462670 Yeast Cell Factories: Construction of Platform Strains and Development of Synthetic Biology Tools
Biological conversion of renewable feedstock into fuels and chemicals has been intensively investigated due to increasing concerns on sustainability and global climate change. Compared with its counterparts, Saccharomyces cerevisiae, the baker’s yeast, is more industrially relevant thanks to its well-studied genetic and physiological background, the availability of a large collection of genetic tools, the compatibility of high-density and large-scale fermentation, the resistance to phage infection, and the high tolerance against toxic inhibitors and products. Therefore, S. cerevisiae is one of the most popular cell factories and has been successfully used in the modern fermentation industry to produce a wide variety of products such as ethanol, organic acids, amino acids, enzymes, and therapeutic proteins.
My Ph.D. work was mainly focused on the construction of yeast cell factories for efficient and cost-effective production of biofuels and chemicals using synthetic biology and metabolic engineering approaches. Notably, a wide variety of products with industrial interests are derived from a few precursor metabolites. For example, lactate, 2,3-butanediol, and iso-butanol are derived from pyruvate, while n-butanol, polyhydroxybutyrate, isoprenoids, fatty acids, fatty alcohols, alkanes, and fatty acid ethyl esters are derived from acetyl-CoA. Therefore, we aim to develop platform yeast cell factories with enhanced supply of these precursor metabolites for efficient production of biofuels and chemicals. To achieve this, it generally involves host genome engineering such as the disruption of the competing pathways and biosynthetic pathway engineering such as the introduction of heterologous pathways. For example, in order to construct an acetyl-CoA overproducing yeast strain, the ethanol and glycerol formation routes were partially or fully blocked and alternative acetyl-CoA biosynthetic pathways, such as the pyruvate dehydrogenase (PDH) from a bacterium and the ATP-dependent citrate lyase (ACL) from an oleaginous yeast, were introduced. The higher efficiency and lower energy requirement of PDH and ACL, when combined with host genome engineering, increased the acetyl-CoA levels by more than three-fold and the production of acetyl-CoA derived product by more than 12-fold.
During my postdoc research, I switched my focus to the development of novel synthetic biology tools to facilitate the development of cell factories. Although a wide variety of cell factories have been constructed using the traditional strategies mentioned above, we are still facing the challenges of constructing efficient cell factories in a short period of time. Therefore, novel synthetic biology tools are demanded to revolutionize cell factory development. In terms of the biosynthetic pathway engineering, we constructed a series of plasmids with different copy numbers, which enabled the fine-tuning of gene expression levels and optimization of multi-gene biosynthetic pathways. In terms of genome engineering, we took advantage of the recently developed CRISPR/Cas9 system to achieve both gain of function (knock-in and gene activation) and loss of function (knock out and gene repression) in yeast. More importantly, we combined gene deletion, gene repression, and gene activation into a single system, in which a full spectrum of gene expression profiles (zero expression, down-regulation, and up-regulation) was achieved. Now we are performing genome-scale studies to develop a rapid and efficient strategy for the construction of cell factories and unveil the synergistic effects between gene deletion, gene repression, and gene activation in yeast.
As a graduate student in the University of Illinois, I have the opportunity to work with and mentor many talented undergraduate and graduate students. Three semesters of TA in the department of Chemical and Biomolecular engineering have given me a better understanding on how to teach effectively in a big classroom, how to mentor students with various academic background, as well as the skills to communicate with students from a wide range of countries. Besides these formal teaching assignments, I have served as a mentor for undergraduate and junior graduate students in the lab, helping them to understand not only the synthetic biology principles and experimental techniques, but also the experimental design and problem-solving skills.
Keywords: Yeast cell factory; Synthetic biology; Genome engineering; CRISPR/Cas9; Plasmid copy number
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