synthesis reportedly requires two further elongations, a second [DELTA]6 desaturation and a peroxisomal chain shortening step. This paper describes cDNAs, fad1 and fad2, isolated from the herbivorous, marine teleost fish (Siganus canaliculatus) with high similarity to mammalian Fad proteins. Functional characterization of the cDNAs by heterologous expression in the yeast Saccharomyces cerevisiae showed that Fad1 was a bifunctional [DELTA]6/[DELTA]5 Fad. Previously, functional dual specificity in vertebrates had been demonstrated for a zebrafish Danio rerio Fad and baboon Fad, so the present report suggests bifunctionality may be more widespread in vertebrates. However, Fad2 conferred on the yeast the ability to convert 22: 5n--3 to DHA indicating that this S. canaliculatus gene encoded an enzyme having [DELTA]4 Fad activity. This is a unique report of a Fad with [DELTA]4 activity in any vertebrate species and indicates that there are two possible mechanisms for DHA biosynthesis, a direct route involving elongation of EPA to 22: 5n--3 followed by [DELTA]4 desaturation, as well as the more complicated pathway as described above. [DELTA]4 desaturase I bifunctional [DELTA]6/[DELTA]5 desaturase | polyunsaturated fatty acid biosynthesis | Siganus canaliculatus | teleost doi/ 10.1073/pnas.1008429107

The CuAAC reaction (click chemistry) has been used in conjunction with solid-phase synthesis to produce catalytically active hairpin ribozymes around 100 nucleotides in length. Cross-strand ligation through neighboring nucleobases was successful in covalently linking presynthesized RNA strands with high efficiency (transligation). In an alternative strategy, intrastrand click ligation was employed to produce a functional hammerhead ribozyme containing a novel nucleic acid backbone mimic at the catalytic site (cisligation). The ability to synthesize long RNA strands by a combination of solid-phase synthesis and click ligation is an important addition to RNA chemistry. It is compatible with a plethora of site-specific modifications and is applicable to the synthesis of many biologically important RNA molecules. chemical ligation | click RNA ligation | CuAAC | triazole backbone | biocompatible doi/ 10.1073/pnas.1006447107

A common strategy of metabolic engineering is to increase the endogenous supply of precursor metabolites to improve pathway productivity. The ability to further enhance heterologous production of a desired compound may be limited by the inherent capacity of the imported pathway to accommodate high precursor supply. Here, we present engineered diterpenoid biosynthesis as a case where insufficient downstream pathway capacity limits high-level levopimaradiene production in Escherichia coli. To increase levopimaradiene synthesis, we amplified the flux toward isopentenyl diphosphate and dimethylallyl diphosphate precursors and reprogrammed the rate-limiting downstream pathway by generating combinatorial mutations in geranylgeranyl diphosphate synthase and levopimaradiene synthase. The mutant library contained pathway variants that not only increased diterpenoid production but also tuned the selectivity toward levopimaradiene. The most productive pathway, combining precursor flux amplification and mutant synthases, conferred approximately 2,600-fold increase in levopimaradiene levels. A maximum titer of approximately 700 mg/L was subsequently obtained by cultivation in a bench-scale bioreactor. The present study highlights the importance of engineering proteins along with pathways as a key strategy in achieving microbial biosynthesis and overproduction of pharmaceutical and chemical products. GGPP synthase | levopimaradiene synthase | metabolic engineering | Escherichia coli | molecular reprogramming doi/ 10.1073/pnas.1006138107

Alkylresorcinols and alkylpyrones, which have a polar aromatic ring and a hydrophobic alkyl chain, are phenolic lipids found in plants, fungi, and bacteria. In the Gram-negative bacterium Azotobacter vinelandii, phenolic lipids in the membrane of dormant cysts are essential for encystment. The aromatic moieties of the phenolic lipids in A. vinelandii are synthesized by two type III polyketide synthases (PKSs), ArsB and ArsC, which are encoded by the ars operon. However, details of the synthesis of hydrophobic acyl chains, which might serve as starter substrates for the type III polyketide synthases (PKSs), were unknown. Here, we show that two type I fatty acid synthases (FASs), ArsA and ArsD, which are members of the ars operon, are responsible for the biosynthesis of C22-C26 fatty acids from malonyI-CoA. In vivo and in vitro reconstitution of phenolic lipid synthesis systems with the Ars enzymes suggested that the [C.sub.22]-[C.sub.26] fatty acids produced by ArsA and ArsD remained attached to the ACP domain of ArsA and were transferred hand-to-hand to the active-site cysteine residues of ArsB and ArsC. The type Ill PKSs then used the fatty acids as starter substrates and carried out two or three extensions with malonyl-CoA to yield the phenolic lipids. The phenolic lipids in A. vinelandii were thus found to be synthesized solely from malonyI-CoA by the four members of the ars operon. This is the first demonstration that a type I FAS interacts directly with a type III PKS through substrate transfer. Azotobacter vinelandii | alkylresorcinol | alkylpyrone long-chain fatty acid | cyst

Drosophila melanogaster produces sexually dimorphic cuticular pheromones that are a key component of the courtship behavior leading to copulation. These molecules are hydrocarbons, with lengths of 23 and 25 carbons in males (mainly with one double bond) and 27 and 29 carbons in females (mainly with two double bonds). Here, we describe an elongase gene, eloF, with female-biased expression. The 771-bp ORF encodes a 257-aa protein that shows the highest sequence identity with mouse SSC1 elongase (33%). The activity of the cDNA expressed in yeast was elongation of saturated and unsaturated fatty acids up to C30. RNAi knockdown in Drosophila led to a dramatic modification of female hydrocarbons, with decreased C29 dienes and increased C25 dienes accompanied by a modification of several courtship parameters: an increase in copulation latency and a decrease in both copulation attempts and copulation. Feminization of the hydrocarbon profile in males by using targeted expression of the transformer gene resulted in high expression levels of eloF, suggesting that the gene is under the control of the sex-determination hierarchy. There is no expression of eloF in Drosophila simulans, which synthesize only C23 and C25 hydrocarbons. These results strongly support the hypothesis that eloF is a crucial enzyme for female pheromone biosynthesis and courtship behavior in D. melanogaster. pheromones | RNAi

This paper reports a computational method for describing the conformational flexibility of very large biomolecular complexes using a reduced number of degrees of freedom. It is called the substructure synthesis method, and the basic concept is to treat the motions of a given structure as a collection of those of an assemblage of substructures. The choice of substructures is arbitrary and sometimes quite natural, such as domains, subunits, or even large segments of biomolecular complexes. To start, a group of low-frequency substructure modes is determined, for instance by normal mode analysis, to represent the motions of the substructure. Next, a desired number of substructures are joined together by a set of constraints to enforce geometric compatibility at the interface of adjacent substructures, and the modes for the assembled structure can then be' synthesized from the substructure modes by applying the Rayleigh-Ritz principle. Such a procedure is computationally much more desirable than solving the full eigenvalue problem for the whole assembled structure. Furthermore, to show the applicability to biomolecular complexes, the method is used to study F-actin, a large filamentous molecular complex involved in many cellular functions. The results demonstrate that the method is capable of studying the motions of very large molecular complexes that are otherwise completely beyond the reach of any conventional methods. conformational flexibility | elastic deformation | large conformational change | normal mode analysis

2-C-methylerythritol 4-phosphate has been established recently as an intermediate of the deoxyxylulose phosphate pathway used for biosynthesis of terpenoids in plants and in many microorganisms. We show that an enzyme isolated from cell extract of Escherichia coli converts 2-C-methylerythritol 4-phosphate into 4-diphospho-cytidyl-2-C-methylerythritol by reaction with CTP. The enzyme is specified by the hitherto unannotated ORF ygbP of E. coli. The cognate protein was obtained in pure form from a recombinant hyperexpression strain of E. coli harboring a plasmid with the ygbP gene under the control of a T5 promoter and lac operator. By using the recombinant enzyme, 4-diphosphocytidyl-[2-[sup.14]C]2-C-methyl-erythritol was prepared from [2-[sup.14]C]2-C-methylerythritol 4-phosphate. The radiolabeled 4-diphosphocytidyl-2-C-methylerythritol was shown to be efficiently incorporated into carotenoids by isolated chromoplasts of Capsicum annuum. The E. coli ygbP gene appears to be part of a small operon also comprising the unannotated ygbB gene. Genes with similarity to ygbP and ygbB are present in the genomes of many microorganisms, and their occurrence appears to be correlated with that of the deoxyxylulose pathway of terpenoid biosynthesis. Moreover, several microorganisms have genes specifying putative fusion proteins with ygbP and ygbB domains, suggesting that both the YgbP protein and the YgbB protein are involved in the deoxyxylulose pathway. A germ from Arabidopsis thaliana with similarity to ygbP carries a putative plastid import sequence, which is well in line with the assumed localization of the deoxyxylulose pathway in the plastid compartment of plants.

A three-plasmid system for heterologous expression of 6-deoxyerythronolide B synthase (DEBS) has been developed to facilitate combinatorial biosynthesis of polyketides made by type I modular polyketide synthases (PKSs). The eryA PKS genes encoding the three DEBS subunits were individually cloned into three compatible Streptomyces vectors carrying mutually selectable antibiotic resistance markers. A strain of Streptomyces lividans transformed with all three plasmids produced 6-deoxyerythronolide B at a level similar to that of a strain transformed with a single plasmid containing all three genes. The utility of this system in combinatorial biosynthesis was demonstrated through production of a library of modified polyketide macrolactones by using versions of each plasmid constructed to contain defined mutations. Combinations of these vector sets were introduced into S. lividans, resulting in strains producing a wide range of 6-deoxyerythronolide B analogs. This method can be extended to any modular PKS and has the potential to produce thousands of novel natural products, including ones derived from further modification of the PKS products by tailoring enzymes.