Engineering Rhodosporidium toruloides for limonene production

Background Limonene is a widely used monoterpene in the production of food, pharmaceuticals, biofuels, etc. The objective of this work was to engineer Rhodosporidium toruloides as a cell factory for the production of limonene. Results By overexpressing the limonene synthase (LS), neryl pyrophosphate synthase (NPPS)/geranyl pyrophosphate synthase and the native hydroxy-methyl-glutaryl-CoA reductase (HMGR), we established a baseline for limonene production based on the mevalonate route in Rhodosporidium toruloides. To further enhance the limonene titer, the acetoacetyl-CoA thiolase/HMGR (EfMvaE) and mevalonate synthase (EfMvaS) from Enterococcus faecalis, the mevalonate kinase from Methanosarcina mazei (MmMK) and the chimeric enzyme NPPS-LS were introduced in the carotenogenesis-deficient strain. The resulting strains produced a maximum limonene titer of 393.5 mg/L. Conclusion In this study, we successfully engineered the carotenogenesis yeast R. toruloides to produce limonene. This is the first report on engineering R. toruloides toward limonene production based on NPP and the fusion protein SltNPPS-CltLS. The results demonstrated that R. toruloides is viable for limonene production, which would provide insights into microbial production of valuable monoterpenes.

Computer-Aid Directed Evolution of GPPS and PS Enzymes

Pinene, a natural active monoterpene, is widely used as a flavoring agent, perfume, medicine, and biofuel. Although genetically engineered microorganisms have successfully produced pinene, to date, the biological yield of pinene is much lower than that of semiterpenes (isoprene) and sesquiterpenes (farnesene). In addition to the low heterologous expression of geranyl pyrophosphate synthase (GPPS) and pinene synthase (PS), cytotoxicity due to accumulation of the monoterpene also limits the production of pinene in microorganisms. In this study, we attempted to use two strategies to increase the biological yield of pinene. By deleting the random coils of GPPS and PS alone or in combination, a strain with a 335% yield increase was obtained. Additionally, upon computer-guided molecular modeling and docking of GPPS with isopentenyl pyrophosphate (IPP), its substrate, the key sites located within the catalytic pocket for substrate binding, was predicted. After screening, a strain harboring the T273R mutation of GPPS was selected among a batch of mutations of the key sites with a 154% increase in pinene yield.

Expression analysis of key enzymes involved in the accumulation of iridoid in Rehmannia glutinosa

As the traditional Chinese herb, Rehmannia glutinosa (R. glutinosa) has significant effects on health. The main active ingredient of R. glutinosa is iridoid. In this study, root, stem and leaf of R. glutinosa at five different growth stages were collected, the content of iridoid in R. glutinosa was determinated, and the expression of key enzymes in R. glutinosa was analyzed by quantitative real-time PCR (q-PCR). We found that the content of iridoid was increased continuously during the first three growth stages (I-III) of R. glutinosa. It reached the maximum value in root and leaf at growth stage III of R. glutinosa. However, the content of iridoid was not lower in leaf, compared with root. 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR), Geranyl pyrophosphate synthase (GPPS), Geraniol 10-hydroxylase (G10H) and 10-hydroxygeraniol oxidoreductase (10HGO) were the key enzymes involved in the synthesis of iridoid in plant, but their expression pattern or level was different in various tissues and stages of R. glutinosa. All appeared tissue specificity and their expression was related to the accumulation of iridoid in R. glutinosa. Thus, DXR, GPPS, G10H and 10HGO might take part in the synthesis of iridoid in R. glutinosa and could be differently regulated during the growth and development of R. glutinosa, which would provide theoretical basis for the research on secondary metabolism of iridoid in R. glutinosa.