In Vitro Multienzyme Pathway Assembly for Isoprenoids and Isoprenoid Precursors Production

Ph.DDOCTOR OF PHILOSOPH

Site-directed mutagenesis of structural hot spots for enhanced solubility of deoxyxylulose phosphate pathway enzymes

Increasing the metabolic flux through a biochemical pathway is highly desirable for metabolic engineering. One strategy is to enhance the solubility of overexpressed pace-making enzymes. Accurate theoretical prediction of target mutation sites is instrumental to reduce the experimental efforts and speed up the optimization process. In this study, the rate-limiting steps along the non-mevalonate (DXP) pathway, namely E. coli Dxs and IspG, were used as the model enzymes to learn and develop a set of bioinformatics tools that would enable rational optimization of enzyme solubility. TANGO prediction was first used to identify the aggregation-prone regions (APRs), and then SIFT analysis was carried out to eliminate the non-tolerable amino acids in the APRs. Preliminary results have shown that 5 out of 8 tested mutations have resulted in an increase in Dxs solubility. Similarly, 7 out of 12 IspG mutants have displayed enhanced solubility. Importantly, the in vivo activities of the more soluble mutants were improved. Taken together, the solubility of both Dxs and IspG were enhanced by ~2-fold, by targeted single amino acid mutation. The study demonstrated rapid improvement of enzyme solubility by combinations of computational tools. The information gained would be useful for rational engineering of over-expressed pathway enzymes and improve pathway efficiencies

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

Engineering an artificial pathway for Cis-Α-irone biosynthesis

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Plasma phospholipid arachidonic acid in relation to non-alcoholic fatty liver disease:Mendelian randomization study

Objectives: The role of plasma phospholipid arachidonic acid (AA) in the development of non-alcoholic fatty liver disease (NALFD), cirrhosis, and liver cancer remains unclear. This study aimed to determine the causality of the associations of plasma phospholipid AA with NALFD, cirrhosis, and liver cancer using Mendelian ran-domization analysis. Methods: Nine independent single-nucleotide polymorphisms associated with plasma phospholipid AA at the genome-wide significance were used as instrumental variables. Summary-level data for three outcomes were obtained from 1) a genome-wide association study for NAFLD, 2) the UK Biobank study, and 3) the Finn -Gen study. The sensitivity analysis excluding the pleiotropic variant rs174547 in the FADS1 gene was per-formed. Estimates from different sources were combined using the fixed-effects meta-analysis method. Results: Per standard deviation increase in AA levels, the combined odds ratio was 1.06 (95% confidence inter-val, 1.02-1.11; P = 0.008) for NAFLD, 1.05 (95% confidence interval, 1.01-1.09; P = 0.009) for cirrhosis, and 0.99 (95% confidence interval, 0.94-1.05; P = 0.765) for liver cancer. The associations remained stable in the sensitivity analysis excluding rs174547. Conclusions: This study suggests potential causal associations of high levels of plasma phospholipid AA with the risk of NAFLD and cirrhosis.De två sista författarna delar sistaförfattarskapet.</p

Engineering an artificial pathway for Cis-alpha-irone biosynthesis

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Structural understanding of fungal terpene synthases for terpene product cyclization

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Antioxidants, Minerals, and Vitamins in Relation to Crohn’s disease and Ulcerative Colitis:A Mendelian Randomization Study

Background Evidence for antioxidants, minerals and vitamins in relation to the risk of Crohn's disease (CD) and ulcerative colitis (UC) is limited and inconsistent. This mendelian randomization (MR) study aimed to examine the causal associations of circulating levels of antioxidants, minerals and vitamins with CD and UC. Methods Single-nucleotide polymorphisms associated with antioxidants (beta-carotene, lycopene and uric acid), minerals (copper, calcium, iron, magnesium, phosphorus, zinc and selenium), and vitamins (folate, vitamins A, B6, B12, C, D, E and K1) were employed as instrumental variables. Genetic associations with CD and UC were extracted from the UK Biobank, the FinnGen study and the International Inflammatory Bowel Disease Genetics Consortium. The inverse variance weighted method and sensitivity analyses were performed. Results Genetically predicted higher lycopene (OR = 0.94, 95% CI: 0.91–0.97), vitamins D (OR = 0.65, 95% CI: 0.54–0.79) and K1 (OR = 0.93, 95% CI: 0.90–0.97) levels were inversely associated with CD risk, whereas genetically predicted higher magnesium (OR = 1.53, 95% CI: 1.23–1.90) levels were positively associated with CD risk. Higher levels of genetically predicted lycopene (OR = 0.91, 95% CI: 0.88–0.95), phosphorus (OR = 0.69, 95% CI: 0.58–0.82), selenium (OR = 0.91, 95% CI: 0.85–0.97), zinc (OR = 0.91, 95% CI: 0.89–0.94), folate (OR = 0.71, 95% CI: 0.56–0.92) and vitamin E (OR = 0.78, 95% CI: 0.69–0.88) were associated with reduced UC risk, whereas genetically predicted high levels of calcium (OR = 1.46, 95% CI: 1.22–1.76) and magnesium (OR = 1.24, 95% CI: 1.03–1.49) were associated with increased risk of UC. Conclusions Our study provided evidence that circulating levels of antioxidants, minerals and vitamins might be causally linked to the development of IBD

Therapeutic role of interleukin-1 receptor antagonist in pancreatic diseases: mendelian randomization study

BackgroundThe interleukin-1 pathway has been linked to pancreatic diseases. We applied the Mendelian randomization approach to explore whether higher interleukin-1 receptor antagonist (IL-1RA) levels reduce the risk of acute and chronic pancreatitis and pancreatic cancer.MethodsGenetic variants associated with blood IL-1RA levels at the genome-wide significance level and located 5MB downstream or upstream of the IL1RN gene were extracted from a genome-wide meta-analysis of 21,758 participants. After pruning, genetic variants without linkage disequilibrium were used as genetic instrument for IL-1RA. Summary-level data on acute and chronic pancreatitis and pancreatic cancer were obtained from the UK Biobank and FinnGen studies. The associations were meta-analyzed for one outcome from two sources.ResultsGenetically predicted higher levels of IL-1RA were associated with a lower risk of acute and chronic pancreatitis and pancreatic cancer. In the meta-analysis of UK Biobank and FinnGen, the combined odds ratio was 0.87 (95% confidence interval [CI] 0.77-0.97, P=0.003) for acute pancreatitis, 0.73 (95% CI 0.65-0.82, P=2.93×10-8) for chronic pancreatitis, and 0.86 (95% CI 0.77-0.96, P=0.009) for pancreatic cancer per one standard deviation increment in genetically predicted levels of IL-1RA.ConclusionThis study suggests a protective role of IL-1RA in three major pancreatic diseases, which hints the therapeutic potentials of IL-1RA in pancreatic diseases