Transcriptomic and Proteomic Characterizations of the Molecular Response to Blue Light and Salicylic Acid in Haematococcus pluvialis

Haematococcus pluvialis accumulates a large amount of astaxanthin under various stresses, e.g., blue light and salicylic acid (SA). However, the metabolic response of H. pluvialis to blue light and SA is still unclear. We investigate the effects of blue light and SA on the metabolic response in H. pluvialis using both transcriptomic and proteomic sequencing analyses. The largest numbers of differentially expressed proteins (DEPs; 324) and differentially expressed genes (DEGs; 13,555) were identified on day 2 and day 7 of the treatment with blue light irradiation (150 μmol photons m−2s−1), respectively. With the addition of SA (2.5 mg/L), a total of 63 DEPs and 11,638 DEGs were revealed on day 2 and day 7, respectively. We further analyzed the molecular response in five metabolic pathways related to astaxanthin synthesis, including the astaxanthin synthesis pathway, the fatty acid synthesis pathway, the heme synthesis pathway, the reactive oxygen species (ROS) clearance pathway, and the cell wall biosynthesis pathway. Results show that blue light causes a significant down-regulation of the expression of key genes involved in astaxanthin synthesis and significantly increases the expression of heme oxygenase, which shows decreased expression by the treatment with SA. Our study provides novel insights into the production of astaxanthin by H. pluvialis treated with blue light and SA.

Transcription Factor-Based Biosensor for Dynamic Control in Yeast for Natural Product Synthesis

The synthesis of natural products in yeast has gained remarkable achievements with intensive metabolic engineering efforts. In particular, transcription factor (TF)-based biosensors for dynamic control of gene circuits could facilitate strain evaluation, high-throughput screening (HTS), and adaptive laboratory evolution (ALE) for natural product synthesis. In this review, we summarized recent developments of several TF-based biosensors for core intermediates in natural product synthesis through three important pathways, i.e., fatty acid synthesis pathway, shikimate pathway, and methylerythritol-4-phosphate (MEP)/mevalonate (MVA) pathway. Moreover, we have shown how these biosensors are implemented in synthetic circuits for dynamic control of natural product synthesis and also discussed the design/evaluation principles for improved biosensor performance.

Propionic Acid-Based PET Imaging of the Fatty Acid Synthesis Pathway in Prostate Cancer

BackgroundThe aim of this study was to evaluate the potential value of 2-[18F]fluoropropionic acid ([18F]FPA) for PET imaging of prostate cancer (PCa) and to confirm the correlation between [18F]FPA accumulation and fatty acid synthase (FASN) levels in PCa models. The results of the first [18F]FPA PET study of a PCa patient are reported.MethodsA PET imaging comparison of [18F]FDG and [18F]FPA was performed in LNCaP, PC-3 and DU145 tumors. Additionally, in vivo blocking experiments in those models were conducted with orlistat. Western blotting staining of FASN were performed in the those xenograft tumors.ResultsThe uptake of [18F]FPA in the LNCaP and PC-3 tumors was higher than that of [18F]FDG (P<0.05 and P<0.05), while [18F]FDG was significantly superior to [18F]FPA in detecting DU145 tumors (P<0.05). Grayscale scanning showed that FASN expression in the LNCaP and PC-3 tumors was 33.3% and 10.3% higher than that in the DU145 tumors, respectively. The accumulation (% ID/g) of [18F]FPA in the LNCaP , PC-3 and DU145 tumors decreased by 27.6, 40.5 and 11.7%, respectively, after treatment with orlistat. The [18F]FPA showed higher tumor/background ratios than [18F]FDG in the first PCa patient (P<0.05).ConclusionsThe [18F]FPA uptake in PCa models was positively correlated with FASN expression and could be reduced after administration of a single FASN inhibitor. In addition, the [18F]FPA is a potential broad-spectrum PET imaging agent, and the imaging effect of [18F]FPA may be superior to [18F]FDG in human PCa.

Raman-guided subcellular pharmaco-metabolomics for metastatic melanoma cells

Non-invasively probing metabolites within single live cells is highly desired but challenging. Here we utilize Raman spectro-microscopy for spatial mapping of metabolites within single cells, with the specific goal of identifying druggable metabolic susceptibilities from a series of patient-derived melanoma cell lines. Each cell line represents a different characteristic level of cancer cell de-differentiation. First, with Raman spectroscopy, followed by stimulated Raman scattering (SRS) microscopy and transcriptomics analysis, we identify the fatty acid synthesis pathway as a druggable susceptibility for differentiated melanocytic cells. We then utilize hyperspectral-SRS imaging of intracellular lipid droplets to identify a previously unknown susceptibility of lipid mono-unsaturation within de-differentiated mesenchymal cells with innate resistance to BRAF inhibition. Drugging this target leads to cellular apoptosis accompanied by the formation of phase-separated intracellular membrane domains. The integration of subcellular Raman spectro-microscopy with lipidomics and transcriptomics suggests possible lipid regulatory mechanisms underlying this pharmacological treatment. Our method should provide a general approach in spatially-resolved single cell metabolomics studies.

Genetic Regulation of the Bacterial Omega-3 Polyunsaturated Fatty Acid Biosynthesis Pathway

ABSTRACT A characteristic among many marine Gammaproteobacteria is the biosynthesis and incorporation of omega-3 polyunsaturated fatty acids into membrane phospholipids. The biosynthesis of eicosapentaenoic acid (EPA) and/or docosahexaenoic acid (DHA) is mediated by a polyketide/fatty acid synthase mechanism encoded by a set of five genes, pfaABCDE. This unique fatty acid synthesis pathway coexists with the principal type II dissociated fatty acid synthesis pathway, which is responsible for the biosynthesis of core saturated, monounsaturated, and hydroxylated fatty acids used in phospholipid and lipid A biosynthesis. In this work, a genetic approach was undertaken to elucidate genetic regulation of the pfa genes in the model marine bacterium Photobacterium profundum SS9. Using a reporter gene fusion, we showed that expression of the pfa operon is downregulated in response to exogenous fatty acids, particularly long-chain monounsaturated fatty acids. This regulation occurs independently of the canonical fatty acid regulators, FabR and FadR, present in P. profundum SS9. Transposon mutagenesis and screening of a library of mutants identified a novel transcriptional regulator, which we have designated pfaF, to be responsible for the observed regulation of the pfa operon in P. profundum SS9. Gel mobility shift and DNase I footprinting assays confirmed that PfaF binds the pfaA promoter and identified the PfaF binding site. IMPORTANCE The production of long-chain omega-3 polyunsaturated fatty acids (PUFA) by marine Gammaproteobacteria, particularly those from deep-sea environments, has been known for decades. These unique fatty acids are produced by a polyketide-type mechanism and subsequently incorporated into the phospholipid membrane. While much research has focused on the biosynthesis genes, their products, and the phylogenetic distribution of these gene clusters, no prior studies have detailed the genetic regulation of this pathway. This study describes how this pathway is regulated under various culture conditions and has identified and characterized a fatty acid-responsive transcriptional regulator specific to PUFA biosynthesis.