UNIVERSITY OF CALIFORNIA Los Angeles Rewriting central metabolism for carbon conservation ...

UNIVERSITY OF CALIFORNIA Los Angeles Rewriting central metabolism for carbon conservation ...

124 Pages · 2015 · 3.96 MB · English

biorefinery. Most approaches have focused on optimizing naturally occurring pathways to improve titer, productivity, and yield. However, certain inherent hibernation. Arch. Biochem. Biophys. 408, 279–285 (2002). 49. Kelley-loughnane, N. et al. Purification, kinetic studies, and homology model of

UNIVERSITY OF CALIFORNIA Los Angeles Rewriting central metabolism for carbon conservation ... free download

UCLA UCLA Electronic Theses and Dissertations Title Rewriting central metabolism for carbon conservation Permalink https://escholarshiporg/uc/item/57r7z8zc Author Bogorad, Igor Walter Publication Date 2015 Peer reviewed|Thesis/dissertation eScholarshiporg Powered by the California Digital Library University of California UNIVERSITY OF CALIFORNIA Los Angeles Rewriting central metabolism for carbon conservation A dissertation submitted in partial satisfaction of the requirements for the d egree Doctor of Philosophy in Bioengineering by Igor Bogorad 2015 © Copyright by Igor Bogorad 2015 ii ABSTRACT OF THE DISSERTATION Rewriting central metabolism for carbon conservation by Igor Bogorad Doctor of Philosophy in Bioengineering University of California, Los Angeles, 2015 Professor James C Liao, Chair The efficient use of carbon sources is a core objective in metabolic engineering and biorefinery Most approaches have focused on optimizing naturally occurring pathways to improve titer, productivity, and yield However, certain inherent limitations cannot be surpassed if natural pathways are used Here we designed two synthetic metabolic pathways, Non Oxidative Glycolysis (NOG) a nd Methanol C ondensation Cycle (MCC) for the utilization of sugar and methanol, respectively We also created a methanol auxotrophic Escherichia coli strain that depends on methanol for growth The first project, NOG, was designed to address an intrinsic carbon ineffi ciency in all sugar based biorefinery The inefficiency in the Embe n iii Meyerhof Parnas (EMP) pathway (commonly called glycolysis), results in one third of carbon being lost as CO 2 during the synthesis of acety lCoA To bypass the limits of this pathway , we redesigned central metabolism to obtain better carbon conservation The NOG pathway was designed and engineered into E coli that avoid s CO 2 loss in the conversion of suga r to acetyl CoA This represented a 50% carbon improvement compared to the classical EMP pathway While sugars can be consumed by nearly all organisms, other high energy carbon sources are of high interest The second project focuses on the utilization of methanol which is a cheap C 1 molecule that is more energy rich than sugars Methanol is produced commercially from methane in natural gas However, current technologies for methanol conversion require high temperature and pressure Natural biological pathways for methanol utilization are carbon and ATP inefficient We designed the MCC and to allow conversion of methanol to higher alcohols with theoretical 100% carbon conservation at room temperature and ambient pressure This pathways was demonstrated using an in vitro cell free system Since methylotrophs (organisms that can grow on methanol) are difficult to engineer, the conversion of methanol to higher value chemicals has been difficult Furthermore, decades of research have failed to create a methylotrophic E coli strain In the third project we created a methanol auxotrophic E coli strain that is dependent on methanol for growth This strain will be used to improve methanol dehydrogenase , a limiting step in methanol conversion iv The dissertation of Igor Bogorad is approved ______________________ Yi Tang ______________________ Tatiana Segura ______________________ Sebeeha Merchant ______________________ James Liao, Committee Chair University of California, Los Angeles 2015 v To my loving family vi Table of Contents 1 Introduction 1 2 Synthetic non oxidative glycolysis enables complete carbon conservation 3 21 Abstract 3 22 Main Text 3 23 Methods 10 24 Figures 15 25 Supplementary material 19 3 Building carbon –carbon bonds using a biocatalytic methanol condensation cycle 37 31 Abstract 37 32 Significance Statement 38 33 Introduction 39 34 Results 41 35 Discussion 46 36 Materials and Methods 47 37 Figures 50 38 Supplementary Figures 59 39 Supplementary Tables 65 4 M ethanol Assimilation in E coli 68 41 Introduction

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