Metabolic Engineering Acetyl-CoA Pathway To Estabolish Saccharomyces Cerevisiae Platform

Acetyl-CoA is an important precursor molecule inside the microbial cells, and it directly involved in nearly 200 biochemical reactions, including the Krebs cycle, lipid metabolism, amino acid metabolism, and terpenoids metabolism. Using metabolic engineering and synthetic biology strategy modified different metabolic pathways to produce acetyl-CoA derived chemicals is a popular research up to now. In this study, we used 3 hydroxy propionic acid, fatty acid and beta-carotene as the targeted products. By engineering different metabolic pathways in Saccharomyces cerevisiae, we try to establish a platform cell factory to produce acetyl-CoA derived products.The chemical 3-hydroxypropionic acid (3HP) is an important building block and is ranked among the top third of the 12 platform chemicals selected by the U.S. Department of Energy. The bifunctionality of 3HP makes it a versatile platform chemical for numerous applications, including the production of acrylic acid, acryl amide, malonic acid, and 1,3-propanediol. Recently, there has been great interest in producing 3HP at the industrial scale from renewable sources. Several biosynthetic pathways have been proposed for producing 3HP, and the conversion of glycerol to 3HP via glycerol dehydratase and aldehyde dehydrogenase has been extensively investigated. Utilizing the malonyl-CoA pathway to produce 3HP is expected to have some advantages. For example, this pathway is vitamin-B12-independent. Various C5 and C6 sugars derived from lignocellulosic biomass can be used as raw materials for 3HP production since acetyl-CoA is a common intermediate of sugar metabolism. A high conversion yield of glucose is expected because 3HP production from glucose is energetically well balanced. In this study, a part of the 3HP/4-hydroxybutyrate cycle from Metallosphaera sedula was first introduced for 3HP production. To study the basic biochemical information of this pathway, an in vitro-reconstituted system was initially established using acetyl-CoA as the substrate. The results indicated that the 3HP formation was sensitive to acetyl-CoA carboxylase (ACC) and malonyl-CoA reductase (MCR), but not malonate semialdehyde reductase (MSR). In addition, the competition between 3HP formation and fatty acid production was analyzed in vitro. In vivo, we evaluated 3HP production in E. coli and in 3 yeast strains (S. cerevisiae, K. pastoris, and Y. lipolytica). Subsequently, an E. coli strain was further engineered to produce 3HP by fine-tuning MCR expression by testing a series of promoters. Finally, several metabolic-engineering strategies were further performed for 3HP overproduction, including increasing the level of precursor molecules and the cofactor NADPH, as well as blocking fatty acid synthesis. These findings would be valuable for future engineering studies involving the malonyl-CoA pathway for enhanced the production of 3HP or 3HP derived products.Fatty acids and their derivatives (fatty acid esters, alkanes and fatty alcohols) are thought to be an excellent candidate of renewable biofuels. In previous research, the in vitro system of fatty acid synthesis was established in our lab. Based on this system, the limiting step of fatty acid synthesis and basic biochemical properties were discovered. Then we extend this work into to S. cerevisiae, the most used microbial in industrial fermentation, to build a strong "cell factory". There are several differences between S. cerevisiae and the commonly used engineering strain, Escherichia coli. The iterative process is performed by the fatty-acid-synthase complex, which is a large, barrel-shaped complex that contains six copies of alpha and beta subunits (FAS2 and FAS1). The final products of fatty-acid biosynthesis are fatty acyl-CoAs. The ACC and ACS are tightly regulated in S. cerevisiae. There are many genes are responsible for degradation of free fatty acids in S. cerevisiae. Deletion of FAA1, FAA4, and POX1 increased the production of fatty acids from 48 mg/L to 106 mg/L. In order to release fatty acids, we overexpressed the thioesterases TesA'(TesA without the N-terminal membrane signal peptide), Cinnamomum camphorum TE, PTE1 (intrinsic acyl-CoA thioesterase in S. cerevisiae which located in peroxisomes), and PTE1'(PTE1 without the carboxy-terminal peroxisomal signal peptide). Overexpression of the intact S. cerevisiae thioesterase PTE1 gene yielded a relatively modest increase (from 77 mg/L to 115 mg/L) in fatty-acid production. In parallel, improvimg the supply of precursor acetyl-CoA can increase fatty acid level to 124 mg/L. However, overexpression of ACC1 has little effect on fatty acids biosynthesis. In order to evaluate the effect of ACC1 in fatty-acid synthesis, a cell-free system was developed. Addition purified ACC1 from yeast was titrated into the cell-free extract with acetyl-CoA, fatty-acid production was not detectable. However, when the equimolar purified E. coli ACC1 was then added into the yeast cell-free extract, there were detectable fatty acids accumulated. This result clearly indicated that the activity of yeast ACC1 was too low to support in vitro fatty acid synthesis. Three MS methods were applied to identify the phosphorylation sites. As it has been reported that phosphorylation of ACC1 may influent its activity, so phosphorylation sites of ACC1 was further identified. After searching the ProteinPilot (AB SCIEX) database,55 phosphorylation sites were discovered. All predicted phosphorylation sites from the database and the above three MS experiments were further identified by using this MIDAS method. Finally, fourteen phosphorylation sites were confirmed in ACC1. There were three sites in the BC domain, one site in the BCCP domain, one in the CT domain, and nine sites in the connectional region. We found that none of the phosphorylation sites were close to the active site. Although the regulatory mechanisms remain unclear, our results provide rationale for future studies to target this critical step.As a kind of valuable carotenoids, beta-carotene have numerous applications, such as food colorants, nutrient supplements, cosmetic and pharmaceutical. Recently, there has been great interest in producing beta-carotene in engineered organism from renewable sources, instead of through the traditional chemical synthesis. In this study, a series of galactose induced GAL promoters were tested in S. cerevisiae and chosen for beta-carotene biosynthesis pathway construction. Then three heterologous genes from Blakeslea trispora, responsible for the biosynthesis of beta-carotene, were introduced into S. cerevisiae to produce beta-carotene. Combination of those metabolic engineering strategies, the engineered strain could produce 45 mg/L of beta carotene, and produce 29 mg/L of lycopene at the same time in shake flask fermentation. Then through the optimization of carbon source, the engineered strain can produce 149 mg/L of total carotenoids (64 mg/L beta-carotene and 85 mg/L lycopene). Afterwards, in order to further improve the accumulation of beta carotene, we set up an absolute quantitative methods to detect the intermediate. And the MVA module was balanced by monitoring the metabolic intermediates and adjusting the expression level of targeted protein. We believe that this construction strategy is also suitable for other terpene overproduction in S. cerevisiae.