Breeding Capsicum chinense Lines with High Levels of Capsaicinoids and Capsinoids in the Fruit

Capsaicinoids, which cause a hot sensation when eaten, are uniquely present in pepper (Capsicum sp.) and are biosynthesized by combining vanillyl amine with branched fatty acids. A mutation in the gene encoding putative aminotransferase (pAMT)—the enzyme that normally biosynthesizes the capsaicinoid precursor vanillyl amine—leads instead to the biosynthesis of vanillyl alcohol, which combines with branched fatty acids to form capsinoids. Here, we report a method for increasing the capsaicinoid and capsinoid contents using quantitative trait locus (QTL) alleles involved in capsaicinoid biosynthesis in the pericarps of extremely spicy peppers. QTLs for capsinoid contents were detected on chromosome 6 and 10 using an F2 population from ‘SNU11–001’ and ‘Bhut Jolokia (BJ)’ (‘SJ’). ‘SNU11–001’ contains high capsinoid contents and ‘BJ’ contains high capsaicinoid contents in both the placenta and pericarp. These QTLs overlapped QTL regions associated with pungency in the pericarp. ‘BJ’ was crossed also with ‘Habanero’ (‘HB’), which contains capsaicinoids mainly in the placenta, and the resulting (‘HJ’) F2 and F3 offspring with ‘BJ’ genotypes were selected based on QTL markers and the pericarp pungency phenotype. Similarly, F2 and F3 offspring with high capsinoid contents in the pericarp were selected in ‘SJ’ with reference to ‘BJ’ genotypes at the QTLs. Through continuous self-pollination, ‘SJ’ and ‘BJ’ lines with high capsinoid and capsaicinoid contents, respectively, in both the placenta and pericarp were developed. This study is the first to show that lines containing high levels of capsinoids and capsaicinoids can be bred using pericarp capsaicinoid biosynthesis genes.

Production of 10-methyl branched fatty acids in yeast

Background Despite the environmental value of biobased lubricants, they account for less than 2% of global lubricant use due to poor thermo-oxidative stability arising from the presence of unsaturated double bonds. Methyl branched fatty acids (BFAs), particularly those with branching near the acyl-chain mid-point, are a high-performance alternative to existing vegetable oils because of their low melting temperature and full saturation. Results We cloned and characterized two pathways to produce 10-methyl BFAs isolated from actinomycetes and γ-proteobacteria. In the two-step bfa pathway of actinomycetes, BfaB methylates Δ9 unsaturated fatty acids to form 10-methylene BFAs, and subsequently, BfaA reduces the double bond to produce a fully saturated 10-methyl branched fatty acid. A BfaA-B fusion enzyme increased the conversion efficiency of 10-methyl BFAs. The ten-methyl palmitate production (tmp) pathway of γ-proteobacteria produces a 10-methylene intermediate, but the TmpA putative reductase was not active in E. coli or yeast. Comparison of BfaB and TmpB activities revealed a range of substrate specificities from C14-C20 fatty acids unsaturated at the Δ9, Δ10 or Δ11 position. We demonstrated efficient production of 10-methylene and 10-methyl BFAs in S. cerevisiae by secretion of free fatty acids and in Y. lipolytica as triacylglycerides, which accumulated to levels more than 35% of total cellular fatty acids. Conclusions We report here the characterization of a set of enzymes that can produce position-specific methylene and methyl branched fatty acids. Yeast expression of bfa enzymes can provide a platform for the large-scale production of branched fatty acids suitable for industrial and consumer applications.