Enhancing Terpene yield from sugars via novel routes to 1-deoxy-d-xylulose 5-phosphate.

Terpene synthesis in the majority of bacterial species, together with plant plastids, takes place via the 1-deoxy-d-xylulose 5-phosphate (DXP) pathway. The first step of this pathway involves the condensation of pyruvate and glyceraldehyde 3-phosphate by DXP synthase (Dxs), with one-sixth of the carbon lost as CO2. A hypothetical novel route from a pentose phosphate to DXP (nDXP) could enable a more direct pathway from C5 sugars to terpenes and also circumvent regulatory mechanisms that control Dxs, but there is no enzyme known that can convert a sugar into its 1-deoxy equivalent. Employing a selection for complementation of a dxs deletion in Escherichia coli grown on xylose as the sole carbon source, we uncovered two candidate nDXP genes. Complementation was achieved either via overexpression of the wild-type E. coli yajO gene, annotated as a putative xylose reductase, or via various mutations in the native ribB gene. In vitro analysis performed with purified YajO and mutant RibB proteins revealed that DXP was synthesized in both cases from ribulose 5-phosphate (Ru5P). We demonstrate the utility of these genes for microbial terpene biosynthesis by engineering the DXP pathway in E. coli for production of the sesquiterpene bisabolene, a candidate biodiesel. To further improve flux into the pathway from Ru5P, nDXP enzymes were expressed as fusions to DXP reductase (Dxr), the second enzyme in the DXP pathway. Expression of a Dxr-RibB(G108S) fusion improved bisabolene titers more than 4-fold and alleviated accumulation of intracellular DXP.

Use of nonionic surfactants for improvement of terpene production in Saccharomyces cerevisiae.

To facilitate enzyme and pathway engineering, a selection was developed for improved sesquiterpene titers in Saccharomyces cerevisiae. α-Bisabolene, a candidate advanced biofuel, was found to protect yeast against the disruptive action of nonionic surfactants such as Tween 20 (T20). An experiment employing competition between two strains of yeast, one of which makes twice as much bisabolene as the other, demonstrated that growth in the presence of T20 provided sufficient selective pressure to enrich the high-titer strain to form 97% of the population. Following this, various methods were used to mutagenize the bisabolene synthase (BIS) coding sequence, coupled with selection by subculturing in the presence of T20. Mutagenesis targeting the BIS active site did not yield an improvement in bisabolene titers, although mutants were found which made a mixture of α-bisabolene and ß-farnesene, another candidate biofuel. Based on evidence that the 3' end of the BIS mRNA may be unstable in yeast, we randomly recoded the last 20 amino acids of the enzyme and, following selection in T20, found a variant which increased specific production of bisabolene by more than 30%. Since T20 could enrich a mixed population, efficiently removing strains that produced little or no bisabolene, we investigated whether it could also be applied to sustain high product titers in a monoculture for an extended period. Cultures grown in the presence of T20 for 14 days produced bisabolene at titers up to 4-fold higher than cultures grown with an overlay of dodecane, used to sequester the terpene product, and 20-fold higher than cultures grown without dodecane.

Mutations in Escherichia coli aceE and ribB genes allow survival of strains defective in the first step of the isoprenoid biosynthesis pathway.

A functional 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway is required for isoprenoid biosynthesis and hence survival in Escherichia coli and most other bacteria. In the first two steps of the pathway, MEP is produced from the central metabolic intermediates pyruvate and glyceraldehyde 3-phosphate via 1-deoxy-D-xylulose 5-phosphate (DXP) by the activity of the enzymes DXP synthase (DXS) and DXP reductoisomerase (DXR). Because the MEP pathway is absent from humans, it was proposed as a promising new target to develop new antibiotics. However, the lethal phenotype caused by the deletion of DXS or DXR was found to be suppressed with a relatively high efficiency by unidentified mutations. Here we report that several mutations in the unrelated genes aceE and ribB rescue growth of DXS-defective mutants because the encoded enzymes allowed the production of sufficient DXP in vivo. Together, this work unveils the diversity of mechanisms that can evolve in bacteria to circumvent a blockage of the first step of the MEP pathway.

A murine model of isolated cardiac steatosis leads to cardiomyopathy.

Lipid accumulation in the heart is associated with obesity and diabetes mellitus and may play an important role in the pathogenesis of heart failure seen in this patient population. Stored triglycerides are synthesized by the enzyme diacylglycerol acyl transferase (DGAT). We hypothesized that forced expression of DGAT1 in the cardiac myocyte would result in increased lipid accumulation and heart dysfunction. A cardiac myocyte-selective DGAT1 transgenic mouse was created and demonstrated increased lipid accumulation in the absence of hyperglycemia, plasma dyslipidemia or differences in body weight. Over time, expression of DGAT1 in the heart resulted in the development of a significant cardiomyopathy. Echocardiography revealed diastolic dysfunction with increased early mitral inflow velocity to late mitral inflow velocity ratio and decreased deceleration time, suggesting a restrictive pattern in the transgenic mice. Moderate systolic dysfunction was also seen at 52 weeks. Histological analysis showed increased cardiac fibrosis and increased expression of procollagen type 1A, matrix metalloproteinase 2, and tissue inhibitor of matrix metalloproteinase 2 in the transgenic mice. Mitochondrial biogenesis was reduced in the transgenic hearts, as was expression of cytochrome c oxidase 1 and cytochrome c. Expression of key transcription factors important in the regulation of mitochondrial biogenesis were reduced. These findings suggest that triglyceride accumulation, in the absence of systemic metabolic derangement, results in cardiac dysfunction and decreased mitochondrial biogenesis.

Cloning of casbene and neocembrene synthases from Euphorbiaceae plants and expression in Saccharomyces cerevisiae.

A large number of diterpenes have been isolated from Euphorbiaceae plants, many of which are of interest due to toxicity or potential therapeutic activity. Specific Euphorbiaceae diterpenes of medical interest include the latent HIV-1 activator prostratin (and related 12-deoxyphorbol esters), the analgesic resiniferatoxin, and the anticancer drug candidate ingenol 3-angelate. In spite of the large number of diterpenes isolated from these plants and the similarity of their core structures, there is little known about their biosynthetic pathways. Other than the enzymes involved in gibberellin biosynthesis, the only diterpene synthase isolated to date from the Euphorbiaceae has been casbene synthase, responsible for biosynthesis of a macrocyclic diterpene in the castor bean (Ricinus communis). Here, we have selected five Euphorbiaceae species in which to investigate terpene biosynthesis and report on the distribution of diterpene synthases within this family. We have discovered genes encoding putative casbene synthases in all of our selected Euphorbiaceae species and have demonstrated high-level casbene production through expression of four of these genes in a metabolically engineered strain of Saccharomyces cerevisiae. The only other diterpene synthase found among the five plants was a neocembrene synthase from R. communis (this being the first report of a neocembrene synthase gene). Based on the prevalence of casbene synthases, the lack of other candidates, and the structure of the casbene skeleton, we consider it likely that casbene is the precursor to a large number of Euphorbiaceae diterpenes. Casbene production levels of 31 mg/L were achieved in S. cerevisiae and we discuss strategies to further increase production by maximizing flux through the mevalonate pathway.

Atrial natriuretic peptide suppresses endothelin gene expression and proliferation in cardiac fibroblasts through a GATA4-dependent mechanism.

AIMS: Atrial natriuretic peptide (ANP) is a hormone that has both antihypertrophic and antifibrotic properties in the heart. We hypothesized that myocyte-derived ANP inhibits endothelin (ET) gene expression in fibroblasts. METHODS AND RESULTS: We have investigated the mechanism(s) involved in the antiproliferative effect of ANP on cardiac fibroblasts in a cell culture model. We found that cardiac myocytes inhibited DNA synthesis in co-cultured cardiac fibroblasts as did treatment with the ET-1 antagonist BQ610. The effect of co-culture was reversed by antibody directed against ANP or the ANP receptor antagonist HS-142-1. ANP inhibited the expression of the ET-1 gene and ET-1 gene promoter activity in cultured fibroblasts. The site of the inhibition was localized to a GATA-binding site positioned between -132 and -135 upstream from the transcription start site. GATA4 expression was demonstrated in cardiac fibroblasts, GATA4 bound the ET-1 promoter both in vitro and in vivo, and siRNA-mediated knockdown of GATA4 inhibited ET-1 expression. ET-1 treatment resulted in increased levels of phospho-serine(105) GATA4 in cardiac fibroblasts and this induction was partially suppressed by co-treatment with ANP. CONCLUSION: Collectively, these findings suggest that locally produced ET-1 serves as an autocrine stimulator of fibroblast proliferation, that ANP produced in neighbouring myocytes serves as a paracrine inhibitor of this proliferation, and that the latter effect operates through a reduction in GATA4 phosphorylation and coincident reduction in GATA4-dependent transcriptional activity.

Endothelin-stimulated human B-type natriuretic peptide gene expression is mediated by Yin Yang 1 in association with histone deacetylase 2.

Increased B-type natriuretic peptide (BNP) gene expression is regarded as one of the hallmarks of cardiac myocyte hypertrophy. Here we demonstrate that both basal- and endothelin-1-dependent stimulation of human (h) BNP gene transcription requires the presence of an intact Yin Yang 1 (YY1) binding site positioned at -62 bp relative to the transcription start site. Mutation of this site reduced both basal and stimulated hBNP promoter activity. This site was shown to bind YY1 both in vitro and within the context of the intact cell. The latter interaction increased after endothelin-1 treatment. Exposure to endothelin-1 also resulted in increased nuclear localization of YY1 and a reduction in acetylation of the YY1 protein. Overexpression of wild-type YY1 increased both basal and endothelin-stimulated hBNP promoter activity, whereas a carboxy-terminal deletion mutant of YY1 was devoid of activity. Treatment with the histone deacetylase inhibitor trichostatin A resulted in decreased hBNP reporter activity. YY1 was shown to associate with histone deacetylase 2, and histone deacetylase 2 was shown to associate directly with the hBNP promoter in the intact cell. Collectively these findings demonstrate that YY1 plays an important role in regulating the transcriptional activity of the hBNP gene promoter. These data suggest a model in which YY1 activates hBNP transcription through interaction with histone deacetylase 2.

Expression of the vitamin d receptor is increased in the hypertrophic heart.

The liganded vitamin D receptor (VDR) is thought to play an important role in controlling cardiac function. Specifically, this system has been implicated as playing an antihypertrophic role in the heart. Despite this, studies of VDR in the heart have been limited in number and scope. In the present study, we used a combination of real-time polymerase chain reaction, Western blot analysis, immunofluorescence, and transient transfection analysis to document the presence of functional VDR in both the myocytes and fibroblasts of the heart, as well as in the intact ventricular myocardium. We also demonstrated the presence of 1-alpha-hydroxylase and 24-hydroxylase in the heart, 2 enzymes involved in the synthesis and metabolism of 1,25 dihydroxyvitamin D. VDR is shown to interact directly with the human B-type natriuretic peptide gene promoter, a surrogate marker of the transcriptional response to hypertrophy. Of note, induction of myocyte hypertrophy either in vitro or in vivo leads to an increase in VDR mRNA and protein levels. Collectively, these findings suggest that the key components required for a functional 1,25 dihydroxyvitamin D-dependent signaling system are present in the heart and that this putatively antihypertrophic system is amplified in the setting of cardiac hypertrophy.