The impact of genome evolution on the allotetraploid Nicotiana rustica - an intriguing story of enhanced alkaloid production.

BACKGROUND: Nicotiana rustica (Aztec tobacco), like common tobacco (Nicotiana tabacum), is an allotetraploid formed through a recent hybridization event; however, it originated from completely different progenitor species. Here, we report the comparative genome analysis of wild type N. rustica (5 Gb; 2n = 4x = 48) with its three putative diploid progenitors (2.3-3 Gb; 2n = 2x =24), Nicotiana undulata, Nicotiana paniculata and Nicotiana knightiana. RESULTS: In total, 41% of N. rustica genome originated from the paternal donor (N. undulata), while 59% originated from the maternal donor (N. paniculata/N. knightiana). Chloroplast genome and gene analyses indicated that N. knightiana is more closely related to N. rustica than N. paniculata. Gene clustering revealed 14,623 ortholog groups common to other Nicotiana species and 207 unique to N. rustica. Genome sequence analysis indicated that N. knightiana is more closely related to N. rustica than N. paniculata, and that the higher nicotine content of N. rustica leaves is the result of the progenitor genomes combination and of a more active transport of nicotine to the shoot. CONCLUSIONS: The availability of four new Nicotiana genome sequences provide insights into how speciation impacts plant metabolism, and in particular alkaloid transport and accumulation, and will contribute to better understanding the evolution of Nicotiana species.

Genomic impact of cigarette smoke, with application to three smoking-related diseases.

There is considerable evidence that inhaled toxicants such as cigarette smoke can cause both irreversible changes to the genetic material (DNA mutations) and putatively reversible changes to the epigenetic landscape (changes in the DNA methylation and chromatin modification state). The diseases that are believed to involve genetic and epigenetic perturbations include lung cancer, chronic obstructive pulmonary disease (COPD), and cardiovascular disease (CVD), all of which are strongly linked epidemiologically to cigarette smoking. In this review, we highlight the significance of genomics and epigenomics in these major smoking-related diseases. We also summarize the in vitro and in vivo findings on the specific perturbations that smoke and its constituent compounds can inflict upon the genome, particularly on the pulmonary system. Finally, we review state-of-the-art genomics and new techniques such as high-throughput sequencing and genome-wide chromatin assays, rapidly evolving techniques which have allowed epigenetic changes to be characterized at the genome level. These techniques have the potential to significantly improve our understanding of the specific mechanisms by which exposure to environmental chemicals causes disease. Such mechanistic knowledge provides a variety of opportunities for enhanced product safety assessment and the discovery of novel therapeutic interventions.

FabG, an NADPH-dependent 3-ketoacyl reductase of Pseudomonas aeruginosa, provides precursors for medium-chain-length poly-3-hydroxyalkanoate biosynthesis in Escherichia coli.

Escherichia coli hosts expressing fabG of Pseudomonas aeruginosa showed 3-ketoacyl coenzyme A (CoA) reductase activity toward R-3-hydroxyoctanoyl-CoA. Furthermore, E. coli recombinants carrying the poly-3-hydroxyalkanoate (PHA) polymerase-encoding gene phaC in addition to fabG accumulated medium-chain-length PHAs (mcl-PHAs) from alkanoates. When E. coli fadB or fadA mutants, which are deficient in steps downstream or upstream of the 3-ketoacyl-CoA formation step during beta-oxidation, respectively, were transformed with fabG, higher levels of PHA were synthesized in E. coli fadA, whereas similar levels of PHA were found in E. coli fadB, compared with those of the corresponding mutants carrying phaC alone. These results strongly suggest that FabG of P. aeruginosa is able to reduce mcl-3-ketoacyl-CoAs generated by the beta-oxidation to 3-hydroxyacyl-CoAs to provide precursors for the PHA polymerase.

Properties of engineered poly-3-hydroxyalkanoates produced in recombinant Escherichia coli strains.

To prepare medium-chain-length poly-3-hydroxyalkanoates (PHAs) with altered physical properties, we generated recombinant Escherichia coli strains that synthesized PHAs with altered monomer compositions. Experiments with different substrates (fatty acids with different chain lengths) or different E. coli hosts failed to produce PHAs with altered physical properties. Therefore, we engineered a new potential PHA synthetic pathway, in which ketoacyl-coenzyme A (CoA) intermediates derived from the beta-oxidation cycle are accumulated and led to the PHA polymerase precursor R-3-hydroxyalkanoates in E. coli hosts. By introducing the poly-3-hydroxybutyrate acetoacetyl-CoA reductase (PhbB) from Ralstonia eutropha and blocking the ketoacyl-CoA degradation step of the beta-oxidation, the ketoacyl-CoA intermediate was accumulated and reduced to the PHA precursor. Introduction of the phbB gene not only caused significant changes in the monomer composition but also caused changes of the physical properties of the PHA, such as increase of polymer size and loss of the melting point. The present study demonstrates that pathway engineering can be a useful approach for producing PHAs with engineered physical properties.