The Coordinated Upregulated Expression of Genes Involved in MEP, Chlorophyll, Carotenoid and Tocopherol Pathways, Mirrored the Corresponding Metabolite Contents in Rice Leaves during De-Etiolation

Light is an essential regulator of many developmental processes in higher plants. We investigated the effect of 4-hydroxy-3-methylbut-2-enyl diphosphate reductase 1/2 genes (OsHDR1/2) and isopentenyl diphosphate isomerase 1/2 genes (OsIPPI1/2) on the biosynthesis of chlorophylls, carotenoids, and phytosterols in 14-day-old etiolated rice (Oyza sativa L.) leaves during de-etiolation. However, little is known about the effect of isoprenoid biosynthesis genes on the corresponding metabolites during the de-etiolation of etiolated rice leaves. The results showed that the levels of α-tocopherol were significantly increased in de-etiolated rice leaves. Similar to 1-deoxy-D-xylulose-5-phosphate synthase 3 gene (OsDXS3), both OsDXS1 and OsDXS2 genes encode functional 1-deoxy-D-xylulose-5-phosphate synthase (DXS) activities. Their expression patterns and the synthesis of chlorophyll, carotenoid, and tocopherol metabolites suggested that OsDXS1 is responsible for the biosynthesis of plastidial isoprenoids in de-etiolated rice leaves. The expression analysis of isoprenoid biosynthesis genes revealed that the coordinated expression of the MEP (2-C-methyl-D-erythritol 4-phosphate) pathway, chlorophyll, carotenoid, and tocopherol pathway genes mirrored the changes in the levels of the corresponding metabolites during de-etiolation. The underpinning mechanistic basis of coordinated light-upregulated gene expression was elucidated during the de-etiolation process, specifically the role of light-responsive cis-regulatory motifs in the promoter region of these genes. In silico promoter analysis showed that the light-responsive cis-regulatory elements presented in all the promoter regions of each light-upregulated gene, providing an important link between observed phenotype during de-etiolation and the molecular machinery controlling expression of these genes.

Stable isotope and chemical inhibition analyses suggested the existence of a non-mevalonate-like pathway in the yeast Yarrowia lipolytica

Methyl erythritol phosphate (MEP) is the metabolite found in the MEP pathway for isoprenoid biosynthesis, which is known to be utilized by plants, algae, and bacteria. In this study, an unprecedented observation was found in the oleaginous yeast Yarrowia lipolytica, in which one of the chromatographic peaks was annotated as MEP when cultivated in the nitrogen limiting condition. This finding raised an interesting hypothesis of whether Y. lipolytica utilizes the MEP pathway for isoprenoid biosynthesis or not, because there is no report of yeast harboring the MEP pathway. Three independent approaches were used to investigate the existence of the MEP pathway in Y. lipolytica; the spiking of the authentic standard, the MEP pathway inhibitor, and the 13C labeling incorporation analysis. The study suggested that the mevalonate and MEP pathways co-exist in Y. lipolytica and the nitrogen limiting condition triggers the utilization of the MEP pathway in Y. lipolytica.

Cross-talk between the methylerythritol phosphate and mevalonic acid pathways of isoprenoid biosynthesis in poplar

Plants use two distinct isoprenoid biosynthesis pathways: the methylerythritol phosphate (MEP) pathway and mevalonic acid (MVA) pathway. 1-deoxy-D-xylulose5-phosphate synthase (DXS) and 1-deoxy-D-xylulose5-phosphate reductoisomerase (DXR) are the rate-limiting enzymes in the MEP pathway, and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) is a key regulatory enzyme in the MVA pathway. Previously, overexpression of Populus trichocarpa PtDXR in Nanlin 895 poplar was found to upregulate resistance to salt and drought stresses, and the transgenic poplars showed improved growth. In the present study, PtHMGR overexpressors (OEs) exhibited higher expression levels of DXS, DXR, 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase (HDS), and 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase (HDR) and lower expression levels of 2-C-methyl-d-erythritol4-phosphate cytidylyltransferase (MCT), 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (CMK), and 3-hydroxy-3-methylglutaryl-coenzyme A synthase (HMGS) than non-transgenic poplars (NT). However, the poplar PtDXR-OEs showed upregulated expression levels of MEP-related genes and downregulated expression of MVA-related genes. Moreover, overexpression of PtDXR and PtHMGR in poplars caused changes in MVA-derived trans-zeatin-riboside (TZR), isopentenyl adenosine (IPA), castasterone (CS) and 6-deoxocastasterone (DCS), as well as MEP-derived carotenoids, gibberellins (GAs), and abscisic acid (ABA). In PtHMGR-OEs, greater accumulation of geranyl diphosphate synthase (GPS) and geranyl pyrophosphate synthase (GPPS) transcript levels in the MEP pathway led to accumulation of MEP-derived isoprenoids, while upregulation of farnesyl diphosphate synthase (FPS) expression in the MVA pathway contributed to increased levels of MVA-derived isoprenoids. Similarly, in PtDXR-OEs, increased GPS and GPPS transcript levels in the MEP pathway boosted MEP-derived isoprenoid levels and changes in FPS expression affected MVA-derived isoprenoid yields. From these results, we can conclude that cross-talk exists between the MVA and MEP pathways.

Growth-uncoupled isoprenoid synthesis in Rhodobacter sphaeroides

Background: Microbial cell factories are usually engineered and employed for cultivations that combine product synthesis with growth. Such a strategy inevitably invests part of the substrate pool towards the generation of biomass and cellular maintenance. Hence, engineering strains for the formation of a specific product under non-growth conditions would allow to reach higher product yields. In this respect, isoprenoid biosynthesis represents an extensively studied example of growth-coupled synthesis with rather unexplored potential for growth-independent production. Rhodobacter sphaeroides is a model bacterium for isoprenoid biosynthesis, either via the native 2-methyl-D-erythritol 4-phosphate (MEP) pathway or the heterologous mevalonate (MVA) pathway, and for poly-β-hydroxybutyrate (PHB) biosynthesis.Results: This study investigates the use of this bacterium for growth-independent production of isoprenoids, with amorpha-4,11-diene as reporter molecule. For this purpose, we employed the recently developed Cas9-based genome editing tool for R. sphaeroides to rapidly construct single and double deletion mutant strains of the MEP and PHB pathways, and we subsequently transformed the strains with the amorphadiene producing plasmid. Furthermore, we employed 13C-metabolic flux ratio analysis to monitor the changes in the isoprenoid metabolic fluxes under different cultivation conditions. We demonstrated that active flux via both isoprenoid pathways while inactivating PHB synthesis maximizes growth-coupled isoprenoid synthesis. On the other hand, the strain that showed the highest growth-independent isoprenoid yield and productivity, combined the plasmid-based heterologous expression of the orthogonal MVA pathway with the inactivation of the native MEP and PHB production pathways.Conclusions: Apart from proposing a microbial cell factory for growth-independent isoprenoid synthesis, this work provides novel insights about the interaction of MEP and MVA pathways under different growth-conditions.