DESIGN AND DEVELOPMENT OF MOLECULAR TOOLS FOR THE COMBINATORIAL MULTIVARIANT-MODULAR APPROACH TO ENGINEERING METABOLIC PATHWAYS IN E.COLI

Ph.DDOCTOR OF PHILOSOPH

Experimental design-aided systematic pathway optimization of glucose uptake and deoxyxylulose phosphate pathway for improved amorphadiene production

Artemisinin is a potent antimalarial drug; however, it suffers from unstable and insufficient supply from plant source. Here, we established a novel multivariate-modular approach based on experimental design for systematic pathway optimization that succeeded in improving the production of amorphadiene (AD), the precursor of artemisinin, in Escherichia coli. It was initially found that the AD production was limited by the imbalance of glyceraldehyde 3-phosphate (GAP) and pyruvate (PYR), the two precursors of the 1-deoxy-d-xylulose-5-phosphate (DXP) pathway. Furthermore, it was identified that GAP and PYR could be balanced by replacing the phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) with the ATP-dependent galactose permease and glucose kinase system (GGS) and this resulted in fivefold increase in AD titer (11 to 60 mg/L). Subsequently, the experimental design-aided systematic pathway optimization (EDASPO) method was applied to systematically optimize the transcriptional expressions of eight critical genes in the glucose uptake and the DXP and AD synthesis pathways. These genes were classified into four modules and simultaneously controlled by T7 promoter or its variants. A regression model was generated using the four-module experimental data and predicted the optimal expression ratios among these modules, resulting in another threefold increase in AD titer (60 to 201 mg/L). This EDASPO method may be useful for the optimization of other pathways and products beyond the scope of this study.Singapore-MIT Alliance for Research and Technology (SMART

Development of a microRNA Panel for Classification of Abnormal Mammograms for Breast Cancer

Mammography is extensively used for breast cancer screening but has high false-positive rates. Here, prospectively collected blood samples were used to identify circulating microRNA (miRNA) biomarkers to discriminate between malignant and benign breast lesions among women with abnormal mammograms. The Discovery cohort comprised 72 patients with breast cancer and 197 patients with benign breast lesions, while the Validation cohort had 73 and 196 cancer and benign cases, respectively. Absolute expression levels of 324 miRNAs were determined using RT-qPCR. miRNA biomarker panels were identified by: (1) determining differential expression between malignant and benign breast lesions, (2) focusing on top differentially expressed miRNAs, and (3) building panels from an unbiased search among all expressed miRNAs. Two-fold cross-validation incorporating a feature selection algorithm and logistic regression was performed. A six-miRNA biomarker panel identified by the third strategy, had an area under the curve (AUC) of 0.785 and 0.774 in the Discovery and Validation cohorts, respectively, and an AUC of 0.881 when differentiating between cases versus those with benign lesions or healthy individuals with normal mammograms. Biomarker panel scores increased with tumor size, stage and number of lymph nodes involved. Our work demonstrates that circulating miRNA signatures can potentially be used with mammography to differentiate between patients with malignant and benign breast lesions

Enhancing solubility of deoxyxylulose phosphate pathway enzymes for microbial isoprenoid production

Background: Recombinant proteins are routinely overexpressed in metabolic engineering. It is well known that some over-expressed heterologous recombinant enzymes are insoluble with little or no enzymatic activity. This study examined the solubility of over-expressed homologous enzymes of the deoxyxylulose phosphate pathway (DXP) and the impact of inclusion body formation on metabolic engineering of microbes. Results: Four enzymes of this pathway (DXS, ISPG, ISPH and ISPA), but not all, were highly insoluble, regardless of the expression systems used. Insoluble dxs (the committed enzyme of DXP pathway) was found to be inactive. Expressions of fusion tags did not significantly improve the solubility of dxs. However, hypertonic media containing sorbitol, an osmolyte, successfully doubled the solubility of dxs, with the concomitant improvement in microbial production of the metabolite, DXP. Similarly, sorbitol significantly improved the production of soluble and functional ERG12, the committed enzyme in the mevalonate pathway. Conclusion: This study demonstrated the unanticipated findings that some over-expressed homologous enzymes of the DXP pathway were highly insoluble, forming inclusion bodies, which affected metabolite formation. Sorbitol was found to increase both the solubility and function of some of these over-expressed enzymes, a strategy to increase the production of secondary metabolites.Singapore–MIT Alliance for Research and Technology (Flagship Research Project

A Spatial-Temporal Transformer Architecture Using Multi-Channel Signals for Sleep Stage Classification

Sleep stage classification is a fundamental task in diagnosing and monitoring sleep diseases. There are 2 challenges that remain open: (1) Since most methods only rely on input from a single channel, the spatial-temporal relationship of sleep signals has not been fully explored. (2) Lack of sleep data makes models hard to train from scratch. Here, we propose a vision Transformer-based architecture to process multi-channel polysomnogram signals. The method is an end-to-end framework that consists of a spatial encoder, a temporal encoder, and an MLP head classifier. The spatial encoder using a pre-trained Vision Transformer captures spatial information from multiple PSG channels. The temporal encoder utilizing the self-attention mechanism understands transitions between nearby epochs. In addition, we introduce a tailored image generation method to extract features within multi-channel and reshape them for transfer learning. We validate our method on 3 datasets and outperform the state-of-the-art algorithms. Our method fully explores the spatial-temporal relationship among different brain regions and addresses the problem of data insufficiency in clinical environments. Benefiting from reformulating the problem as image classification, the method could be applied to other 1D-signal problems in the future.ISSN:1534-4320ISSN:1558-021

Mobile phase gradient used for the separation of DXP intermediates in the UPLC method.

*<p>Aqueous solution: 15 mM acetic acid and 10 mM tributylamine.</p

Analytical performance of the SPE UPLC-MS method.

*<p>Concentration of CDP-MEP for intra-day/inter-day variation was 0.25 µM.</p

Overexpression of ispG to alleviate efflux of MEC for lycopene production.

<p>SIDF: BL21 Gold (DE3) harboring pET-SIDF and pACLYC; SIDFG: BL21 Gold (DE3) harboring pET-SIDFG and pACLYC. (A) Lycopene production as a function of time; (B) Extracellular MEC concentration as a function of time; (C) Intracellular MEC concentration as a function of time; (D) Intracellular HMBPP concentration as a function of time. Presented data were average of triplicates and standard errors were drawn on the plot.</p

Novel reference genes for quantifying transcriptional responses of Escherichia coli to protein overexpression by quantitative PCR

Background: Accurate interpretation of quantitative PCR (qPCR) data requires normalization using constitutively expressed reference genes. Ribosomal RNA is often used as a reference gene for transcriptional studies in E. coli. However, the choice of reliable reference genes has not been systematically validated. The objective of this study is to identify a set of reliable reference genes for transcription analysis in recombinant protein over-expression studies in E. coli. Results: In this study, the meta-analysis of 240 sets of single-channel Affymetrix microarray data representing over-expressions of 63 distinct recombinant proteins in various E. coli strains identified twenty candidate reference genes that were stably expressed across all conditions. The expression of these twenty genes and two commonly used reference genes, rrsA encoding ribosomal RNA 16S and ihfB, was quantified by qPCR in E. coli cells over-expressing four genes of the 1-Deoxy-D-Xylulose 5-Phosphate pathway. From these results, two independent statistical algorithms identified three novel reference genes cysG, hcaT, and idnT but not rrsA and ihfB as highly invariant in two E. coli strains, across different growth temperatures and induction conditions. Transcriptomic data normalized by the geometric average of these three genes demonstrated that genes of the lycopene synthetic pathway maintained steady expression upon enzyme overexpression. In contrast, the use of rrsA or ihfB as reference genes led to the mis-interpretation that lycopene pathway genes were regulated during enzyme over-expression. Conclusion: This study identified cysG/hcaT/idnT to be reliable novel reference genes for transcription analysis in recombinant protein producing E. coli.Singapore-MIT Allianc