Background The oleaginous, carotenogenic yeast Rhodotorula toruloides has been increasingly explored as a platform organism for the production of terpenoids and fatty acid derivatives. Fatty alcohols, a fatty acid derivative widely used in the production of detergents and surfactants, can be produced microbially with the expression of a heterologous fatty acyl-CoA reductase. Due to its high lipid production, R. toruloides has high potential for fatty alcohol production, and in this study several metabolic engineering approaches were investigated to improve the titer of this product. Results Fatty acyl-CoA reductase from Marinobacter aqueolei was co-expressed with SpCas9 in R. toruloides IFO0880 and a panel of gene overexpressions and Cas9-mediated gene deletions were explored to increase the fatty alcohol production. Two overexpression targets (ACL1 and ACC1, improving cytosolic acetyl-CoA and malonyl-CoA production, respectively) and two deletion targets (the acyltransferases DGA1 and LRO1) resulted in significant (1.8 to 4.4-fold) increases to the fatty alcohol titer in culture tubes. Combinatorial exploration of these modifications in bioreactor fermentation culminated in a 3.7 g/L fatty alcohol titer in the LRO1Δ mutant. As LRO1 deletion was not found to be beneficial for fatty alcohol production in other yeasts, a lipidomic comparison of the DGA1 and LRO1 knockout mutants was performed, finding that DGA1 is the primary acyltransferase responsible for triacylglyceride production in R. toruloides, while LRO1 disruption simultaneously improved fatty alcohol production, increased diacylglyceride and triacylglyceride production, and increased glucose consumption. Conclusions The fatty alcohol titer of fatty acyl-CoA reductase-expressing R. toruloides was significantly improved through the deletion of LRO1, or the deletion of DGA1 combined with overexpression of ACC1 and ACL1. Disruption of LRO1 surprisingly increased both lipid and fatty alcohol production, creating a possible avenue for future study of the lipid metabolism of this yeast.

Peru is home to six of the main genetic clusters of cocoa (Theobroma cacao L.) and is an important exporter of fine-flavor cocoa varieties. Two varieties of Peruvian native cocoa, in high demand for the fine chocolate market, grow in the regions of Bagua (Amazonas) and Quillabamba (Cusco). The main chemical and organoleptic characteristics of these two varieties were determined. Cocoa pastes and chocolates made with cocoa from these regions were subjected to chemical proximate analysis and determination of fatty acid profile, theobromine, caffeine, total phenolics, total flavonoids, antioxidant activity, and main sensory attributes. A multiple factor analysis (MFA) was applied to the data. Samples from Quillabamba differ from those of Bagua mainly by having a higher fat and lower theobromine content. The main sensory attributes detected for Bagua samples that differ from, those of Quillabamba were fruity, acidic, astringent, and bitter notes.

Rhodotorula toruloides has been increasingly explored as a host for bioproduction of lipids, fatty acid derivatives, and terpenoids. Various genetic tools have been developed, but neither a centromere nor an autonomously replicating sequence (ARS), both necessary elements for stable episomal plasmid maintenance, have yet been reported. In this study, Cleavage Under Targets and Release Using Nuclease (CUT&RUN), a method used for genome-wide mapping DNA-protein interactions, was used to identify R. toruloides IFO0880 genomic regions associated with the centromeric histone H3 protein Cse4, a marker of centromeric DNA. Fifteen putative centromeres ranging from 8 to 19 kb in length were identified and analyzed, and four were tested for, but did not show, ARS activity. These centromeric sequences contained below average GC content, corresponded to transcriptional cold-spots, were primarily nonrepetitive, and shared some vestigial transposon-related sequences but otherwise did not show significant sequence conservation. Future efforts to identify an ARS in this yeast can utilize these centromeric DNA sequences to improve the stability of episomal plasmids derived from putative ARS elements.