Advancing CRISPR technologies to engineer yeast metabolism
Doktorsavhandling, 2019

The advent of genetic engineering tools has initiated an era of manipulating microorganisms for the production of valuable compounds for our society. Precise engineering of these microbes commonly requires introducing genetic modifications such as gene deletion, overexpression, and accurate regulation in order to enhance the production of the compound of interest. In this context, the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 technology, adapted from the prokaryotic adaptive immune system, has revolutionized our ability to manipulate a broad range of living organisms. In contrast to other methods, this technology works like a molecular pair of scissors (Cas9) which is guided by a programmable RNA (gRNA) molecule binding at a specific location in the DNA. The programmability and time-efficiency offered by this technology have in the recent years been successfully exploited in rewiring the metabolic network to enhance the production of metabolites used in various areas of industrial biotechnology.
 

In this thesis, I present several studies applying the technological diversity provided by CRISPR in the context of building efficient yeast cell factories for the production of oleochemicals -sustainable substitutes for plant derived lipids. Since oleochemicals derive from lipid products, the main engineering strategies presented essentially focus on fatty acid metabolism and its precursors. First, we exploited CRISPR/Cas9 endonuclease capacity to extensively remodel yeast lipid metabolism. We showed that the disruption of several metabolic fluxes allows to overcome the main limiting steps in fatty acid biosynthesis and favors the production of free fatty acids and triacylglycerols, two important precursors for the production of oleochemicals. Second, we harnessed the ability to precisely regulate genes using the catalytically deactivated form of the Cas9 protein (dCas9) coupled to transcription factors for fine-tuning the expression of genes involved in lipid biogenesis. Additionally, we proposed a framework for dCas9-based applications based on computational techniques for predicting key genes potentially favoring the production of yeast endogenous metabolites. Finally, we expanded the CRISPR repertoire by building new tools to accelerate yeast cell factory design. We exploited a Type I CRISPR-associated endoribonuclease for multiplex genome engineering and transcriptional regulation via processing an RNA transcript into multiple gRNAs, and we developed a computational tool for designing gRNAs targeting multiple loci at once. In summary, the work presented in this thesis provides various ways to efficiently engineer yeast metabolism by exploiting the diversity of CRISPR technologies, as well as new tools to the community for future engineering strategies.

oleochemicals

metabolic engineering

Saccharomyces cerevisiae

CRISPR

Lecture hall KA, Kemihuset, Kemigården 4
Opponent: Prof. Tom Ellis

Författare

Raphael Ferreira

Chalmers, Biologi och bioteknik, Systembiologi

Advancing biotechnology with CRISPR/Cas9: recent applications and patent landscape

Journal of Industrial Microbiology and Biotechnology,; Vol. 45(2018)p. 467-480

Artikel i vetenskaplig tidskrift

Redirection of lipid flux toward phospholipids in yeast increases fatty acid turnover and secretion

Proceedings of the National Academy of Sciences of the United States of America,; Vol. 115(2018)p. 1262-1267

Artikel i vetenskaplig tidskrift

Metabolic engineering of Saccharomyces cerevisiae for overproduction of triacylglycerols

Metabolic Engineering Communications,; Vol. 6(2018)p. 22-27

Artikel i vetenskaplig tidskrift

Transcriptional reprogramming in yeast using dCas9 and combinatorial gRNA strategies

Microbial Cell Factories,; Vol. 16(2017)p. 46-

Artikel i vetenskaplig tidskrift

Ferreira, R., Skrekas, C., Hedin, A., Sanchez, BJ., Nielsen, J., and David, F. Model-assisted fine-tuning of central carbon metabolism in Saccharomyces cerevisiae through dCas9-based regulation

Multiplexed CRISPR/Cas9 Genome Editing and Gene Regulation Using Csy4 in Saccharomyces cerevisiae

ACS Synthetic Biology,; Vol. 7(2018)p. 10-15

Artikel i vetenskaplig tidskrift

Exploiting off-targeting in guide-RNAs for CRISPR systems for simultaneous editing of multiple genes

FEBS Letters,; Vol. 591(2017)p. 3288-3295

Artikel i vetenskaplig tidskrift

Humanity has been harnessing microbes since Neolithic times, e.g. brewing beer or wine using yeast. Following the discovery of the DNA structure, scientists have endeavored to genetically reprogram some of these microbes to enhance the production of various compounds beneficial for our society, e.g. insulin, monoclonal antibodies, and biofuels, as well as finding new ways for producing them. Engineering strategies are commonly performed by introducing genetic modifications into the genome of the host, e.g. gene deletions and regulations, and the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 technology has recently been revolutionizing our ability to perform such genetic manipulations. In contrast to other methods, this technology works like a molecular pair of scissors (Cas9) which is guided by a programmable RNA (a guide-RNA or gRNA) molecule binding at a specific location in the DNA.
 
In this thesis, I present different examples where the technological diversity provided by CRISPR can be applied to engineer Saccharomyces cerevisiae (baker’s yeast) into an efficient producer of oleochemicals - sustainable substitutes for petrochemicals and fuels. Since these compounds are derived from vegetable and animal oil, the presented strategies throughout the thesis focus on fatty acid metabolism and its precursors. Firstly, we exploited CRISPR/Cas9 to remove several genes involved in the yeast lipid metabolism to ultimately rewire it towards the accumulation of free fatty acids and triacylglycerols, two important precursors utilized for production of oleochemicals. Secondly, we harnessed the ability to precisely regulate genes using a deactivated form of the Cas9 (dCas9) protein coupled to transcription factors for controlling the expression of different genes involved in lipid biogenesis. We also propose a computational framework for dCas9-based strategies, which allows predicting genes to regulate in order to rewire the yeast metabolism towards the production of specific metabolites. Finally, we expanded the CRISPR repertoire by building new tools developed to facilitate yeast engineering.
 
The work presented in this thesis offers various ways to efficiently engineer yeast metabolism by exploiting the diversity of CRISPR technologies, and also provides new tools to the community for future engineering strategies.

Drivkrafter

Hållbar utveckling

Ämneskategorier

Biokemi och molekylärbiologi

Mikrobiologi

Bioinformatik och systembiologi

Infrastruktur

Chalmers infrastruktur för masspektrometri

Styrkeområden

Livsvetenskaper och teknik (2010-2018)

ISBN

978-91-7905-148-8

Doktorsavhandlingar vid Chalmers tekniska högskola. Ny serie: 4615

Utgivare

Chalmers tekniska högskola

Lecture hall KA, Kemihuset, Kemigården 4

Opponent: Prof. Tom Ellis

Mer information

Senast uppdaterat

2019-07-30