Preliminary Study Of Measuring Zwf-knockout Escherichia Coli Central Metabolic Flux Under Anaerobic Condition Using ¹³C Labeling Technique

This paper adopts the 13C labeling technique combined with Nuclear Magnetic Resonance (NMR) measurement technique as quantitive analysis method to identify the central metabolic flux distributions of zwf gene knockout Escherichia coli under anaerobic enviroment. This project will provide a more accurate bird's view about metabolic flux distributions under zwf gene knockout condition and will help to locate more protential gene manipulation point for strain production improvement of succinic acid. Due to the limited time, unavailability of equipments and chemicals, this paper only covers preliminary experiments to figure out the procedures and culture parameters of the zwf gene knockout E.coli prophase NMR chemostat experiment under anaerobic culture condition. Problems such as substrates labeling strategy, minimal measured metabolite set, NMR spectrum analysis, etc. are theoretically analyzed and settled properly.Considering the complexisity of determinating substrate labeling stragety, the selection of labeling pattern in this paper is qualitively decided under the guidance of combined optimization of high NMR spectrum resolution and strong signal intensity. The minimal measured metabolite set, on the other hand, is quantitive analyzed and determined with the objective of the relative optimal combination of minimizing the number of NMR measured intermediate and improving the credibility of calculated metabolic flux distribution. The resolution contrast of different metabolite NMR basic solutions, resolution power of the different combination of metabolites, and economical element are the three aspects taken into consideration for the minimal metabolite set candidate selection. Qualitative anlysis is given.In the aspect of NMR spectrum analysis, NUTs, which is designed for 2D NMR spectrum analysis, is adopted to calculate the isotopomer distribution vector (IDV) of minimal metabolite set from the NMR Spectrum. The credibility of obtained IDV will pave the way for the later accurate central metabolic flux distribution calculation.The calculation of the central metabolic flux distribution in this paper can be devided into three steps. First of all, all associated isotopomer mapping matrixs of target metabolic network should be calculated. Secondly, all the stoichometric equations should be listed as the flux solution space bound restrictions. The extreme solutions of the flux solution space then need to be calculated based on above mentioned stoichomitric equations and mixed integer linear programming. Finally, use the minimizing the difference between simulated IDV and calculated IDV of minimal metabolite set as the optimization objective to solve the central metabolic flux distribution of zwf gene knockout Escherichia coli under anaerobic enviroment.