Files in this item

FilesDescriptionFormat

application/pdf

application/pdfLIAN-DISSERTATION-2015.pdf (6MB)Restricted Access
(no description provided)PDF

Description

Title:Engineering Saccharomyces cerevisiae with enhanced supply of precursor metabolites for efficient production of fuels and chemicals
Author(s):Lian, Jiazhang
Director of Research:Zhao, Huimin
Doctoral Committee Chair(s):Zhao, Huimin
Doctoral Committee Member(s):Rao, Christopher; Schroeder, Charles M.; Jin, Yong-Su
Department / Program:Chemical & Biomolecular Engineering
Discipline:Chemical Engineering
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):Pyruvate
Acetyl coenzyme A (Acetyl-CoA)
Acyl-CoA
Plasmid Copy Number
Clustered regularly-interspaced short palindromic repeats (CRISPR/Cas9)
Yeast
Biofuel
Metabolic Engineering
Synthetic Biology
Abstract:Saccharomyces cerevisiae has been widely established as a platform microorganism for industrial production of a wide variety of products including but not limited to ethanol, organic acids, amino acids, enzymes, and therapeutic proteins, owing to its high tolerance to harsh industrial conditions, such as low pH, high sugar concentration and growth inhibitors in the biomass hydrolysate, as well as resistance to phage infection. However, one of the major challenges for high level production of value-added compounds other than ethanol is the strong fluxes for ethanol formation even under aerobic condition, a phenomenon known as Crabtree effect. Notably, a wide variety of products with industrial interest are derived from a few precursor metabolites, such as 2,3-butanediol (BDO) and iso-butanol from pyruvate, n-butanol, polyhydroxybutyrate, and isoprenoids from acetyl-CoA, and free fatty acids, fatty alcohols, and fatty acid ethyl esters from long chain acyl-CoAs. Therefore, metabolic engineering and synthetic biology approaches were applied to engineer S. cerevisiae with enhanced supply of these precursor metabolites for efficient production of fuels and chemicals. More importantly, these engineering efforts can be integrated to construct a platform yeast cell factory, since pyruvate is the direct precursor for acetyl-CoA generation and enhanced acetyl-CoA levels will provide a driving force for acyl-CoAs pool engineering. To construct a pyruvate overproducing yeast strain, three structural genes encoding pyruvate decarboxylases, PDC1, PDC5, and PDC6 were deleted to completely eliminate ethanol production. Followed by overexpression of MTH1 and adaptive evolution, the resultant Pdc- yeast strain grew on glucose as the sole carbon source with pyruvate as the major product. Subsequent introduction of a BDO biosynthetic pathway resulted in the production of BDO at a yield over 70% of the theoretical value and a titer higher than 100 g/L using fed-batch fermentation. To engineer acetyl-CoA pool in S. cerevisiae, alternative acetyl-CoA biosynthesis routes were characterized using synthetic biology approaches and metabolic engineering was applied to redirect the metabolic fluxes towards acetyl-CoA biosynthesis. Acetyl-CoA biosynthetic pathways from Escherichia coli (pyruvate dehydrogenase, PDH) and Yarrowia lipolytica (ATP-dependent citrate lyase, ACL) were found to enable the growth of the Acs- (acs1Δ acs2Δ) strain on glucose as the sole source. To construct a functional PDH in the cytosol of yeast, different lipoylation pathways were introduced and engineered. Besides the naturally existing scavenging pathway and de novo biosynthetic pathway, we also designed a semi-synthetic lipoylation pathway based on the acyl-ACP synthetase (AasS). The scavenging pathway resulted in a functional PDH that enabled the growth of Acs- strain to a similar level of the wild-type strain. The de novo biosynthetic lipoylation pathway was hindered by the difficulty in reconstituting a functional type II fatty acid synthase (FAS) in the cytosol to provide the precursor (octanoyl-ACP) for protein lipoylation. The introduction of the semi-synthetic lipoylation pathway (VhAasS-cytoACP1-cytoPPT2) resulted in functional PDH and rescued the growth of the Acs- strain when octanoic acid was supplementated. Based on these results, a de novo biosynthetic lipoylation pathway was re-designed and proposed. Then these heterologous biosynthetic pathways were combined with host engineering to design and construct acetyl-CoA overproducing yeast strains. By deleting ADH1, ADH4, GPD1, and GPD2 involved in ethanol and glycerol formation, the glycolytic flux was redirected towards this precursor metabolite, resulting in a 4 fold improvement in n-butanol production. Subsequent introduction of alternative acetyl-CoA biosynthetic pathways, the production of n-butanol was further increased. Although significant improvement of n-butanol production was achieved, the final titer was still much lower than that of ethanol and the resultant yeast strains suffered from accumulation of the toxic intermediates such as acetaldehyde and acetate. Therefore, more efforts were put into the engineering of acetyl-CoA pools in the Pdc- strain, where ethanol production was completely eliminated and pyruvate was accumulated to high levels. To engineer acyl-CoAs levels in S. cerevisiae, a new biosynthesis platform based on the reversal of β-oxidation cycle was constructed using synthetic biology approaches. Compared with the conical FAS, which is ATP, ACP, and NADPH dependent, the reversed β-oxidation pathway is featured for its ATP and ACP independence and CoA and NADH dependence. The energetic benefits of ATP independence and availability of CoA and NADH versus ACP and NADPH confer advantages for acyl-CoAs biosynthesis. Reversed β-oxidation pathways were constructed and found to produce n-butanol, decanoic acid, and ethyl decanoate, indicating the functional reversal of β-oxidation cycle at least 4 turns. To facilitate the engineering in S. cerevisiae, new synthetic biology tools was also developed. First, plasmids with step-wise increased copy numbers (20-100 copies per cell) were constructed by engineering the expression level of selection marker proteins, including both auxotrophic and dominant markers. More importantly, the copy number of the plasmids with engineered dominant markers (5-100 copies per cell) showed a positive correlation with the concentration of antibiotics supplemented to the growth media. Based on this finding, a new and simplest synthetic biology approach named induced pathway optimization by antibiotic doses (iPOAD) was developed to optimize the performance of multi-gene biosynthetic pathways by different combination of antibiotic concentrations in S. cerevisiae. To demonstrate this approach, iPOAD was applied to optimize the lycopene and n-butanol biosynthetic pathways, with the production of lycopene and n-butanol increased by 10- and 100-fold, respectively. Finally, the iPOAD optimized pathway was integrated to chromosomes to increase the strain stability and eliminate the requirement of antibiotic supplementation, by taking advantage of the iPOAD and CRISPR-Cas9 technologies for multiplex pathway integration.
Issue Date:2015-07-14
Type:Thesis
URI:http://hdl.handle.net/2142/88274
Rights Information:Copyright 2015 Jiazhang Lian
Date Available in IDEALS:2015-09-29
Date Deposited:August 201


This item appears in the following Collection(s)

Item Statistics