Metabolic engineering of cofactor supply and product removal for a FAS II System in Saccharomyces cerevisiae

Universidade do Minho Escola de Engenharia Inês de Oliveira Abreu Metabolic Engineering of Co-Factor Supply and Product Removal for a FAS II System in Saccharomyces cerevisiae outubro de 2024 UMinho | 2024 Inês Abreu Metabolic Engineering of Co-Factor Supply and Product Removal for a FAS II System in Saccharomyces cerevisiae University of Minho School of Engineering Inês de Oliveira Abreu Metabolic Engineering of Cofactor Supply and Product Removal for a FAS II System in Saccharomyces cerevisiae Master’s Dissertation in Chemical and Biological Engineering Dissertation supervised by Professor Doctor Lucília Maria Alves Ribeiro Domingues Professor Doctor Björn Fredrik Johansson october 2024 Copyright and Terms of Use for Third Party Work This dissertation reports on academic work that can be used by third parties as long as the internationally accepted standards and good practices are respected concerning copyright and related rights. This work can thereafter be used under the terms established in the license below. Readers needing authorization conditions not provided for in the indicated licensing should contact the author through the RepositóriUM of the University of Minho. License granted to users of this work: CC BY-NC-ND https://creativecommons.org/licenses/by-nc-nd/4.0/ i Acknowledgements I’d like to thank my supervisors, Professors Lucília Domingues and Björn Johansson for the orientation provided for the realization of this project and sharing of scientific knowledge. My deepest gratitude also goes to my colleagues and friends from LGM: Patrícia Ataíde, Beatriz Leite, Clara Arantes, Inês Ribeiro, Cláudia Barata and Faezeh Ghasemi; it was a pleasure to have such fun, kind and reliable lab mates. A special thanks to Humberto Pereira, whose guidance, insights and sense of humor have been invaluable throughout this project. To Jeh, Fabs and Érica, who have been like sisters to me - thank you for growing alongside me. To Francisco (”coleguinha”), for his unwavering support and endless patience — I’ll be there for you, ’cause you’re there for me too . And to my friends from uni, who made these hellish five years worth it. Finally, my deepest appreciation goes to my family for being part of this journey, and especially to those whose support truly carried me through. And to you, T. ii Statement of Integrity I hereby declare having conducted this academic work with integrity. I confirm that I have not used plagiarism or any form of undue use of information or falsification of results along the process leading to its elaboration. I further declare that I have fully acknowledged the Code of Ethical Conduct of the University of Minho. University of Minho, Braga, december 2024 Inês de Oliveira Abreu iii Abstract Metabolic Engineering of Cofactor Supply and Product Removal for a FAS II System in Saccharomyces cerevisiae The global demand for fatty acids (FA) is increasing, raising concerns regarding the environmental impact of sustaining such supply chains. Fatty acids are integral to sectors such as pharmaceuticals, food production, and biofuels. Utilizing microorganisms as cell factories offers a sustainable and innovative approach to produce high-value compounds, reducing dependence on traditional resources. Saccharomyces cerevisiae is widely used in industry due to its robustness, adaptability, and ease of genetic manipulation. Fatty acids in cells are sourced either from exogenous intake or through de novo synthesis. S. cerevisiae employs a type I Fatty Acid Synthase (FAS) system for FA synthesis, whereas prokaryotes rely on a type II FAS system. In yeasts (type I system), all catalytic steps are housed within two multifunctional protein subunits, whereas type II systems rely on separate monofunctional enzymes for each catalytic step. This compartmentalization in type II systems enables the synthesis of a broader range of fatty acids and allows for easier genetic manipulation. This project aimed to enhance fatty acid synthesis in a type II FAS system expressed in S. cerevisiae by increasing metabolic flux through the oxidative pentose phosphate pathway or by promoting product removal. To achieve this, yeast strains with deletions in the FAS1 and FAS2 genes were engineered by deleting the PGI1 gene, which encodes phosphoglucose isomerase, as well as transforming these strains with a plasmid vector enabling simultaneous expression of twelve FASII genes from Escherichia coli and Arabidopsis thaliana . An attempt was made to construct a plasmid expressing a diacylglycerol acyltransferase, an enzyme catalyzing the final and rate-limiting step in triacylglycerol synthesis. The PGI1 deletion in the base strains was successful; however, the transformation of the FASII system did not restore fatty acid synthesis in these strains. Fluorescence analysis of lipid bodies and growth profile studies revealed severely deficient growth. Moreover, construction of a plasmid to express DGA1 was unsuccessful, preventing further investigation into the effects of DGA1 overexpression in these strains. Keywords: metabolic engineering, Saccharomyces cerevisiae , fatty acid synthesis iv Resumo Engenharia Metabólica do Fornecimento de Co-fator e Remoção de Produto num Sistema FAS II em Saccharomyces cerevisiae A procura por ácidos gordos tem aumentado e a manutenção da oferta desses produtos tem um impacto grave no planeta, visto que desempenham papéis cruciais em indústrias como a farmacêutica, alimentar e de combustíveis. O uso de microrganismos como fábricas celulares oferece uma abordagem inovadora e sustentável para produzir compostos de alto valor, reduzindo a dependência de matérias-primas tradicionais. Saccharomyces cerevisiae , amplamente usado na indústria pela sua resistência, adaptabilidade e facilidade de manipulação genética, pode obter ácidos gordos de fontes externas ou síntese de novo . Este microrganismo possui um sistema de síntese de ácidos gordos tipo I, em que todas as etapas catalíticas ocorrem numa única cadeia polipeptídica, ao contrário do sistema tipo II dos procariotas, onde cada etapa é catalizada por enzimas monofuncionais, promovendo maior diversidade de ácidos gordos e facilidade de manipulação genética. Este projeto visou melhorar a síntese de ácidos gordos num sistema FASII expressado em S. cerevisiae . Leveduras que já possuíam deleções ao nível dos genes FAS1 e FAS2 foram transformadas com um vetor que expressa doze genes do sistema FASII de Escherichia coli e Arabidopsis thaliana . Deletou-se o gene PGI1 , que codifica uma fosfoglucose isomerase, aumentando o fluxo metabólico para a via oxidativa da pentose fosfato. Outra estratégia consistiu na remoção produtos, ao construir um plasmídeo que expressasse uma aciltransferase de diacilglicerol, codificada pelo gene DGA1 . A deleção do gene PGI1 foi bem-sucedida, mas a transformação com o sistema FASII não recuperou a capacidade das estirpes sintetizarem ácidos gordos. Análises dos corpos lipídicos e curvas de crescimento mostraram que as células possuem um crescimento bastante lento. Além disso, não foi possível construir um plasmídeo que expressasse DGA1 , impossibilitando estudos sobre o efeito da sobreexpressão desse gene na acumulação de ácidos gordos por estas estirpes. Palavras-chave: engenharia metabólica, Saccharomyces cerevisiae , síntese de ácidos gordos v Contents I Main Body of the Dissertation 1 1 Introduction 2 1.1 Fatty acids and their relevance in day-to-day life . . . . . . . . . . . . . . . . . . . . 2 1.1.1 Fatty acids and nutrition . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 1.1.2 Fatty acids in the energy industry . . . . . . . . . . . . . . . . . . . . . . . 4 1.2 Yeastsascellfactories ................................ 5 1.3 Production of fatty acids in the cell . . . . . . . . . . . . . . . . . . . . . . . . . . 6 1.3.1 The Biochemistry of Fatty Acid Assembly . . . . . . . . . . . . . . . . . . . 7 1.3.2 TheFASISystem .............................. 8 1.3.3 TheFASIISystem .............................. 9 1.3.4 Regulation of the fatty acid synthesis . . . . . . . . . . . . . . . . . . . . . 10 1.4 Enhancing the Production of Fatty Acids by S. cerevisiae ............... 11 1.4.1 Enhancing precursor supply (push strategies) . . . . . . . . . . . . . . . . . 12 1.4.2 Downstream pathway engineering strategies (pull strategies) . . . . . . . . . . 14 1.5 Objectives ...................................... 17 1.5.1 Push strategy for FA synthesis with gene deletions and complementation of yeastsFAS.................................. 17 1.5.2 Pull strategy for FA synthesis with gene cloning . . . . . . . . . . . . . . . . 17 1.5.3 Analysis of fatty acid production . . . . . . . . . . . . . . . . . . . . . . . . 17 2 Materials and Methods 18 2.1 Yeast and bacterial strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 2.2 Culturemedia .................................... 19 2.3 Plasmid and genomic DNA extractions . . . . . . . . . . . . . . . . . . . . . . . . . 21 vi List of Abbreviations Amp - Ampicillin DGAT - Diacylglycerol Acyltransferase FA - Fatty acids FAS - Fatty Acid Synthase GFP - Green FLuorescent Protein HMyr - Myristic Acid LB - Lysogeny Broth Miller Medium LD - Lipid Droplets LiAc/SS carrier DNA/PEG - Lithium Acetate, Single Stranded carrier DNA, Polyethylene Glycol NADPH - Nicotinamide Adenine Dinucleotide Phosphate NAT - Nourseothricin PBS - Phosphate Buffer Solution PGI - Phosphoglucose Isomerase PP(P) - Pentose Phosphate (Pathway) PUFA - Polyunsaturated Fatty Acids SC - Synthetic Complete Medium TAG - Tryacylglycerol TE - Tris/EDTA YPD - Yeast extract-Peptone-Dextrose YPFD - Yeast extract-Peptone-Fructose-Dextrose YPGES - Yeast extract-Peptone-Glycerol-Ethanol YPK - Yeast Pathway Kit YNB - Yeast Nitrogen Base xiii Part I Main Body of the Dissertation 1 Chapter 1 Introduction 1.1 Fatty acids and their relevance in day-to-day life Fatty acids are energy-rich molecules that play an indispensable role in society, being present in everyday products for domestic use, cosmetics, food, pharmaceuticals, and fuels, for example (Cerone and Smith, 2021). In 2023, the global market for fatty acids was estimated at 30.97 billion USD (United States dollars), and it is speculated to reach 48.42 billion USD by 2032 (Figure 1)(Research, 2023). Figure 1: Fatty acid market size from 2022 to 2032. Data retrieved from the website https://www.precedenceresearch.com/fatty-acids-market. The first recorded use of vegetable oils and animal fats dates back to Mesopotamia (7000 BC) and ancient Egypt (5000 BC), where they were used as body oils and lotions for personal hygiene or in mummification rituals around 2000 BC. Over time, new techniques for essential oil production were introduced, such as distillation or seed pressing. These methods underwent significant evolution during the advent of the industrial revolution, with the implementation of organic chemistry, and their use followed the path of large-scale production. Nowadays, the consumption of fats and oils is primarily sustained by the extraction of lipids from vegetable sources (Arhar et al., 2021; Cerone and Smith, 2021). Overall, the world fat consumption for food usage is increasing and the demand for biodiesel production 2 from fats and oils is also growing fast. This constant increase in demand and the fact that marine and vegetable sources are not adequate to meet the high level of fatty acids required worldwide, on top of climate change, have determined the necessity for the search for renewable and sustainable sources for fatty acids (Cerone and Smith, 2021). 1.1.1 Fatty acids and nutrition Polyunsaturated fatty acids (PUFAs) are hydrocarbon chains with two or more double bonds. PUFAs are classified as either n-3 (omega-3) or n-6 (omega-6) based on the position of the first double bond from the methyl end of the fatty acid. They are primarily found in fish, such as cod, halibut, mackerel, and salmon (Cerone and Smith, 2021). Key PUFAs include α-linoleic acid (ALA), an n-6 PUFA found in vegetable oils, nuts, and seeds, and docosahexaenoic acid (DHA), an n-3 PUFA found in meats and eggs (Table 1). Table 1: Structural types of the main polyunsaturated fatty acids (PUFAs) present in foods and biological tissues. Adapted from A. M. Minihane and J. A. Lovegrove (2006) Fatty Acid Structural Type Abbreviation Linoleic acid C18:2-cis (n-6) LA α-linoleic acid C18:3-cis (n-3) ALNA γ-linoleic acid C18:3-cis (n-6) GLA Eicosapentaenoic acid C20:5-cis (n-3) EPA Docosapentaenoic acid C22:5-cis (n-3) DPA Docosahexaenoic acid C22:6-cis (n-3) DHA Due to changes in the human diet—shifting from a diet rich in omega-3 polyunsaturated fatty acids (PUFAs) and a balanced omega-6/omega-3 ratio to one that is high in saturated fatty acids (SFAs) and contains more omega-6—researchers are increasingly interested in producing healthier meat with a higher PUFA-to-SFA ratio and a more favorable omega-6 to omega-3 balance (Cerone and Smith, 2021). Plants have been considered a fundamental source of omega-3 fatty acids, such as alphalinolenic acid (18:3) – ALA, which is present in leaves and oleaginous seeds (walnuts, flax seeds, canola oils and leafy green plants). Additionally, microalgae biomass can also be highly purified to obtain pure chemicals for pharmaceutical applications such as the production of omega-3 food supplements (Cerone and Smith, 2021; Minihane and Lovegrove, 2006; Pyle, 2022; El-Hage Scialabba, 2022). 3 1.1.2 Fatty acids in the energy industry Taking the example of fuels, with population growth and the widespread use of automobiles, there is a concern about the detrimental effects of fossil fuel contributions to climate change. In this context, a greener alternative emerged: biofuels. Coupled with research efforts in this area, various policies have been implemented in recent years to stimulate global biofuel production (Hu et al., 2019; Ebadian et al., 2020). Some of this policies include market-pull policies, which boost market adoption of established biofuels, and technology-push policies, which fund early-stage biofuel technology development to support commercialization (Ebadian et al., 2020). Moreover, international political instability has affected access to fossil fuels, resulting in serious socio-economic consequences (Mattanovich et al., 2014). The process of biodiesel production comprehends a transesterification reaction where a triglyceride reacts with an alcohol to form esters and glycerol (Figure 2). This reaction usually occurs in the presence of a strong alkaline catalyst, like sodium hydroxide, for example. During the transesterification process, the viscosity of the biodiesel is reduced compared to the vegetable oil (Topi, 2020). Figure 2: Transesterification reaction of triglycerides with MeOH to Fatty Acids Methyl Esters. Image obtained from D. Topi (2020). The production of biofuels such as biodiesel relies on natural oils and fats that are common in human food as raw materials, which generates resource scarcity problems (Miranda et al., 2020; Zhou et al., 2014). Besides that, the supply can be further compromised by the quantity and quality of harvests, slow growth of plants and seasonal peaks (rather than a continuous supply) (Arhar et al., 2021). Therefore, more reliable alternatives would be necessary to solve an issue that arose as a solution to a pre-existing problem: considering the current demand, how to meet the population’s needs without compromising the planet’s sustainability? Even though microbial lipids were considered a highly promising source of next-generation biodiesel, factors such as high processing costs and lack of competitiveness compared to vegetable oil hinder its industrial application. The production cost of microbial lipids was estimated to range from $1300 to $9000 4 per ton of oil, primarily depending on the raw materials and culture modes. The minimum selling price of microbial lipids was near to the current price of oil, being estimated to be in the range of $0.8 to $4.4 per kilogram (Lei et al., 2024). For the time being, microbial lipids cannot compete with the current industry as there are hindrances in terms of yield, cost of production/extraction and other factors. However there is motivation to innovate and further pursue the development of bio-based industries. 1.2 Yeasts as cell factories The expression ”cell factory” originated in 1970, due to the development and expansion of biotechnology and engineering. In the 1990s, this concept became more widely used (Nikel and Mattanovich, 2021). ”Microbial cell factory,” on the other hand, is a term that refers to the use of microorganisms as hosts to obtain industrially interesting products and inexpensive renewable raw materials. There are microorganisms that function as chassis and are more widely used for these purposes, as their metabolic networks have been extensively studied, namely Escherichia coli , Saccharomyces cerevisiae , and Bacillus sp. (Qian et al., 2023). The synthesis of molecular compounds can be achieved through renewable sources, using well-studied microorganisms as ”cell factories” (Nikel and Mattanovich, 2021; Mattanovich et al., 2014). With the advent of green chemistry and society’s attention focused on climate change and environmental protection, there is a growing need to produce fatty acids in large quantities without putting pressure on ecosystems. Microalgae, yeasts, fungi, and bacteria have proven to be excellent fat producers. Research in this field employs genetics, metabolic engineering, and biochemistry to modify these organisms to produce industrially relevant products, leveraging their rapid growth, adaptability, and ease of genetic manipulation. The pursuit of metabolic engineering for building cell factories is the future to overcome fatty acid scarcity (Cerone and Smith, 2021). Yeasts exhibits rapid growth, tolerance to environmental stresses and strong robustness, is less susceptible to viral infections, making it compatible with cultivation in bioreactors and large-scale production. Additionally, they allow for various genetic manipulation methods and optimization of recombinant protein production. Because yeasts are eukaryotic microorganisms, they can perform post-translational modifications on proteins. S. cerevisiae has been a well-studied model organism and has been implemented in the production of recombinant drugs and industrial enzymes (Miranda et al., 2020; Pereira et al., 2022; Yang et al., 2024; Pereira et al., 2014). There are also oleaginous microorganisms, such as Yarrowia lipolytica , that can accumulate more 5 than 20% of their dry cell weight as lipids in the form of droplets inside the cell. ”To date, over 160 yeast species have been reported in the scientific literature with lipid contents greater than or equal to 20% (w/w)” (Abeln and Chuck, 2021). Some of the species that have been thoroughly studied are Yarrowia lipolytica , Rhodotorula toruloides , Cutaneotrichosporon oleaginosus , Lipomyces starkeyi and Rhodotorula glutinis , for example (Abeln and Chuck, 2021; Legodi and Moganedi, 2023; Arhar et al., 2021; Miranda et al., 2020). However, Y. lipolytica , for example, is not as tolerant to industrial harsh conditions as S. cerevisiae (Baptista et al., 2021). Even though S. cerevisiae is regarded as a non-oleaginous yeast, there is an industrial wild-type strain, D5A, that was shown to accumulate over 20% of lipids from glucose when growth is nitrogen-limited. This shows that oleaginous microorganisms’ genus may not solely depend on phenotype and that S. cerevisiae can be a good candidate for lipid production (Arhar et al., 2021; He et al., 2018). 1.3 Production of fatty acids in the cell “The International Lipid Classification and Nomenclature Committee (ILCNC) classified lipids into eight categories—namely: fatty acids, glycerolipids (e.g., triacylglycerols, TAGs), glycerophospholipids (GPLs; fatty acid-glycerol-phosphate ester), sphingolipids, sterol lipids, prenol lipids, saccharolipids, and polyketides” (Fahy et al., 2009). Fatty acids (FA) play major structural and functional roles for the biology of the cell. Some examples of such functions include acting as “building blocks” of cell membranes, storage materials – energy supply – within the cells and signaling. The FA in cells derive either from exogenous sources or from de novo synthesis (De Carvalho and Caramujo, 2018; Lenhinger et al., 2017; Saini et al., 2021). 6 1.3.1 The Biochemistry of Fatty Acid Assembly The process of fatty acid synthesis in prokaryotes and eukaryotes differs in the molecular structures of the enzymes employed for this purpose, nevertheless, the reaction mechanisms are the same (Figure 3). These processes begin with carboxilation of an acetyl-CoA molecule by the addition of CO2to malonyl-CoA. This is possible through the action of the enzyme acetyl-CoA carboxylase (ACC). This is the first committed step toward FA synthesis and, therefore, a limiting factor. Then, a ketoacyl synthase condenses a malonylCoA with acetyl-CoA (with the release of CO2) forming acetoacetyl-ACP/CoA. This constitutes the first step of the elongation cycle. Elongation occurs by cycling through a four-step process: condensation, reduction, dehydration and reduction, catalyzed by ketoacyl reductase, dehydratase, and enoyl reductase, respectively. An acyl carrier protein (ACP) acts as a carrier for acyl intermediates during fatty acid chain elongation. This iterative process occurs using malonyl-CoA as the provider of 2C units until the carbon chain length of the FA reaches 16 or 18, which are the most common types of FA produced by yeasts FAS (Tehlivets et al., 2007; Zhou et al., 2014). Figure 3: Reaction mechanism for fatty acid production and elongation. Image adapted from Tehlivets et al. (2007). 7 1.3.2 The FAS I System S. cerevisiae expresses a type I FAS system: in fungi, FA synthesis is carried by two multifunctional polypeptides, that can be found exclusively in the cytosol (De Carvalho and Caramujo, 2018; Ridgway and McLeod, 2015; Lenhinger et al., 2017). Each of these polypeptides encloses several domains that play specific roles in the mechanism (Lomakin et al., 2007; Pozdniakova et al., 2023; Ridgway and McLeod, 2015). Figure 4 consists of a 3D representation of FAS I, with the intention of illustrating the complexity and functionality of these enzymes. Figure 4: Molecular 3D structure of fungal FAS I. Highlights of the top views of the enzyme with the αhexamer and β-trimer; respective domains from the αand βchains. Image obtained from I. B. Lomakin, Y. Xiong, and T. A. Steitz (2007). The type I FAS in yeast comprises two subunits, α-subunit Fas2 and β-subunit Fas1, encoded by the genes FAS2 and FAS1 , respectively. These subunits are organized as a hexameric α6β6 molecular complex of 2.6 MDa. Fas1 consists of an acetyl transferase (AT), enoyl reductase (ER), dehydratase (DH) and malonyl-palmitoyl transferase (MPT), while Fas2 contains an acyl carrier protein (ACP), 3-ketoreductase (KR), 3-ketosynthase (KS) and the phosphopantheteinyl transferase (PPT). This phosphopantetheine moiety is located at the C-terminus of the α-subunit and serves to activate yeasts FAS, and transport reaction intermediates through the various catalytic centers of the complex (Ridgway and McLeod, 2015; Hu et al., 2019; Lomakin et al., 2007; Tehlivets et al., 2007). The reaction is initiated by transferring an acetyl primer and malonyl elongation substrate to the acyl carrier protein - carried out by an acetyl transferase and a malonyl/palmitoyl transferase. Then, a ketoacyl synthase condenses these molecules to acetoacetyl-ACP, following the normal reaction mechanism for FA synthesis. The final product will be shuttled by ACP from ER to MPT where it is transferred to CoA and then released (Tehlivets et al., 2007; Ridgway and McLeod, 2015). 8 1.3.3 The FAS II System The FAS II system is more commonly present in bacteria, organelles of algae, protists but also in mitochondria and chloroplasts of eukaryotic cells. This system is composed of dispersed monofunctional polypeptides, each catalyzing a single reaction and encoded by a separate gene (Tehlivets et al., 2007; Ridgway and McLeod, 2015; White et al., 2005; Marrakchi et al., 2002). This pathway can produce a wide range of fatty acid structures, since it’s more flexible. Not only does it produce chains with different lengths, it also produces unsaturated fatty acids, isoand anteisobranched-chain fatty acids and hydroxy fatty acids. In prokaryotes, the molecules produced by this system comprise of FA for cell membranes, biotin, lipoic acid and the quorum-sensing acylhomoserine lactones (Ridgway and McLeod, 2015; White et al., 2005). For E. coli , ACP is a protein encoded by the acpP gene and is converted to its active form by [ACP]synthase (AcpS). AcpS transfers the 4-phosphopantetheine prosthetic group from CoA to apo-ACP. ACP undergoes rapid turnover in vivo, and the hydrolysis of the prosthetic group is accomplished by a Mn2+-dependent phosphodiesterase (AcpH). The malonyl group of malonyl-CoA is transferred to ACP by malonyl-CoA: ACP transacylase (FabD). All the subsequent intermediates in FAS are attached to the terminal sulphydryl of ACP, making this protein cofactor a partner in all subsequent reactions. β-Ketoacyl-ACP synthase III (FabH) catalyzes the first condensation step in the pathway using acetyl-CoA as the primer and malonyl-ACP as the acceptor. The acetoacetyl-ACP formed by FabH then begins the elongation cycle, that consists of four core enzyme activities that progressively elongate the acyl chain attached to ACP by two carbons through each revolution. β-Ketoacyl-ACP reductase (FabG) is a NADPH-dependent reductase that originates β-hydroxyacyl-ACP. This intermediate is then dehydrated by the β-hydroxyacyl-ACP dehydratases (either FabA or FabZ). The enoyl-ACP is reduced by the NADH-dependent enoyl-ACP reductase (FabI) to complete the cycle. There are two other isoforms of this enzyme (FabK and FabL) found in bacteria. The FabK group has an additional flavin cofactor and is found in gram-positive bacteria. Subsequent rounds of elongation are initiated by the condensing enzymes FabB or FabF. These enzymes condense the growing acyl-ACP with malonyl-ACP to extend the fatty acid chain by two carbons (Marrakchi et al., 2002; White et al., 2005). Figure 5 allows the comparison between FAS I and FAS II systems. 9 not further increase secreted FFA levels (Scharnewski et al., 2008). Leber et al. (2015) included a deletion of the fatty acyl-CoA synthetase gene FAT1 , creating a faa1∆faa4∆fat1∆strain that secreted 60% more long-chain FFA relative to the faa1∆faa4∆parent strain. Preventing both FFA activation and β-oxidation can further increase extracellular FA levels (Fernandez-Moya and Da Silva, 2017; Leber et al., 2015). In strains expressing a heterologous TE, combined deletions of FAA4 and POX1 increased the FFA secreted to the medium by 54% over the FAA4 deletion alone. Li et al. (2014) found that deletion of FAA1 , FAA4 and POX1 increased the FFA by 31% relative to deletion of FAA1 and FAA4 (Fernandez-Moya and Da Silva, 2017; Leber et al., 2015; Li et al., 2014; Runguphan and Keasling, 2014). Increasing synthesis of FAs via diaglycerol acyltransferases expression Yeasts store fatty acids as neutral lipids, in form of triaglycerols (TAGs) and steryl esters (SEs), forming lipid droplets. Diacylglycerol acyltransferase (DGAT) catalyzes the acyl-CoA-dependent acylation of sn-1,2diacylglycerol to produce triacylglycerol (TAG). Therefore, the DGA1 gene along with Lro1 (encoding for a phospholipid:diacylglycerol acyltransferase, an enzyme that enzyme specifically transfers acyl groups from the sn-2 position of a phospholipid to diacylglycerol (DAG), thus forming an sn-1-lysophospholipid) are the major contributors for TAG synthesis. SEs are synthesized by Are1 and Are2, using fatty acyl-CoA and sterol. Lipid droplet formation is not essential for the survival of the cell, since the deletion of the genes responsible for synthesizing these neutral lipids ( DGA1 , LRO1 , ARE1 , and ARE2 ) does not harm yeast (Greer et al., 2015; Fernandez-Moya and Da Silva, 2017; Grillitsch et al., 2011). It has been shown that the combination of overexpressing Dga1 and Tgl3 (a major TAG lipase) led to the highest FFA production (Dahlqvist et al., 2000). There have also been attempts at exploring over-expression of a type 1 plant diacylglycerol acyltranferase (DGA1) to increase lipid content in S. cerevisiae (Greer et al., 2015; Knoshaug et al., 2018). The work of Knoshaug et al. (2018) showed that yeast strains with Snf1 knocked out and over-expression of DGA1 boosted lipid accumulation from 20% to 50% (Knoshaug et al., 2018). In 2013, another attempt at increasing lipid production in S. cerevisiae aimed to overexpress a specific variant of diacylglycerol acyltransferase, known as Dga1∆Np, lacking the N-terminal 29 amino acids. The researchers investigated the effects of this overexpression in a mutant strain lacking the DGA1 gene and found that it significantly enhances lipid accumulation, achieving up to 45% lipid content in glucose-rich media. It was also concluded that the deletion of the 3’ terminal region of the DGA1 gene, which not only allows Dga1∆Np to function more effectively but also modulates the expression of adjacent genes involved in lipid metabolism. The findings suggest that engineering yeast strains with Dga1∆Np can serve as a promising strategy for optimizing lipid production (Kamisaka et al., 2013). 16 1.5 Objectives The aim of this study consisted in exploring the effects of redirection of co-factor supply and product removal in fatty acid synthesis in S. cerevisiae strains that are lacking the native FAS I system and express a hybrid FAS II system with genes from E. coli and A. thaliana instead. 1.5.1 Push strategy for FA synthesis with gene deletions and complementation of yeasts FAS Knowing that NADPH is a necessary cofactor in fatty acid production, can a deletion of the PGI1 gene allow for the accumulation of this co-factor and, subsquently, increase FA synthesis? The PGI1 gene was deleted from S. cerevisiae strains that already had deletions in FAS1 and FAS2 . The new strains would then be transformed with a pTA1_FASIIb plasmid. Additionally, overexpression of the ZWF1 gene would be achieved with transformation with a M8_ZWF1 plasmid. 1.5.2 Pull strategy for FA synthesis with gene cloning The effects of a pull strategy would be studied by constructing a vector expressing a diacylglycerol acyltransferase, encoded by the DGA1 gene. For this objective, the construction of a pTA9_TDH3_DGA1_TEF1 plasmid vector would be employed and later expressed in S. cerevisiae . 1.5.3 Analysis of fatty acid production The new mutants would be cultivated and their growth profiles would be analyzed, as well as the production of fatty acids, to assess the efects of the metabolic engineering strategies specified above. For this purpose, image-based quantitative analysis of lipid droplets would be performed by observation of cells by fluorescence microscopy and flow cytometry. 17 Chapter 2 Materials and Methods 2.1 Yeast and bacterial strains Escherichia coli strain XL1-Blue (Stratagene, La Jolla, CA, USA) was used for plasmid transformation, storage and later extraction. The Saccharomyces cerevisiae strain CEN.PK2-1C was used as the starting point for strain construction in previous work. The CEN∆fas1∆fas2 (µ1836, ∆fas1,2) and CEN∆fas2 (µ1767, ∆fas2) yeast mutants were already present in the host’s lab strain collection and the deletions were performed with the Cre/loxP method. They were used as chassis for further engineering, regarding the PGI1 deletion and M8_ZWF1 and pTA1_FASIIb plasmid transformation (Pozdniakova et al., 2023). CEN.PK113-7D was used as a control strain for various experiments as it does not present any kind of auxotrophy. CEN.PK113-5D was used for assembly by homologous recombination of the plasmid pTA9_TDH3_DGA1_TEF1. Table 2 compiles all the strains constructed during the work that was carried out. All strains with an attributed ID were stored on glycerol 50% at -80ºC. 18 Table 2: Identification of all the chassis strains and subsequent mutants, as well as control strains for this work. Strains IOA5 and IOA6 were not able to grow without exogenous fatty acids, therefore, they underwent an adaptation period on YPFD to be able to grow without myristic acid. However, IOA5 did not make the cut because of culture contamination, and only IOA6 was successfully adapted, which explains the * indicator on the ID, that refers to the final adapted strain Strain Nomenclature Genotype Unique ID Source CEN.PK113-7D No auxotrophy µ876 - CEN.PK2-1C MATa ura3_52 his3_∆1leu2_3112 trp1_289, MAL2_8c SUC2 µ922 - CEN.PK113-5D ura3-52 HIS3 LEU2 TRP1 MAL2-8c SUC2 µ878 - ∆fas1,2 CEN.PK2-1C ∆fas1∆fas2 µ1836 Submitted ∆fas2 CEN.PK2-1C ∆fas2 µ1767 Submitted IOA1 CEN.PK2-1C ∆fas1∆fas2 (∆PGI1::natMX4) µ1907 This study IOA2 CEN.PK2-1C ∆fas2 (∆PGI1::natMX4) µ1908 This study IOA3 CEN.PK2-1C (∆PGI1::natMX4) µ1909 This study IOA4 CEN.PK113-7D (∆PGI1::natMX4) µ1911 This study IOA5 CEN.PK2-1C ∆fas1∆fas2 (∆PGI1::natMX4) + pTA1_FASIIb µ1942 This study IOA6 CEN.PK2-1C ∆fas2 (∆PGI1::natMX4) + pTA1_FASIIb µ1950* This study IOA7 CEN.PK2-1C (∆PGI1::natMX4) + pTA1_FASIIb - This study IOA8 CEN.PK113-7D (∆PGI1::natMX4) + pTA1_FASIIb - This study IOA9 CEN.PK2-1C ∆fas1∆fas2 (∆PGI1::natMX4) + M8_ZWF1 - This study IOA10 CEN.PK2-1C ∆fas2 (∆PGI1::natMX4) + M8_ZWF1 - This study IOA11 CEN.PK2-1C (∆PGI1::natMX4) + M8_ZWF1 - This study IOA12 CEN.PK113-7D (∆PGI1::natMX4) + M8_ZWF1 - This study IOA13 CEN.PK2-1C ∆fas1∆fas2 + pTA1_FASIIb - This study IOA14 CEN.PK2-1C ∆fas2 + pTA1_FASIIb - This study 2.2 Culture media CEN.PK2-1C, CEN.PK113-7D, and CEN.PK113-5D were grown on YPD rich media containing 1% (w/v) yeast extract, 2% (w/v) peptone, and 2% (w/v) glucose. For the PGI1 deletion, ∆fas1,2 and ∆fas2 were initially pre-inoculated in liquid YPFD medium composed of 2% (w/v) peptone, 1% (w/v) yeast extract, 2% (w/v) fructose, and 0.1% (w/v) glucose, supplemented with 0.5% (w/v) myristic acid (Hmyr) and 1% (w/v) Tween 40 (polysorbate 40) – referred to as “YPFD HMyr” (Boles et al., 1993; Pereira, 2013). For the selection of PGI1 deletion strains, solid YPFD HMyr was prepared with 200 µL/mL nourseothricin – termed “YPFD HMyr NAT.” To investigate the respiratory capacity of the transformants, media containing ethanol was also prepared – designated “YPGES.” This media contains 2% (w/v) peptone, 1% (w/v) yeast extract, 1% (w/v) sodium succinate, 2.08% (w/v) glycerol, 2.75% (w/v) 95% ethanol, and 0.5% (w/v) myristic acid, along with 1% (w/v) polysorbate 40, with a pH of 5.5 (Weger et al., 2002). For the subse19 quent plasmid transformations, transformants were selected on synthetic complete media (SC) with the omission of amino acids based on the marker present in the plasmid. Specifically, pTA1_FASIIb contained a leucine marker; therefore, cells were grown on synthetic complete media with yeast nitrogen base and myristic acid, supplemented with 2% (w/v) fructose and 0.1% (w/v) glucose, and a mixture of drop-out histidine, uracil, and tryptophan – referred to as “SC Fruct.+0.1%Gluc HMyr HUT.” For the PGI1 deleted mutants transformed with M8_ZWF1, the same media was used, except for the aminoacid mix that consisted of histidine, leucine, and tryptophan, since the plasmid had a selection marker for uracil – termed “SC Fruct.+0.1%Gluc HMyr HLT.” The composition of the drop-out mix can be found in Appendix A. The compositions of the media explained above are summarized in Table 3. For bacterial cultures, cells were cultivated in Lysogeny Broth Miller medium (“LB”): 1% (w/v) tryptone, 0.5% (w/v) yeast extract, and 1% (w/v) sodium chloride, with 0.1 g/L ampicillin added when needed – referred to as “LB Amp.” Table 3: Media composition in g/L for yeast growth Reagent (g/L) YPD (HMyr) YPFD (HMyr) YPGES (HMyr) SC+Fruct+Gluc HMyr SC Supplier Yeast Extract 10 10 10 - - PanReac AppliChem Peptone 20 20 20 - - Grisp Glucose 20 1 - 1 - Scharlau Myristic acid (if needed) 0.456 0.456 0.456 0.456 - Sigma Tween 40 10 10 10 10 - Acros Organics Agar (if needed) 20 20 20 20 20 LabChem Nourseothricin - 0.2 - - - Jena Bioscience Sodium succinate - - 10 - - Sigma Sodium chloride - - 10 - - PanReact AppliChem Ammonium sulphate - - - 5 5 PanReact AppliChem Glycerol - - 20.8 - - Sigma Ethanol - - 20.6 - - Sigma YNB (without amminoacids or ammonium sulphate) - - - 1.7 1.7 BD ”Drop-out” (mg/L) - - - 1.37 1.37 - Hystidine (mg/L) - - - 80 80 Formedium Uracyl (mg/L) - - - 80 80 Formedium Tryptophan (mg/L) - - - 80 80 Formedium Leucine (mg/L) - - - 400 40 Formedium 20 2.3 Plasmid and genomic DNA extractions For DNA extraction from bacterial cell cultures, plasmid DNA was extracted using the NZYMiniprep kit (NZYTech), following the manufacturer’s protocol. For plasmid rescue in yeast, the same Miniprep kit was used with an additional pre-treatment step to disrupt the yeast cell wall. Yeast biomass was scraped from a fresh plate and resuspended in 1 mL of water. The cells were then centrifuged, and the supernatant was discarded. The recommended amount of resuspension buffer was added to the sample, which was then vortexed. After that, 200 µL of zirconiumsilica beads were added, and the sample was vortexed for about one minute using a Vortex Genie 2 (Scientific Industries, Inc.). The lysis buffer was added as quickly as possible to minimize DNA damage caused by nucleases released during cell wall disruption. The rest of the protocol was followed without further changes, and the DNA was stored at -20°C. For genomic DNA extraction from yeast, the cells were first washed in water. In an Eppendorf tube, 150 µL of TE (10x) (Tris-EDTA), phenol:chloroform alcohol (25:24:1), and zirconium-silica beads were added to the biomass. This mixture was vortexed for five minutes. Afterward, the sample was centrifuged for ten minutes at 14,000 rpm. The supernatant, containing the DNA, was then stored at -20°C. 2.4 PCR conditions and methods All PCR conditions were calculated using the WebPCR free tool (https://pydna.pythonanywhere.com). This tool provides PCR conditions, including melting temperature (Tm), extension time, and the size of the PCR product, expressed in base pairs (bp). After centrifugation, the PCR tubes were placed in the thermocycler (T100; Bio-Rad) with the temperature program obtained with the WebPCR simulation. Following the reaction, the PCR products were run on agarose gels composed of 1% Tris-acetate-EDTA (TAE) buffer (1x, Bio-Rad), 10 g/L agarose (Grispr), and 0.5 µg/mL MidoriGreen (Nippon Genetics). A 1x loading buffer was incorporated with each PCR product,consisting of 2% FICOLL 400, 0.0025 % Xylene Cyanol FF, 0.3 % VAHINE Yellow, 0.024% Orange G. The molecular weight marker used was Gene Ruler 1 kb (Thermo Scientific). Electrophoresis was performed at 200 V for 20 minutes, and the DNA bands were visualized and captured under UV light using the GenoSmart UV transilluminator (VWR). Colony PCRs from yeast were performed by collecting a small amount of biomass (approximately 1 mm) and adding it to an Eppendorf tube with 20 µL of 1x TE buffer and approximately 50 mg of zirconium-silica beads. This mixture was subjected to one minute of vortexing using the Vortex Genie and 21 then incubated at 95°C for ten minutes. The DNA samples were centrifuged at maximum speed for one minute, and the supernatant was used for fragment amplification. For each PCR, the reaction mixture had the following composition: 50% master mix (MM), 2.5% forward primer, 2.5% reverse primer, and 35% ultrapure H2O. For all reactions, 1 µL of DNA template was used. If the template was from a plasmid extraction from E. coli , the DNA samples were diluted 1:100 with ultrapure sterile water before collecting 1 µL for use as the PCR template. A negative control was always performed to make sure the PCR reaction mix was not contaminated: this consisted of all reaction components except the template DNA which was substituted by ultrapure water. For Taq polymerase reactions, the master mix was 2x concentrated and consisted of 2x Taq polymerase buffer, 0.05 U/µL Taq polymerase, 4 mM MgCl2, and 0.4 mM of each dNTP. For PCR reactions requiring proofreading accuracy, Supreme polymerase (NZYTech) was used. The Supreme master mix (2x) contained 2x PCR buffer, 0.05 U/µL Supreme polymerase, and 0.25 mM of each dNTP. 2.5 Yeast and bacterial transformations For each bacterial transformation, calcium-competent E. coli XL1-Blue cells were initially thawed on ice, followed by the addition of 10 µL of DNA for every 200 µL of cells. The cells were stored at -80°C in 200 µL aliquots and prepared according to the protocol by Inoue et al. (1990). After gently mixing the components and incubating them on ice for thirty minutes, the cells were subjected to heat shock in a 42°C water bath for exactly 45 seconds. The samples were cooled on ice for two minutes to accelerate heat transfer, followed by the addition of pre-warmed LB medium to allow for recovery at 37°C for one hour in 2 mL Eppendorf tubes. The culture was centrifuged for thirty seconds at 12,000g, and the supernatant was discarded, leaving a residual volume of 200-300 µL. The final pellet was resuspended, and 50 µL of transformed cells were used to inoculate an LB Amp plate (Inoue et al., 1990). In the case of S. cerevisiae transformation, the “LiAc/SS carrier DNA/PEG method” (Gietz and Schiestl, 2007; Pham et al., 2011) was followed for the genome deletions and plasmid transformation. The day before the transformation, a 5 mL pre-inoculum was prepared and incubated overnight (approximately 19 to 32 hours before the experiment, depending on the strain’s growth rate). On the next day, the OD640nm of the pre-inoculum was measured, and based on the cell count, part of the pre-inoculum was used to start a new culture with an initial OD of 0.17. The cells were incubated at 30°C with shaking at 200 rpm until the cell density reached approximately 0.689, completing at least two cell divisions. Cells were 22 then harvested and kept on ice overnight. The following day, the inoculum was centrifuged at 3000g for 5 minutes, washed in 25 mL of sterile water, and resuspended in 1 mL of sterile water. The cells were transferred to a 1.5 mL Eppendorf tube, centrifuged for 20 seconds, and resuspended in 1 mL of sterile water by vortexing. Approximately 1.0 × 108cells was transferred to 1.5 mL Eppendorf tubes, centrifuged, and the supernatant was removed. To each pellet, 300 µL of a mix containing 50% w/v polyethylene glycol (PEG), 1 M lithium acetate, and previously boiled single-stranded carrier DNA (ssDNA) was added, along with 60 µL of DNA. The mixture was resuspended by vortexing and incubated at 42°C in a thermoblock for 40 minutes. Cells were recovered by centrifugation at maximum speed for 30 seconds. When the selection marker was an antibiotic, cells were resuspended in 1 mL of growth medium and allowed to recover for about 4 hours at 30°C. After recovery, cells were centrifuged, and 200 µL of sterile ultrapure water was added to each tube to resuspend the culture. Finally, 1/10 of the cells were plated on selection media using the ”samba method” and incubated for 4 days at 30°C. For strains whose selection process was based in acquired auxotrophy, this step was skipped directly into plating. 2.6 Plasmid assembly by the Yeast Pathway Kit The Yeast Pathway Kit (YPK) was used to construct the pTA9_TDH3_DGA1_TEF1 plasmid. The YPK metabolic pathway assembly strategy takes advantage of the observation that natural intergenic sequences from genes expressed in tandem are both terminators of the upstream gene and promoters for the downstream gene. These intergenic sequences (designated terminator – promoters or TPs) are used both for transcription regulation and for aiding the assembly of multiple gene metabolic pathways from single-gene expression cassettes. TPs from the intergenic sequences upstream of genes such as TEF1 (579 bp) and TDH3 (698 bp) were PCR-amplified from S. cerevisiae chromosomal DNA (Pereira et al., 2016). The pYPKa vector (Figure 9) is a positive selection vector, allowing only host cells with inserted foreign DNA to survive under specific conditions, while those with uncut or self-ligated vectors do not grow (Choi Young-Jun and Lee, 2002). By cloning genes into pYPKa, E. coli can be transformed to primarily obtain plasmids containing the desired genes. New plasmids can then be assembled via homologous recombination in S. cerevisiae , incorporating multiple genes in the correct order, thereby allowing for coordinated regulation and expression. 23 Figure 9: Schematic representation of assembly by the YPK strategy. The promoter must be amplified with primers ”577_crp585-557” and ”567_pCAPsAjiIF”; the terminator is amplified with the primers ”568_pCAPsAjiIRe” and ”578_crp42-70” and, lastly, the open reading frame (ORF) that is used for insertion of the desired gene requires amplification with the primers ”468_pCAPs_release_fw” and ”467_pCAPs_release_re”. The red, green, pink, and grey boxes represent the matching sequences, allowing for homologous recombination when constructing any plasmid using these tandemly expressed genes. Images obtained from F. Pereira et al. (2016). For this work, the promoter and terminator were amplified from two different plasmids: pYPKa_E_TEF1 and pYPKa_Z_TDH3, extracted from E. coli . The sequences corresponding to the TDH3 and TEF1 genes were amplified with the primers “577_crp585-557” and “567_pCAPsAjiIF”, “568_pCAPsAjiIR” and “578_crp42-70” respectively. The DGA1 gene was amplified from a sample of S. cerevisiae chromosomal DNA with the primers “1803_fwdDGA1” and “1802_revDGA1”, specifically designed for this purpose (Appendix B). The suicide vector pYPKa was extracted from E. coli and digested overnight at 37°C with AjiI (Thermofisher), leaving two blunt edges in the plasmid at the enzyme’s restriction site. For this step, 17 µL of pYPKa vector, 2 µL of buffer for AjiI and 1 µL of the restriction enzyme were mixed. Inactivation would follow at 65°C for twenty minutes. Next, the linearized plasmid was subjected to a phosphorylation treatment (two hours at 37°C) with alkaline phosphatase to reduce the risk of recircularization. Then, the DNA would be purified with NZYTech’s Gel Pure kit, following the manufacturers protocol. The ligation of DGA1 into pYPKa was possible with T4 Ligase (Thermofisher) and it occured overnight at 4°C. For one ligation protocol, 15 µL of purified linearized pYPKa plasmid; 2 µL of buffer for T4 ligase, 1 µL of T4 ligase and 2 µL of the DGA1 PCR product. The result was a plasmid named pYPKa_A_DGA1, where “A” refers to the restriction site used to linearize the plasmid (AjiI). CEN.PK113-5D was transformed with a linearized pTA9 vector, the TDH3 promoter, the TEF1 terminator and the DGA1 gene (amplified from pYPKa with primers ”468_pCAPs_release_fw” and ”467_pCAPs_release_re”) and the cell’s machinery would be responsible 24 for assembling the correspondent genes into the backbone vector. Yeast cells were transformed according to the protocol described in Section 2.5. 2.7 Growth curves To determine growth profile of the selected strains, cells were cultured in 100 mL Erlenmeyer flasks containing 15 mL of medium at 30°C for 72 hours. The initial OD was adjusted to 0.17. Growth assays involved periodic measurements of cell density (OD640nm). For each biological replicate, the average OD and the standard deviation were calculated. These calculations were performed using Microsoft Excel. To estimate the specific growth rate (µ) of the microbial population, data from the exponential phase of the growth curve were analyzed. The exponential phase represents the period where the population grows at its maximum rate, typically described by Equation 1: ln(OD2(t)) = µ·t+ln(OD1)(1) In practice, the specific growth rate can be obtained in the exponential growth phase by chosing two time poins t1and 2and the respective cell density values, OD1and OD2(Equation 2): µ=ln OD2−ln OD1 (t2−t1)(2) The exponential phase was identified by examining the overall growth curve and selecting three representative time points where the population growth exhibited a linear relationship on a semi-logarithmic plot. These time points were chosen because they fall within the period where the population growth is most closely approximated by an exponential function, as the assumption of constant specific growth rate holds during this phase. Microsoft Excel displays the option of performing the calculations to obtain the equation of the model present in Equation 1. This method assumes that during the selected time interval, the growth rate remains constant, and the effects of nutrient depletion or environmental changes are negligible. Furthermore, the linear regression model assumes that there is minimal measurement error in the population size data. 25 Figure 14: Phenotype testing in YPD; YPD HMyr; YPFD; YPFD HMyr; YPGES; YPGES HMyr for strains IOA1 to IOA4. Position 1: ∆fas1,2; position 2: ∆fas2; position 3: CEN.PK113-7D; position 4: CEN.PK2-1C TRP; positions 5-9: colonies from IOA1; positions 11-15: colonies from IOA3; positions 17-20: colonies from IOA2; positions 21 and 22: colonies from IOA4. For this test, about 1mm of colony was scraped from master plates of the selected clones and dissolved in 100 µL of sterile water; drops of 4 µL of this cell solution were laid on the respective spots in the plates. Cells were incubated for four days at 37 ºC. In Figure 14 it is possible to observe that the ∆fas1,2 and ∆fas2 mutants with the deletion of PGI1 do not grow on glucose without supplementation of exogenous fatty acids (myristic acid), which was the expected outcome (Pozdniakova et al., 2023; Fernandez-Moya et al., 2015). S. cerevisiae uses glucose as the preferable carbon source, being that fermentation is the major pathway for energy production, even under aerobic conditions. However, yeasts can not only metabolize different carbon sources like galactose, fructose, sucrose, and maltose, but can also use nonsugar carbon sources such as ethanol, lactate, glycerol, and acetate. When glucose becomes scarce, ethanol produced during fermentation is used as a carbon source, requiring a shift to respiration (Gasmi et al., 2014; Turcotte et al., 2009). The strains were also subjected to phenotype testing on YPGES to investigate their respiratory capacity. As shown in Figure 3.5, strains ∆fas1,2 and ∆fas2 can grow on YPGES with exogenous fatty acids (YPGES HMyr) but not without them (YPGES), which is in accordance with the results of Podzniakova et al. (2023). The same conclusion is true for strains IOA1 and IOA2. 32 3.2 Gene cloning The mutants that displayed no growth on glucose and the strongest DNA bands in the genotype test were selected for transformation with either pTA1_FASIIb or M8_ZWF1. The clones were selected on synthetic complete media, supplemented with fructose and glucose, as described in Section 2.2 of the Materials and Methods chapter. The mutants containing the plasmid pTA1_FASIIb were selected based on their auxotrophy for leucine (SC Fruct.+0.1% Gluc. HMyr HUT). A positive control for this transformation included the CEN.PK2-1C strain with pTA1. For the transformants with M8_ZWF1, selection was performed using the same medium, but with uracil as the selection marker (SC Fruct.+0.1% Gluc. HMyr HLT). For both experiments, negative controls were prepared by substituting the plasmid DNA in the transformation mix with ultrapure water. Images of the clones from these experiments can be found in Appendix E. While pTA1_FASIIb complemented yeast’s native FAS, the role of M8_ZWF1 was to overexpress the ZWF1 gene to enhance the redirection of co-factors toward fatty acid biosynthesis. ZWF1 encodes glucose6-phosphate dehydrogenase, which catalyzes the first step of the pentose phosphate (PP) pathway, a key pathway for generating NADPH required for fatty acid production. The resulting clones were subjected to phenotype testing on different media, as shown in Figure 15 and 16. Figure 15: Phenotype testing in YPD; YPD HMyr; YPFD; YPFD HMyr; YPGES; YPGES HMyr for strains IOA5 to IOA8, transformed with pTA1_FASIIb. Positions 1 – 4: colonies from IOA5; positions 6 – 9: colonies from IOA6; positions 12 – 15: colonies from IOA7; positions 17 – 20: colonies from IOA8; positions 21 and 22, respectively: CEN.PK113-7D and CEN.PK2-1C. For this test, about 1mm of colony was scraped from master plates of the selected clones and dissolved in 100 µL of sterile water; drops of 4 µL of this cell solution were laid on the respective spots in the plates. Cells were incubated for four days at 37 ºC. 33 Figure 16: Phenotype testing in YPD; YPD HMyr; YPFD; YPFD HMyr and YPGES for strains IOA1 to IOA4 transformed with the plasmid M8_ZWF1. Positions 1 – 4: colonies from IOA9; positions 6 – 9: colonies from IOA10; positions 12 – 15: colonies from IOA11; positions 17 – 20: colonies from IOA12; positions 21 and 22, respectively: CEN.PK113-7D and CEN.PK2-1C. For this test, about 1mm of colony was scraped from the master plates of the selected clones and dissolved in 100 µL of sterile water; drops of 4 µL of this cell solution were laid on the respective spots in the plates. Cells were incubated for four days at 37 ºC. As can be seen in Figure 15 and 16, the transformants were not only unable to grow without myristic acid, but also did not show the ability to metabolize glucose as a carbon source. It was hoped that the cells would be able to metabolize glucose after being complemented with the exogenous FAS II system. Some clones appeared to grow on YPGES. To determine whether these clones truly displayed growth on this medium, further phenotype tests were conducted using YPGES with only 0.5% agarose. This adjustment aimed to rule out the hypothesis that these clones might be obtaining nutrients from the agar present in the medium. These additional tests are also provided in Appendix F. However, it was observed that, similar to other media, the strains did not display any growth. Several hypotheses were proposed to explain these results: 1. The transformation may not have been successful, and these cells might not possess the ptA1_FASIIb plasmid. 2. There could be an issue related to the chassis strain. 3. The cells are heavily engineered, which could result in poor health and display growth difficulties. To test hypothesis number two, two control groups were prepared: strains IOA13 and IOA14. These strains were cultivated in YPD, as shown on Figure 17. Although the growth is very little and span over 2 34 weeks to grow the size of colonies on the picture, this showed that indeed the plasmid should’ve allowed for the metabolization of glucose. Figure 17: Phenotype testing of chassis strains with deletions in genes encoding the FAS system in S. cerevisiae , grown on YPD. The cells were incubated at 30°C for two weeks until the colonies reached the size shown in the image. On the left, six isolated colonies of the ∆fas1,2 strain, transformed with the pTA1_FASIIb plasmid, are displayed (note the presence of fungal and bacterial contamination on the plate). On the right, six isolated colonies of the ∆fas2 strain, also transformed with the same plasmid, are shown. As mentioned before, when glycolysis is disrupted at the level of phosphoglucose isomerase, which catalyzes the interconversion of glucose 6-phosphate and fructose 6-phosphate, glucose-6-phosphate is completely rerouted into the PP pathway. The deletion of PGI1 in S. cerevisiae impedes growth on glucose. However, S. cerevisiae PGI1 mutants can grow on fructose as the primary carbon source, with trace amounts of glucose required for the synthesis of glucose-6-phosphate, a key precursor in phospholipid biosynthesis (Boles et al., 1993; Heux et al., 2008). Heux et al. (2008) studied the PP pathway capacity and NADPH metabolism in different S. cerevisiae strains, focusing on ∆ pgi1 mutants and the effects of expressing the E. coli transhydrogenase udhA. Expression of udhA partially restored growth on glucose by aiding NADPH reoxidation, but not in all strains. For instance, the udhA-expressing V5pgi1 strain did not grow on glucose, suggesting that PP pathway capacity, rather than just NADPH reoxidation, may be a limiting factor and its effectiveness varies depending on the specific genetic background of the yeast strain. It was also noted that the specific G6PDH activity was significantly lower in V5 and S288C strains, so researchers examined whether the growth issue in Udha-expressing pgi1 mutants might be due to a limited capacity in the first step of the PP pathway. To test this hypothesis, ZWF1 was overexpressed in V5pgi1 by in situ replacement of its own promoter by the 35 strong TDH3 promoter and subsequent transformation by a plasmid containing udhA. However, the udhAV5pgi1 strain overexpressing ZWF1 failed to grow on glucose, suggesting that other steps downstream the G6PDH limit the flux through the PP pathway in this strain. This present study also aimed to transform IOA5 and IOA6 strains with the M8_ZWF1 plasmid, but due to time constraints, it was not feasible to complete this experiment within the scope of the project timeline. Similarly to Heux et al.’s findings, this may not be sufficient to restore glucose metabolism, however, it is still relevant as per genetic background of different S. cerevisiae strains may influence the results. Future work should explore overexpressing other PP pathway genes to identify other limiting steps. Further efforts focused on adapting IOA5 and IOA6 to grow without exogenous fatty acids and analyzing their lipid production through fluorescence-based assays. 3.3 Adaptation of transformant cells for growth without exogenous FAs From this point onward, PGI1 deleted strains expressing FASII, strains IOA5 and IOA6 were cultivated in 250 mL Erlenmeyer flasks for six days (see Appendix G) with YPFD to enable growth on media without exogenous fatty acids. A pre-inoculum of 5 mL of the strains was prepared using YPFD HMyr. Consequently, about 100 µL of the cultures were collected and plated on YPFD solid media, as shown in Figure 18. The IOA5 strain became contaminated during the process; therefore, these cells were not selected for further examination. Figure 18: Strain adaptation of IOA5 and IOA6 for growth without exogenous FA. After the growth period was over, 100 µL sample of the liquid adaptation culture was collected and plated on solid YPFD media. On the left, the plates showed that the IOA5 culture was contaminated with bacteria – seen on microscope, no data available – and strain IOA6 showed no visible growth; in the last plate on the right, after 10 days of plating the cells, small colonies were visible. 36 Initially, no colonies were obtained after a 6-day incubation period. Cells were left incubating at 30ºC for more 4 days, until small colonies were observed in the plate of strain IOA6. Seven specimens were collected and submitted for genotype testing. To rule out hypothesis number one from subsection 3.2, four primer combinations were selected to determine whether the pTA1_FASIIb plasmid (Figure 19) was indeed inserted into the cells. The selected primers targeted specific regions of the plasmid, namely, the TEF promoter region of the nourseothricin resistance cassette: “387_A-PGI1” and “394_kanBeta”; Arabidopsis thaliana FATB gene: “948_AthFatA1_YPK_rv” and “949_AthFatA1_YPK_fw”; two different sequence zones encoding for the acyl carrier protein from E. coli : “728_EcacpS_fw” and “1033_rv405”; and, lastly, “728_EcacpS_fw” and “578_crp42-70”. Figure 19: Map of the pTA1_FASIIb plasmid. Image obtained with SnapGene. Figure 20 consists of the agarose gels with the amplification products from the selected regions of the plasmid in question to verify the correct transformation of the strains. The results shown on Figure 20 show that the pTA1_FASIIb plasmid was sucessfully inserted in the cells. The issue regarding the cells phenotype must be related to other metabolic issues, as hypothesized in subsection 3.2. Clones 2 and 8 of IOA6 were selected for further analysis and stored at -80ºC in 50% glycerol. 37 Figure 20: PCR testing of the IO6 strain adapted to grow on YPFD. In the picture below, the negative controls correspond to the wells on the farthest right, although they are not identified in the picture bellow. The different clones (clone 1 to 8) are all in the same order as specified in the pictures above, on consecutive wells. 3.4 Growth profile of the adapted strain IOA6 The IOA6 ( FAS2 and PGI1 deleted strain expressing FASII) strain adapted to grow without myristic acid was then subjected to a 72h period of growth on YPFD, to analyze lipid production. Since these strains appeared to grow very slowly, it was expected that the accumulation of fatty acids was yet very deficient. Figure 21 corresponds to the growth profile of the IOA6 strain as well as a control strain that consisted of a CEN.PK2-1C transformed with the pTA1 plasmid. Figure 21: Growth curves for the IO6 strain and the control strain (CEN.PK2-1C TRP+pTA1) in YPFD medium. Cultures were grown in 15 mL of YPFD for 72 hours at 30°C and 200 rpm in 100 mL Erlenmeyer flasks. A pre-inoculum of both strains was prepared in YPFD and incubated for 2 days under the same conditions, due to the slow growth of the IO6 strain. Only two biological replicates were performed, as the OD of IO6 was insufficient to inoculate a third flask. 38 The biomass production of the IOA6 strain is significantly lower than that of the control strain. Specific growth rates were calculated, and the linear relationships obtained using Microsoft Excel (see Appendix H) were used to determine the specific growth rate for each biological replicate (Table 4). Table 4: Specific growth rates for the IOA6 and the control strain CEN.PK2-1C containing the plasmid pTA1. Strains were adapted to grow without exogenous fatty acids on YPFD Strain Specific growth rate (µ, h-1) R2 IOA6 (Replica 1) 0.0197 0.9391 IOA6 (Replica 2) 0.0197 0.9391 CEN.PK2-1C (Replica 1) 0.2095 0.9741 CEN.PK2-1C (Replica 2) 0.2088 0.9953 Strain IOA6 exhibits a clear deficiency in growth compared to the control. These results align with observations from similar studies. Fernandez-Moya et al. (2015) expressed a heterologous FASII system in S. cerevisiae . In strains where the native FAS2 was deleted, the mutant FASII strain exhibited only half the growth of the native strain used as a control in their study. Pozdniakova et al., who also used the FASII pathway used to this study, reached comparable conclusions. Although employing a heterologous FASII system in S. cerevisiae offers potential for producing a broader range of fatty acids, relying solely on the FASII system, especially with deletions in the native FASI, does not optimize fatty acid production. Further investigation is needed to understand the underlying reasons for these limitations (Pozdniakova et al., 2023; Fernandez-Moya et al., 2015). 3.5 Fluorescence-based cell analysis of lipid production In the yeast S. cerevisiae , as in other eukaryotes, non-polar lipids are stored in structures named lipid droplets (LDs) as a biologically inert form of fatty acids and sterols (Grillitsch et al., 2011). After a 72h incubation period, the same cells were collected into 50 mL sterile Falcon tubes and centrifuged at top speed for about 3 minutes; 1 mL was collected to be used as sample. After BODIPY staining, samples were observed under the microscope (Figure 22). 39 Figure 22: Fluorescence microscopy images of strain IOA6 adapted to grow on YPFD and the control strain CEN.PK2-1C+pTA1, both cultivated in the same medium. Images of the IOA6 (A, B and C, respectively) strain and the control strain CEN.PK2-1C+pTA1 (D, E and F, respectively) were captured using the brightfield channel, GFP channel, and a composite overlay of cells and lipid droplets (LDs). The overlay of cells and LDs was generated using CellProfiler; pink outlines indicate the cells, while yellow outlines indicate the lipid droplets. Images were acquired using a microscope equipped with a 100x/1.3 oil immersion objective lens and a 1.6x magnification setting. The relative area of lipid droplets per cell was calculated after obtaining the measurements from the optimized CellProfiler settings (Table 5). Detection limits and thresholds were optimized to obtain the most accurate outline of cells and LDs possible. Additional information can be found in Appendix I. Table 5: Relative area occupied by lipid droplets (LDs) per cells. Analysis performed on two pictures from strains IOA6 and CEN.PK2-1C+pTA1, after a 72h growth period on YPFD Strain Relative area of LDs per cells (%) IOA6 22.65 CEN.PK2-1C + pTA1 20.02 As observed in the data above, IOA6 seems to produce a similar amount of lipids as the control strain, being that in both cases, lipid droplets account for about 20% of the total area occupied. It should be noted that these results are subject to various sources of error, including sampling and instrumental errors, which can affect the program’s ability to accurately outline the structures being studied, leading to measurement errors in estimating their occupied area. Additionally, debris exhibiting fluorescence may be mistakenly included in the calculations, further contributing to potential inaccuracies. 40 Flow cytometry was performed on the same samples. The same two replicas for each strain were stained with BODIPY and subjected to analysis. The side scatter versus forward scatter plots were analyzed to exclude the events that accounted for debris (smaller objects), excluding the ones with lower values for the forward scatter. After defining the gates corresponding to the stained cells (see Appendix J), the FITC-H::FL1-H histograms were obtained (Figure 23). Figure 23: Histograms depicting BODIPY fluorescence detection for strains IOA6 and CEN.PK2-1C+pTA1, following a 72-hour incubation in YPFD. The FITC-H (FL1-H) parameter was utilized to identify the peaks corresponding to the intensity of the fluorescence signals emitted by the stained cells. (A) Biological replica 1; (B) replica 2. “FITC” (Fluorescein Isothiocyanate) is a fluorescent dye commonly used to label antibodies and other molecules and it emits green fluorescence when excited by a laser. The ”H” refers to the peak height of the fluorescence signal detected by the flow cytometer and is often used for analyzing fluorescence intensity. “FL1” represents a specific fluorescence channel in the flow cytometer, typically used for detecting the emission of dyes like FITC. No statistical studies have been made since there was only two replicas available for study (Table 6). In Figure 23, fluorescence is determined by the rightward shift. Both strains appear to exhibit similar fluorescence levels. The only difference observed is a greater variation in intensity for the IAO6 cells. These results are consistent with those obtained through fluorescence microscopy, indicating that the two strains are comparable. 41 Bibliography Felix Abeln and Christopher J Chuck. The history, state of the art and future prospects for oleaginous yeast research. Microb Cell Fact , 20(1):221, dec 2021. doi: https://doi.org/10.1186/s12934-021-01712-1. Andres Aguilera. Mutations suppressing the effects of a deletion of the phosphoglucose isomerase gene pgi1 in Saccharomyces cerevisiae . Current Genetics , 11(6):429–434, Mar 1987. ISSN 1432-0983. doi: 10.1007/BF00384603. URL https://doi.org/10.1007/BF00384603. Simon Arhar, Gabriela Gogg-Fassolter, Mojca Ogrizović, Klavdija Pačnik, Katharina Schwaiger, Mia Žganjar, Uroš Petrovič, and Klaus Natter. Engineering of Saccharomyces cerevisiae for the accumulation of high amounts of triacylglycerol. Microbial Cell Factories , 20(1):147, Jul 2021. ISSN 1475-2859. doi: 10.1186/s12934-021-01640-0. URL https://doi.org/10.1186/s12934-021-01640-0. Sara L. Baptista, Carlos E. Costa, Joana T. Cunha, Pedro O. Soares, and Lucília Domingues. Metabolic engineering of Saccharomyces cerevisiae for the production of top value chemicals from biorefinery carbohydrates. Biotechnology Advances , 47:107697, 2021. ISSN 0734-9750. doi: https://doi. org/10.1016/j.biotechadv.2021.107697. URL https://www.sciencedirect.com/science/ article/pii/S0734975021000033. E. Boles, W. Lehnert, and F. K. Zimmermann. The role of the nad-dependent glutamate dehydrogenase in restoring growth on glucose of a Saccharomyces cerevisiae phosphoglucose isomerase mutant. Eur J Biochem , 1993. Michela Cerone and Terry K. Smith. A brief journey into the history of and future sources and uses of fatty acids. Frontiers in Nutrition , 8, 2021. ISSN 2296-861X. doi: 10.3389/fnut. 2021.570401. URL https://www.frontiersin.org/journals/nutrition/articles/ 10.3389/fnut.2021.570401. Xiaoxu Chen, Xiaoyu Yang, Yu Shen, Jin Hou, and Xiaoming Bao. Screening phosphorylation site mutations in yeast acetyl-coa carboxylase using malonyl-coa sensor to improve malonyl-coa-derived product. Frontiers in Microbiology , 9, 2018. ISSN 1664-302X. doi: 10.3389/fmicb.2018. 48 00047. URL https://www.frontiersin.org/journals/microbiology/articles/10. 3389/fmicb.2018.00047. Wang Tsung-Tsan Choi Young-Jun and Byong H. Lee. Positive selection vectors. Critical Reviews in Biotechnology , 22(3):225–244, 2002. doi: 10.1080/07388550290789504. URL https://doi.org/10. 1080/07388550290789504. Anders Dahlqvist, Ulf Ståhl, Marit Lenman, Antoni Banas, Michael Lee, Line Sandager, Hans Ronne, and Sten Stymne. Phospholipid:diacylglycerol acyltransferase: An enzyme that catalyzes the acyl-coaindependent formation of triacylglycerol in yeast and plants. Proceedings of the National Academy of Sciences , 97(12):6487–6492, 2000. doi: 10.1073/pnas.120067297. URL https://www.pnas. org/doi/abs/10.1073/pnas.120067297. Carla C. C. R. De Carvalho and Maria José Caramujo. The various roles of fatty acids. Molecules , 23 (10), 2018. ISSN 1420-3049. doi: 10.3390/molecules23102583. URL https://www.mdpi.com/ 1420-3049/23/10/2583. Mahmood Ebadian, Susan van Dyk, James D. McMillan, and Jack Saddler. Biofuels policies that have encouraged their production and use: An international perspective. Energy Policy , 147:111906, 2020. ISSN 0301-4215. doi: https://doi.org/10.1016/j.enpol.2020.111906. URL https://www. sciencedirect.com/science/article/pii/S0301421520306182. Nadia El-Hage Scialabba. Chapter 2 - livestock food and human nutrition. In Nadia El-Hage Scialabba, editor, Managing Healthy Livestock Production and Consumption , pages 29–44. Academic Press, 2022. ISBN 978-0-12-823019-0. doi: https://doi.org/10.1016/B978-0-12-823019-0.00012-X. URL https://www.sciencedirect.com/science/article/pii/B978012823019000012X. Eoin Fahy, Shankar Subramaniam, Robert C. Murphy, Masahiro Nishijima, Christian R.H. Raetz, Takao Shimizu, Friedrich Spener, Gerrit van Meer, Michael J.O. Wakelam, and Edward A. Dennis. Update of the lipid maps comprehensive classification system for lipids¹. Journal of Lipid Research , 50:S9–S14, Apr 2009. ISSN 0022-2275. doi: 10.1194/jlr.R800095-JLR200. URL https://doi. org/10.1194/jlr.R800095-JLR200. Ruben Fernandez-Moya and Nancy A. Da Silva. Engineering Saccharomyces cerevisiae for high-level synthesis of fatty acids and derived products. FEMS Yeast Research , 17(7):fox071, 09 2017. ISSN 1567-1356. doi: 10.1093/femsyr/fox071. URL https://doi.org/10.1093/femsyr/fox071. 49 Ruben Fernandez-Moya, Christopher Leber, Javier Cardenas, and Nancy A. Da Silva. Functional replacement of the Saccharomyces cerevisiae fatty acid synthase with a bacterial type ii system allows flexible product profiles. Biotechnology and Bioengineering , 112(12):2618–2623, 2015. doi: https://doi. org/10.1002/bit.25679. URL https://analyticalsciencejournals.onlinelibrary. wiley.com/doi/abs/10.1002/bit.25679. Najla Gasmi, Pierre-Etienne Jacques, Natalia Klimova, Xiao Guo, Alessandra Ricciardi, François Robert, and Bernard Turcotte. The Switch from Fermentation to Respiration in Saccharomyces cerevisiae Is Regulated by the Ert1 Transcriptional Activator/Repressor. Genetics , 198(2):547–560, 08 2014. ISSN 1943-2631. doi: 10.1534/genetics.114.168609. URL https://doi.org/10.1534/genetics. 114.168609. R. Daniel Gietz and Robert H. Schiestl. High-efficiency yeast transformation using the liac/ss carrier dna/peg method. Nature Protocols , 2(1):31–34, Jan 2007. ISSN 1750-2799. doi: 10.1038/nprot. 2007.13. URL https://doi.org/10.1038/nprot.2007.13. A L Goldstein and J H McCusker. Three new dominant drug resistance cassettes for gene disruption in Saccharomyces Cerevisiae . Yeast , 15(14):1541–1553, oct 1999. ISSN 0749-503X. doi: 10.1002/ (SICI)1097-0061(199910)15:14<1541::AID-YEA476>3.0.CO;2-K. Michael S. Greer, Martin Truksa, Wei Deng, Shiu-Cheung Lung, Guanqun Chen, and Randall J. Weselake. Engineering increased triacylglycerol accumulation in Saccharomyces cerevisiae using a modified type 1 plant diacylglycerol acyltransferase. Applied Microbiology and Biotechnology , 99(5):2243–2253, Mar 2015. ISSN 1432-0614. doi: 10.1007/s00253-014-6284-4. URL https://doi.org/10.1007/ s00253-014-6284-4. Karlheinz Grillitsch, Melanie Connerth, Harald Köfeler, Tabiwang N. Arrey, Benjamin Rietschel, Brigitte Wagner, Michael Karas, and Günther Daum. Lipid particles/droplets of the yeast Saccharomyces cerevisiae revisited: Lipidome meets proteome. Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids , 1811(12):1165–1176, 2011. ISSN 1388-1981. doi: https://doi.org/10.1016/j.bbalip. 2011.07.015. Qiaoning He, Yongfu Yang, Shihui Yang, Bryon S. Donohoe, Stefanie Van Wychen, Min Zhang, Michael E. Himmel, and Eric P. Knoshaug. Oleaginicity of the yeast strain Saccharomyces cerevisiae d5a. Biotechnology for Biofuels , 11(1):258, Sep 2018. ISSN 1754-6834. doi: 10.1186/s13068-018-1256-z. URL https://doi.org/10.1186/s13068-018-1256-z. 50 Stéphanie Heux, Axelle Cadiere, and Sylvie Dequin. Glucose utilization of strains lacking PGI1 and expressing a transhydrogenase suggests differences in the pentose phosphate capacity among Saccharomyces cerevisiae strains. FEMS Yeast Research , 8(2):217–224, 03 2008. ISSN 1567-1356. doi: 10.1111/j. 1567-1364.2007.00330.x. URL https://doi.org/10.1111/j.1567-1364.2007.00330.x. J. Kalervo Hiltunen, Anu M. Mursula, Hanspeter Rottensteiner, Rik K. Wierenga, Alexander J. Kastaniotis, and Aner Gurvitz. The biochemistry of peroxisomal �-oxidation in the yeast Saccharomyces cerevisiae . FEMS Microbiology Reviews , 27(1):35–64, 04 2003. ISSN 0168-6445. doi: 10.1016/S0168-6445(03) 00017-2. URL https://doi.org/10.1016/S0168-6445(03)00017-2. Yating Hu, Zhiwei Zhu, Jens Nielsen, and Verena Siewers. Engineering Saccharomyces cerevisiae cells for production of fatty acid-derived biofuels and chemicals. Open Biol. , 9(5):190049, may 2019. doi: http://dx.doi.org/10.1098/rsob.190049. Hiroaki Inoue, Hiroshi Nojima, and Hiroto Okayama. High efficiency transformation of Escherichia coli with plasmids. Gene , 96(1):23–28, 1990. ISSN 0378-1119. doi: https://doi.org/10.1016/ 0378-1119(90)90336-P. URL https://www.sciencedirect.com/science/article/pii/ 037811199090336P. Yasushi Kamisaka, Kazuyoshi Kimura, Hiroshi Uemura, and Masakazu Yamaoka. Overexpression of the active diacylglycerol acyltransferase variant transforms saccharomyces cerevisiae into an oleaginous yeast. Appl Microbiol Biotechnol , 97(16):7345–7355, apr 2013. doi: https://doi.org/10.1007/ s00253-013-4915-9. Amit Khatri and Vineet Choudhary. Protocol to induce de novo lipid droplets in Saccharomyces cerevisiae . STAR Protocols , 4(4):102732, 2023. ISSN 2666-1667. doi: https://doi.org/10. 1016/j.xpro.2023.102732. URL https://www.sciencedirect.com/science/article/ pii/S2666166723006998. Eric Knoshaug, Stefanie Van Wychen, Arjun Singh, and Min Zhang. Lipid accumulation from glucose and xylose in an engineered, naturally oleaginous strain of Saccharomyces cerevisiae . Biofuel Research Journal , 5(2):800–805, 2018. ISSN 2292-8782. doi: 10.18331/BRJ2018.5.2.3. URL https: //www.biofueljournal.com/article_63201.html. Christopher Leber, Brian Polson, Ruben Fernandez-Moya, and Nancy A. Da Silva. Overproduction and secretion of free fatty acids through disrupted neutral lipid recycle in Saccharomyces cerevisiae . Metabolic 51 Engineering , 28:54–62, 2015. ISSN 1096-7176. doi: https://doi.org/10.1016/j.ymben.2014.11.006. URL https://www.sciencedirect.com/science/article/pii/S109671761400144X. Lesetja M. Legodi and Kgabo L. M. Moganedi. Selection of oleaginous yeasts isolated from sugarcane bagasse as the potential producers of single cell oils and other metabolites. Annals of Microbiology , 73(1):21, Jun 2023. ISSN 1869-2044. doi: 10.1186/s13213-023-01725-8. URL https://doi. org/10.1186/s13213-023-01725-8. Yuxin Lei, Xuemei Wang, Shushuang Sun, Bingyang He, Wenjin Sun, Kexin Wang, Zhengxian Chen, Zhiling Guo, and Zifu Li. A review of lipid accumulation by oleaginous yeasts: Culture mode. Science of The Total Environment , 919:170385, 2024. ISSN 0048-9697. doi: https://doi.org/10.1016/j. scitotenv.2024.170385. URL https://www.sciencedirect.com/science/article/pii/ S0048969724005205. Albert Lenhinger, Michael M. Cox, and David L. Nelson. Lenhinger Principles of Biochemistry (7th ed.) . W. H. Freeman, 2017. Xiaowei Li, Daoyi Guo, Yongbo Cheng, Fayin Zhu, Zixin Deng, and Tiangang Liu. Overproduction of fatty acids in engineered Saccharomyces cerevisiae . Biotechnol. Bioeng. , 111(9):1841–1852, 2014. doi: https://doi.org/10.1002/bit.25239. Ivan B. Lomakin, Yong Xiong, and Thomas A. Steitz. The crystal structure of yeast fatty acid synthase, a cellular machine with eight active sites working together. Cell , 129(2):319–332, 2007. ISSN 00928674. doi: https://doi.org/10.1016/j.cell.2007.03.013. URL https://www.sciencedirect. com/science/article/pii/S0092867407003297. H Marrakchi, Y M Zhang, and C O Rock. Mechanistic diversity and regulation of type II fatty acid synthesis. Biochem Soc Trans , 30(Pt 6):1050–1055, nov 2002. doi: https://doi.org/10.1042/bst0301050. Diethard Mattanovich, Michael Sauer, and Brigitte Gasser. Yeast biotechnology: teaching the old dog new tricks. Microbial Cell Factories , 13(1):34, Mar 2014. ISSN 1475-2859. doi: 10.1186/ 1475-2859-13-34. Yasunari Michinaka, Toshitsugu Shimauchi, Tsunehiro Aki, Toshiaki Nakajima, Seiji Kawamoto, Seiko Shigeta, Osamu Suzuki, and Kazuhisa Ono. Extracellular secretion of free fatty acids by disruption of a fatty acyl-coa synthetase gene in Saccharomyces cerevisiae . Journal of Bioscience and Bioengineering , 52 95(5):435–440, 2003. ISSN 1389-1723. doi: https://doi.org/10.1016/S1389-1723(03)80041-5. URL https://www.sciencedirect.com/science/article/pii/S1389172303800415. A.M. Minihane and J.A. Lovegrove. 5 - health benefits of polyunsaturated fatty acids (pufas). In Christine Williams and Judith Buttriss, editors, Improving the Fat Content of Foods , Woodhead Publishing Series in Food Science, Technology and Nutrition, pages 107–140. Woodhead Publishing, 2006. ISBN 978-1-85573-965-9. doi: https://doi.org/10.1533/9781845691073.1.107. URL https://www.sciencedirect.com/science/article/pii/B9781855739659500054. Catarina Miranda, Sara Bettencourt, Tatiana Pozdniakova, Joana Pereira, Paula Sampaio, Ricardo FrancoDuarte, and Célia Pais. Modified high-throughput nile red fluorescence assay for the rapid screening of oleaginous yeasts using acetic acid as carbon source. BMC Microbiology , 20(1):60, Mar 2020. ISSN 1471-2180. doi: 10.1186/s12866-020-01742-6. URL https://doi.org/10.1186/ s12866-020-01742-6. Margarida Moreira dos Santos, Vijayendran Raghevendran, Peter Kötter, Lisbeth Olsson, and Jens Nielsen. Manipulation of malic enzyme in Saccharomyces cerevisiae for increasing nadph production capacity aerobically in different cellular compartments. Metabolic Engineering , 6(4):352– 363, 2004. ISSN 1096-7176. doi: https://doi.org/10.1016/j.ymben.2004.06.002. URL https: //www.sciencedirect.com/science/article/pii/S1096717604000540. Pablo I Nikel and Diethard Mattanovich. Microbial cell factories: a biotechnology journey across species. Essays Biochem. , 65(2):143–145, jul 2021. doi: https://doi.org/10.1042/EBC20210037. Filipa Pereira. Engineering of core Pentose Metabolism in Saccharomyces cerevisiae for Bio-ethanol Production . PhD thesis, University of Minho, 2013. Filipa Pereira, Flávio Azevedo, Nadia Skorupa Parachin, Bärbel Hahn-Hägerdal, Marie F. Gorwa-Grauslund, and Björn Johansson. Yeast pathway kit: A method for metabolic pathway assembly with automatically simulated executable documentation. ACS Synthetic Biology , 5(5):386–394, 2016. doi: 10.1021/ acssynbio.5b00250. URL https://doi.org/10.1021/acssynbio.5b00250. Francisco B. Pereira, Aloia Romaní, Héctor A. Ruiz, José A. Teixeira, and Lucília Domingues. Industrial robust yeast isolates with great potential for fermentation of lignocellulosic biomass. Bioresource Technology , 161:192–199, 2014. ISSN 0960-8524. doi: https://doi.org/10.1016/j.biortech.2014.03.043. URL https://www.sciencedirect.com/science/article/pii/S0960852414003484. 53 Humberto Pereira, Flávio Azevedo, Lucília Domingues, and Björn Johansson. Expression of Yarrowia lipolytica acetyl-coa carboxylase in saccharomyces cerevisiae and its effect on in-vivo accumulation of malonyl-coa. Computational and Structural Biotechnology Journal , 20:779–787, 2022. ISSN 20010370. doi: https://doi.org/10.1016/j.csbj.2022.01.020. URL https://www.sciencedirect. com/science/article/pii/S2001037022000277. Humberto Pereira, Paulo César Silva, and Björn Johansson. Bacteria and Yeast Colony PCR , pages 209– 221. Springer US, New York, NY, 2023. ISBN 978-1-0716-3358-8. doi: 10.1007/978-1-0716-3358-8_ 17. URL https://doi.org/10.1007/978-1-0716-3358-8_17. Tuan Anh Pham, Shigeyuki Kawai, and Kousaku Murata. Visualization of the synergistic effect of lithium acetate and single-stranded carrier dna on Saccharomyces cerevisiae transformation. Current Genetics , 57(4):233–239, Aug 2011. ISSN 1432-0983. doi: 10.1007/s00294-011-0341-7. URL https: //doi.org/10.1007/s00294-011-0341-7. Pirapan Polburee, Takao Ohashi, Yung-Yu Tsai, Thitinun Sumyai, Noppon Lertwattanasakul, Savitree Limtong, and Kazuhito Fujiyama. Molecular cloning and overexpression of DGA1, an acyl-CoA-dependent diacylglycerol acyltransferase, in the oleaginous yeast Rhodosporidiobolus fluvialis DMKU-RK253. Microbiology (Reading) , 164(1):1–10, nov 2017. doi: https://doi.org/10.1099/mic.0.000584. Tatiana A. Pozdniakova, João P. Cruz, Paulo César Silva, Flávio Azevedo, Pier Parpot, Maria Rosario Domingues, Magnus Carlquist, and Björn Johansson. Optimization of a hybrid bacterial/ Arabidopsis thaliana fatty acid synthase system ii in Saccharomyces cerevisiae . Metabolic Engineering Communications , 17:e00224, 2023. ISSN 2214-0301. doi: https://doi.org/10.1016/j.mec.2023.e00224. URL https://www.sciencedirect.com/science/article/pii/S221403012300007X. Jarmila Princová, Martin Schätz, Ondřej Ťupa, and Martin Převorovský. Analysis of lipid droplet content in fission and budding yeasts using automated image processing. JoVE , (149):e59889, Jul 2019. ISSN 1940-087X. doi: 10.3791/59889. URL https://app.jove.com/59889. Simone Pyle. 2.23 - human gut microbiota and the influence of probiotics, prebiotics, and micronutrients. In Maria Glibetic, editor, Comprehensive Gut Microbiota , pages 271–288. Elsevier, Oxford, 2022. ISBN 978-0-12-822036-8. doi: https://doi.org/10.1016/B978-0-12-819265-8.00076-0. URL https: //www.sciencedirect.com/science/article/pii/B9780128192658000760. Jinyi Qian, Yuzhou Wang, Zijian Hu, Tianqiong Shi, Yuetong Wang, Chao Ye, and He Huang. Bacillus sp. as a microbial cell factory: Advancements and future prospects. Biotechnology Advances , 69: 54 108278, 2023. ISSN 0734-9750. doi: https://doi.org/10.1016/j.biotechadv.2023.108278. URL https://www.sciencedirect.com/science/article/pii/S0734975023001854. Precedence Research. Fatty acids market size, share, and trends 2024 to 2034. https://www. precedenceresearch.com/fatty-acids-market, 2023. Accessed: 2024-10-08. Neale Ridgway and R.S. McLeod. Biochemistry of Lipids, Lipoproteins and Membranes: Sixth Edition . 01 2015. Weerawat Runguphan and Jay D. Keasling. Metabolic engineering of Saccharomyces cerevisiae for production of fatty acid-derived biofuels and chemicals. Metabolic Engineering , 21:103–113, 2014. ISSN 1096-7176. doi: https://doi.org/10.1016/j.ymben.2013.07.003. URL https://www. sciencedirect.com/science/article/pii/S1096717613000670. Ramesh Kumar Saini, Parchuri Prasad, Xiaomin Shang, and Young-Soo Keum. Advances in lipid extraction methods—a review. International Journal of Molecular Sciences , 22(24), 2021. ISSN 1422-0067. doi: 10.3390/ijms222413643. URL https://www.mdpi.com/1422-0067/22/24/13643. Michael Scharnewski, Paweena Pongdontri, Gabriel Mora, Michael Hoppert, and Martin Fulda. Mutants of Saccharomyces cerevisiae deficient in acyl-coa synthetases secrete fatty acids due to interrupted fatty acid recycling. FEBS J. , 275(11):2765–2778, jun 2008. doi: https://doi.org/10.1111/j.1742-4658. 2008.06417.x. Shuobo Shi, Yun Chen, Verena Siewers, and Jens Nielsen. Improving production of malonyl coenzyme aderived metabolites by abolishing snf1-dependent regulation of acc1. mBio , 5(3):10.1128/mbio.01130– 14, 2014a. doi: 10.1128/mbio.01130-14. URL https://journals.asm.org/doi/abs/10. 1128/mbio.01130-14. Shuobo Shi, Juan Octavio Valle-Rodríguez, Verena Siewers, and Jens Nielsen. Engineering of chromosomal wax ester synthase integrated Saccharomyces cerevisiae mutants for improved biosynthesis of fatty acid ethyl esters. Biotechnol Bioeng , 111(9):1740–1747, apr 2014b. doi: https://doi.org/10.1002/bit. 25234. Ga-Hee Shin, Markus Veen, Ulf Stahl, and Christine Lang. Overexpression of genes of the fatty acid biosynthetic pathway leads to accumulation of sterols in Saccharomyces cerevisiae . Yeast , 29(9):371– 383, sep 2012. doi: https://doi.org/10.1002/yea.2916. 55 Oksana Tehlivets, Kim Scheuringer, and Sepp D. Kohlwein. Fatty acid synthesis and elongation in yeast. Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids , 1771(3):255– 270, 2007. ISSN 1388-1981. doi: https://doi.org/10.1016/j.bbalip.2006.07.004. URL https: //www.sciencedirect.com/science/article/pii/S1388198106001995. Regulation of Lipid Metabolism in Yeast. Dritan Topi. Transforming waste vegetable oils to biodiesel, establishing of a waste oil management system in albania. SN Applied Sciences , 2(4):513, Mar 2020. ISSN 2523-3971. doi: 10.1007/ s42452-020-2268-4. URL https://doi.org/10.1007/s42452-020-2268-4. Bernard Turcotte, Xiao Bei Liang, François Robert, and Nitnipa Soontorngun. Transcriptional regulation of nonfermentable carbon utilization in budding yeast. FEMS Yeast Res , 10(1):2–13, jul 2009. S. D. Weger, A. Ganji, K. V. Clemons, J. K. Byron, Y. Minn, and D. A. Stevens. Correlation of the frequency of petite formation by isolates of Saccharomyces cerevisiae with virulence. Medical Mycology , 40(2): 161–168, 01 2002. ISSN 1369-3786. doi: 10.1080/mmy.40.2.161.168. URL https://doi.org/ 10.1080/mmy.40.2.161.168. P Wenz, S Schwank, U Hoja, and H J Schüller. A downstream regulatory element located within the coding sequence mediates autoregulated expression of the yeast fatty acid synthase gene FAS2 by the textitFAS1 gene product. Nucleic Acids Res. , 29(22):4625–4632, nov 2001. doi: https://doi.org/10. 1093/nar/29.22.4625. Stephen W White, Jie Zheng, Yong-Mei Zhang, and Rock. The structural biology of type ii fatty acid biosynthesis. Annu. Rev. Biochem. , 74(1):791–831, 2005. doi: https://doi.org/10.1146/annurev.biochem. 74.082803.133524. Shuo Yang, Liyun Song, Jing Wang, Jianzhi Zhao, Hongting Tang, and Xiaoming Bao. Engineering Saccharomyces cerevisiae for efficient production of recombinant proteins. Engineering Microbiology , 4 (1):100122, 2024. ISSN 2667-3703. doi: https://doi.org/10.1016/j.engmic.2023.100122. URL https://www.sciencedirect.com/science/article/pii/S2667370323000541. Yongjin J. Zhou, Nicolaas A. Buijs, Verena Siewers, and Jens Nielsen. Fatty acid-derived biofuels and chemicals production in Saccharomyces cerevisiae . Frontiers in Bioengineering and Biotechnology , 2, 2014. ISSN 2296-4185. doi: 10.3389/fbioe.2014.00032. URL https://www.frontiersin.org/journals/bioengineering-and-biotechnology/ articles/10.3389/fbioe.2014.00032. 56 Part II Appendices 57 Appendix F Additional experiments regarding phenotype tests The figure bellow corresponds to the further testing on YPGES with 0.5% of agarose for strains IOA5 to IOA8. These strains showed no growth on this media. Figure 33: Phenotype testing in YPGES with and without myristic acid for strains IOA5 to IOA8. Positions 1 and 2: ∆fas1,2 and ∆fas2; position 3: IOA1; position 4: IOA5; position 5: IOA2; position 6: IOA6; position 7: IOA3; position 8: IOA7; position 9: IOA4; position 10: IOA8; position 11: CEN.PK113-7D; position 12: CEN.PK2-1C. For this test, about 1mm of colony was scraped from master plates of the selected clones and dissolved in 100 µL of sterile water; drops of 4 µL of this cell solution were laid on the respective spots in the plates. Cells were incubated for four days at 37 ºC. 64 Appendix G Adaptation of strains IOA5 and IOA6 in YPFD without myristic acid Table 9 shows the optical density measured during the adaptation period on YPFD for strains IOA5 and IOA6. Table 9: Optical density of strains IOA5 and IOA6. Adaptation for growth on YPFD without exogenous fatty acids occurred for a period of 144h in 500mL Erlenmeyer flasks, at 30°C and 200 rpm OD640nm Time (h) IOA5 IOA6 0 0.185 0.205 72 0.780 0.580 144 10.4 0.560 As observed in the Table above, the optical density for strain IOA5 is significantly higher than IOA6, which is not consistent with the fact that these strains both exhibited much longer periods of growth. This was because, after observation under the microscope, it was detected bacterial contamination on the culture of strain IOA5. 65 Appendix H Calculation of the specific growth rate of strain IOA6 The figure bellow consists of the graph plotted with the three time points that exhibited an approximate linear relationship, to determine the specific growth rate in the exponential phase. Figure 34: Plotting of three time points relative to the exponential growth phase of the strains in study, by measurement of cell density throughout incubation time. With the use of Microsoft Excel, the approximate linear relation was calculated for the time points selected, to obtain the specific growth rate of the strains as well as the coefficient of determination (R2), which measures how well a linear regression model fits the data. 66 Appendix I CellProfiler outputs The figures below correspond to the data obtained from the pipeline of the program used to quantify the area of lipid droplets and cells. Figure 35: IOA6 fluorescence microscopy pictures, analyzed with CellProfiler and respective program outputs for labelling and quantification of lipid droplets per cells. There’s the conversion of the coloured picture to gray, and the final outlining of cells and lipid droplets (in purple and yellow, respectively). It is also observable the identification of primary objects according to the definition of the minimum and maximum diameter of cells and lipid droplets and other thresholding parameters specified in Section 2.8.2 of the Materials and Methods Chapter. 67 Figure 36: CEN.PK2-1C+pTA1 control strain fluorescence microscopy pictures, analyzed with CellProfiler and respective program outputs for labelling and quantification of lipid droplets per cell. There’s the conversion of the coloured picture to gray, and the final outlining of cells and lipid droplets (in purple and yellow, respectively). It is also observable the identification of primary objects according to the definition of the minimum and maximum diameter of cells and lipid droplets and other thresholding parameters specified in Section 2.8.2 of the Materials and Methods Chapter. 68 Appendix J Supplementary flow cytometry data The pictures bellow correspond to the forward scatter versus side scatter dot plots after the gating process for selection of the populations to be analyzed by their fluorescence signal. Two biological replicates were analyzed for each IOA6 and CEN.PK2-1C+pTA1. Figure 37: Gate definition process to remove debris included in the flow cytometry analysis, for replica 1. This was achieved with the “Rectangle” tool from Floreada.io that allows for the selection of the desired events. A rectangle excluding the lowest forward scatter values (FSC-H) was drawn on the density plots, around the 1M units from the raw files to exclude unwanted data. 69 Figure 38: Gate definition process to remove debris included in the flow cytometry analysis, for replica 2. This was achieved with the “Rectangle” tool from Floreada.io that allows for the selection of the desired events. A rectangle excluding the lowest forward scatter values (FSC-H) was drawn on the density plots, around the 1M units from the raw files to exclude unwanted data. 70 Appendix K Data relative to the construction of the pTA9_TDH3_DGA1_TEF1 plasmid The figure bellow shows the DNA fragments incorporated into the transformation mix for the construction of the pTA9_TDH3_DGA1_TEF1 plasmid in CEN.PK113-5D. Figure 39: PCR products needed for the pTA9_TDH3_DGA1_TEF1 assembly in CEN.PK113-5D. (A) Amplification of DGA1 from chromosomal DNA from yeast strain CEN.PK2-1C 1:10 diluted. The enzyme Supreme was used for its proofreading ability since it was necessary to make sure the DGA1 gene did not contain any unwanted nucleotides added by Taq Polymerase. (B) PCR products from the promotors TEF1 and TDH3 to flank the DGA1 gene; pTA9 and pYPKa. 71