การปรับปรุงความสามารถในการผลิตลิปิดในยีสต์ โดยเทคนิคการวิเคราะห์เมตาโบลิกฟลักซ์ (ระยะที่ 1-2)
Metabolic Flux Analysis for Improving Lipid Production in Saccharomyces cerevisiae (Phase 1-2)

ดร.วศิมน เรืองเล็ก  (หัวหน้าโครงการ)
Vasimon Ruanglek. (Researcher)
สถาบันพัฒนาและฝึกอบรมโรงงานต้นแบบ
ดร.อาลักษณ์ ทิพยรัตน์
Aluck Thipayarat. (Lecturer)

ทุนหมวดเงินอุดหนุนประจำปี 2547 งบประมาณ 401,430 บาท
ทุนหมวดเงินอุดหนุนประจำปี 2548 งบประมาณ 362,560 บาท

ความสำคัญและที่มาของการวิจัย

Polyunsaturated fatty acids (PUFAs) which are essential compounds in living processes, have entered the pharmaceuticals, nutraceuticals, cosmetics, as well as plastics and lubricant industries with high market demand. These fatty acids are vital components of the plasma membrane, precursors of active signal molecules, and they also help balancing molecular physiology within the cells. Current sources of these fatty acids are from oil seed plants such as safflower, borage, sunflower, corn, and evening primrose as well as cold-climate fish Nonetheless, the commercial production of these fatty acids is limited and far less than the estimated demand. Microbial lipids are good potential substitutes as they can be genetically manipulated and cultivated simply through existing biological processes technology. Many researchers have been putting many efforts to increase the fatty acid production using various approaches. Most of which include changing physical conditions, medium design, process control and optimization, as well as strain improvement through random mutation and specific genetic engineering techniques. Nevertheless, unexpected outcomes are common due to self-adjusting nature of microbial physiology including cell complexity and its metabolic rigidity. Further investigation at intracellular level especially cell metabolic network is therefore compulsory. Thus, besides undergoing the process of genetics alteration, a rational design for feasible cellular metabolism must be taken into account through the use of metabolic engineering technique.
Metabolic engineering offers a systematic analysis tool of metabolic pathways and guides a rational approach in directed improvement of cellular activity through modification of specific biochemical reaction using modeling as underlining technique. The common tools such as metabolic flux analysis (MFA) and metabolic control analysis (MCA) have been reviewed in many literatures. These techniques have been applied to a broad range of problems, including enhancement of product yield and productivity, extension of substrate range, extension of product spectrum and novel products, improvement of cellular properties. In these analyses, the metabolic pathways of microorganism of interest are typically formulated from known pathway information available in biochemistry textbooks and literatures. Due to unavailability of complete biochemical pathways of some hosts of interest, gap filling of these pathways information can be done recently through a new tool called bioinformatics, biological information at genomic level. It therefore allows us to formulate more complete and accurate pathways from genome sequences as metabolic reconstruction. With the additional information obtained from flux analysis, this would lead us to better understanding on the lipid metabolism of this well-known host. A reliable information will then bring about potential pathway design and engineering specifically towards our goal.
Therefore, to obtain reliable flux analysis information, cellular physiology of the host of interest must be studied along with the stoichiometric model simulation in order to compare and testify the accuracy of the constructed physical models generated. Experiments must be set up to verify at the assuming steady state condition while some of both extra- and intracellular metabolites must be monitored as necessary. In this proposal, we will apply chemostat culture technique to study those relationships under various steady state conditions. Flux distribution through the lipid biosynthetic pathways will be quantified and estimated through carbon balance technique. Some important branch points and their nodal rigidity suggested from the metabolic model simulation will be verified through environmental perturbations. This can be performed using another approach through flux comparison among various strains having significantly different lipid profiling. Specific pathway such as Pentose Phosphate pathway, a known majority source of NADPH cofactor that is one important precursor for lipid biosynthesis, will be investigated. Various strains lacking important enzymes in each branch of the pathway will be studied. Such information will disclose how fluxes at branch points in the lipid metabolism are controlled. Physicochemical changes such as temperature, oxygen and nutritional sources will be other approaches that may reveal the interaction and response among different pathways. The information will be used to modify and improve a better metabolic model for lipid production. At the end, some insights lipid metabolism in yeast (perhaps, eukayotes in general) will enable us to rationally alter metabolic pathway at genetic level beyond the limitation of cell metabolic capability for improved cellular properties or phenotype especially the overproduction of certain essential fatty acids.

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Revised: 30 June 2003/13:43:10
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