Team:Chalmers-Gothenburg/Description


Project Description
The Big Picture of Our Project

SHORT PROJECT SUMMARY

We designed...

  • Fatty acid-based compounds (oleochemicals) are important building blocks in the manufacturing of a wide range of industrial products

  • The physicochemical properties of oleochemicals are heavily dependent on the fatty acid chain length.

  • In our system design, the native fatty acid synthase of Saccharomyces cerevisiae is functionally replaced with a bacterial fatty acids synthase pathway from Escherichia coli.

  • This facilitates the tunable regulation of the length of the fatty acid acyl chains by tuning the expression of three thioesterases (the enzymes responsible for terminating the growth of the chain).

  • Tunable gene expression is accomplished using a combination of three transcription factor-based chemical induction systems based on copper ions, tetracycline, and estradiol.

Our progress and results...

  • We successfully integrated 6 out of 8 genes of required genes (but unfortunately did not succeed in integrating the remaining 2), preventing us from testing the fully functional system.

  • All three transcription factor-based induction systems were benchmarked individually, and in combination, using fluorescent proteins. Results indicating that the system is tunable as intended.

  • All parts we designed have been uploaded to the iGEM Parts Registry.


BACKGROUND

  Why fatty acids?


Outlining some of the important derivatizations based on fatty acids as starting molecules. Fatty acid derivates are a heterogeneous group of compounds (with numerous possible modifications), but all share the basic structure of the original fatty acids alkyl carbon chain.
Fatty acids (FA) and their derived compounds (also referred to as oleochemicals) are important building blocks in the manufacturing of a wide range of industrial products including plastics, fuels, paints, detergents, lubricants, personal care-, and pharmaceutical compounds. Their usefulness as base chemicals stems from their versatile chemistry that allows a myriad of derivatizations (Desroches et al., 2012; Dyer et al., 2008; Maisonneuve et al., 2013). The physiochemical properties of oleochemicals are heavily dependent on chain-length and can be influenced by incorporation of branches and other chemical modifications. Control of chain length would allow us to obtain a number of high-value products (Carlsson, 2009; Nosal et al., 2021; Ramos et al., 2009). In the section below we summarize several key oleochemicals and highlight their application areas.
Figure: FAs consist of a polar carboxylic acid with a non-polar aliphatic chain. FAs can be saturated, with non-reactive aliphatic chains (such as stearic- or palmitic acids) or unsaturated, with aliphatic chains bearing double bonds (such as oleic-, linoleic-, linolenic-, and ricinoleic acid, etc.). Particularly unsaturated FAs are of interest with the double bonds being an excellent starting point for modification to functionalize the aliphatic chain.
FAs are good candidates for polymers and can be deoxygenated to produce linear α-olefins (LAO) (Liu et al., 2014). LAOs and other types of olefins are very important platform chemicals and intermediates in many industrial processes. While much research has been dedicated to finding renewable sources of LAOs, they are currently almost exclusively obtained from petroleum resources by oligomerization of ethylene. Shorter chain LAOs (C4-C8) are used in the production of polyethylene (the most common plastic in use today) and longer chain LAOs (C10-C14) are mainly used in the production of surfactants, plasticizers, and lubricants (Chatterjee et al., 2018; Nosal et al., 2021; van der Klis et al., 2012). The annual world consumption of LAOs has been forecast to increase at an average annual rate of 4.0% until 2024 (IHS Markit, 2009).
Figure: Chemical structure of 1-hexene, a typical linear α-olefin. The carbons are numbered in blue, and Greek letters in red are used to indicate the positioning of the double bond.
FAs can be decarboxylated/decarbonylated into straight-chain alkanes, which are structurally and chemically identical to petroleum-derived compounds used in combustible fuels, solvents, and lubricants. Typically, these products are obtained through hydrotreating, a method which has the disadvantages of toxic metal sulfide catalysts and energy intensive high pressure hydrogen (Mäki-Arvela et al., 2007; Santillan‐Jimenez & Crocker, 2012). The most common forms of biodiesels are FA methyl esters (FAME) and FA ethyl esters (FAEE), which have characteristics equivalent to petroleum-derived diesel oil. A major portion of all biodiesels are produced by transesterification of FAs extracted from edible plant oils (Banković-Ilić et al., 2012; Gui et al., 2008; Leung et al., 2010). Currently, fossil fuels supply 84 % of the worlds energy (BP p.l.c, 2021) and the transport sector is the second biggest source of global greenhouse gas emissions, representing around 60 % of global oil production. Consumption of fossil fuels in transportation is predicted to rise to 104 million barrels a day by 2030 (Florini, 2011; Jollands, 2008). While biofuels have been identified as an available and practical solution for decreasing the worlds dependence on petroleum-based fuels, they currently require significant downstream processing to be compatible with modern internal combustion engines (Howard et al., 2013). The increasing large-scale conversion potential of food resources into fuels is further expected to bring global imbalance to the food supply and demand, blurring the line between food and fuel economies as both fields are competing for the same resources (Meyers & Kalaitzandonakes, 2015; Shukla et al., 2019).
The world demand for lubricants and hydraulic fluids is in the reach of several million tonnes and most industrial lubricants on the global market are formulated using petroleum-based feedstocks, such as paraffinic-, naphthenic-, and aromatic oils (also referred to as mineral oils), with about 10-15 % being based on plant oils A major disadvantage of mineral oils are their toxicity and potential for long-term pollution. With studies indicating that a significant portion of lubricants end up in the soil, air, and waterways there is a rapidly developing market for biolubricant technologies less harmful to the environment (Bremmer & Plonsker, 2008; Zaccheria et al., 2016). Lubricants can be directly derived from FAs reacted with alcohols to produce esters, using similar transesterification processes as those used in the production of biodiesels. Several FA esters have been identified as compounds possessing suitable technical and ecological properties (being more easily biodegradable and possessing low aquatic toxicity) for applications as biolubricants, and can potentially replace petroleum-based lubricants in many applications (Akerman et al., 2011; Willing, 2001). Although the use of biolubricants is currently limited when compared to those of mineral oils, the physicochemical properties of FA esters have been shown to cover the technical requirements for the development of high-performance industrial oils and lubricants. Having the potential to be used in industrial applications, such as automotive engines, hydraulic- and metal working fluids, and drilling- and gear oils (Cecilia et al., 2020; Schneider, 2006).
Due to their chemical composition of both polar- and non-polar functional groups, FAs are used to synthesize surfactants - an important class of compounds used in the manufacture of everyday household consumer goods including detergents, personal care products, pharmaceuticals, paints and varnishes (Knepper & Berna, 2003; Smith, 2019). Major industrially produced surfactants include sodium lauryl ether sulfate (SLES), ammonium lauryl sulfate (ALS), and sodium lauryl sulfate (SLS) which are mainly obtained by hydrogenolysis of triglycerides into FAs derived from palm- or coconut oil and subsequent sulfonation (in combination with ethoxylation depending on the product). SLES, ALS and SLS are ubiquitous in products such as makeup, soaps, shampoos, and toothpaste. The surfactants market is projected to reach USD 52.4 billion by 2025 from USD 42.1 billion in 2020 (MarketsandMarkets, 2020) and the supply chains and industrial manufacturing of surfactants are complex, as various inputs (both plant- and petrochemical based) are used for their production. As the use of surfactants are on a similarly large scale as lubricants, there is an increasing concern about the effect of pollution on human health and various ecosystems, especially their toxicity for water living organisms (Olkowska et al., 2011; Ostroumov, 2005). Microorganisms synthesize a wide range of compounds with similar properties as synthetically manufactured surfactants that exhibit more environmentally friendly characteristics. These biosurfactants are investigated as replacement for current technologies, as well as in novel applications (Banat et al., 2010; Marchant & Banat, 2012). While characterized as a structurally diverse group of compounds, with various biosynthetic pathways depending on the producing organism, many are influenced by the cell's overall FA synthesis and the concentration of FAs in the growth medium. The properties of biosurfactants are affected by the chain length of the FAs made available to the cell (Bednarski et al., 2004; Felse et al., 2007; Hollenbach et al., 2020).
Figure: General chemical structure of SLES and mannosylerythritol lipids (a form of biosurfactants produced by Candida antarctica). For SLES, ‘n’ indicates a varying number of ethoxyl groups, where three is the major number in many commercial products.
A major portion of all new drug candidates are lipophilic and incorporate oleochemicals as part of their core structure. It has been found that both small molecule- and protein-based drugs benefit from being conjugated with oleochemicals such steroids and phospholipids, leading to improved pharmacokinetics and bioavailability (Cochrane & Vederas, 2016; Lipinski et al., 1997; Markovic et al., 2020; Van De Waterbeemd et al., 2001). Biosurfactants find use in pharmaceutical and cosmetic preparations, where especially glycolipids and sophorolipids have been of special interest in applications such as drug delivery, wound healing, and as antimicrobial and antibiofilm agents (Ceresa et al., 2021). The design of novel oleochemicals with interesting properties could potentially enhance the development of new drug delivery systems, as seen in the use of lipid nanoparticle delivery systems (LNP) for vaccines during the COVID-19 epidemic. LNPs are composed of blends of FA and other oleochemicals that encapsulate bioactive or pharmaceutical compounds, and are noteworthy used to coat the mRNA strands used in the vaccines developed by Moderna and Pfizer-BioNTech (Pardi et al., 2018; Yew & Misran, 2019).
While humans are unable to synthesize essential FAs, such as linoleic acid and α-linolenic acid, deficiencies are rare due to the availability in a range of foods. However, derivatives of essential FAs have significant clinical applications such as in as dietary supplements. Derivatives of essential FAs are important dietary nutrients and have significant clinical applications. Especially polyunsaturated FAs (PUFA) such as eicosapentaenoic acid and docosahexaenoic acid, are important for the normal development and function of our bodies and needed to maintaining a good health (Dyer et al., 2008; Willis & Marangoni, 1999). Dietary supplementation of PUFAs significantly alleviates the symptoms of many chronic disease but their natural sources are limited, with a major source being fish oils (Das, 2006; Uemura, 2012). Due to the increased consumption and the continued pollution of marine habitats, fish hauls are decreasing (Free et al., 2019), making it highly desirable to find alternative sources that are both economically viable and sustainable.
Figure: Structure of two FAs known to be essential for humans, α-linolenic acid (an omega-3 FA) and linoleic acid (an omega-6 FA). The term omega (ω) is determined by the double bond which is closest to the methyl end of the molecule. For example, the alkyl chain of α-linolenic acid contains of 18 carbons (with three double bonds at carbon number 9, 12, and 15). The omega end of the chain is at carbon 18, and the double bond closest to the omega carbon begins at carbon 15 (18-15=3).

  Environmental impact and the increasing demand for oleochemicals


In the context of an uncertain supply and the environmental impact of using petroleum resources there is an urgent need to find alternative manufacturing processes for oleochemicals. While renewable plant oils have largely replaced petroleum a source for many types of oleochemicals, these are often major drivers of deforestation and biodiversity loss and compete with traditional food-crops for arable land (Carlsson, 2009; Gibbs et al., 2010; Shukla et al., 2019). The majority of plant oils are produced from just four crops: oil palm, soybeans, rapeseed and sunflower, which together account for approximately 79% of the total world production (Ritchie & Roser, 2021). Palm oil (mainly produced in Indonesia and Malaysia) accounts for 30 % of the world total (and is expected to grow by about 4% annually) and is a controversial feedstock material due to high levels of deforestation, and allegations of human rights violations among growers (Gilbert, 2012; Rival & Levang, 2014; Saxon & Roquemore, 2011). Soybean oil is the second-most-widely produced oil in the world (made and used extensively in the United States, Brazil, Argentina, and China). Its rapid market growth has been supported by its use as a feedstock in biodiesel production (Cavalett & Ortega, 2010). The total greenhouse gas emissions from Brazilian soy exports between 2010-2015 was estimated at 223.46 Mt and directly linked to the conversion of natural vegetation into arable land (Escobar et al., 2020; Zalles et al., 2019). The global FAs market is forecast to reach 48.46 billion USD by 2027 and the increasing demand for plant oils urgently promotes the need to find manufacturing methods that are both renewable and sustainable (Reports and Data, 2020). This would significant further the efforts towards a sustainable bio-based economy.

  Challenges using plant oil as a source of oleochemicals


Plant oils consist almost entirely of triglycerides (TAG) esters, containing three FAs with varying chain lengths depending on the source (with 16 and 18 carbons being the most common). The value and application of an oil are determined largely by its FA composition. Due to their natural composition and limitations of current processing methods, the number of FA types that can be separated from plant oil is limited. The specific plant source can greatly affect the properties of the final product, due to it containing a mix of different chain lengths. Most vegetable oils contain just five basic FA structures; palmitic-, stearic-, oleic-, linoleic- and linolenic acid (Dyer et al., 2008). Mixed with these are minor variants with chain lengths ranging between 8 and 24 carbons, containing varying numbers of double bonds, conjugated systems and other functional groups (Carlsson, 2009). Economically viable grades of FAs in many applications are composed of a ranged mixture of different chain lengths, resulting in a problem for their use in more specific applications. In many areas of industry and commercial products, broad cuts of FAs mixtures are employed, rather than chemically pure individual FAs. Developing an effective, environmentally responsible, and economically viable extraction procedure for FAs with specific chain lengths is critical to ensure the success of subsequent derivatization and processing steps, and the sustainability of the entire production procedure.
FA composition based on chain length
Table showing major fatty acids content in both non-edible and edible oils. The number before the colon designates the total number of carbons in the fatty acid chain, the number after the colon represents the number of double bonds. Modified from Dyer et al., 2008.
Crop 14:0 16:0 18:0 18:1 18:2 18:3 20:1
Palm oil (Elaeis oleifera) 5 36 2 50 8
Soybean oil (Glycine max) 11 4 23 54 8
Canola oil (Brassica napus, Brassica rapa, or Brassica juncea) 4 2 60 21 10 1
Sunflower oil (Helianthus annuus) 7 5 19 68
Linseed oil (Linum usitatissimum) 6 2 19 24 47
Conventional manufacturing processes of oleochemicals from plant oils tend to be energy inefficient (requiring high temperatures and pressures), involve high acidity or alkalinity, and utilize a variety of hazardous solvent systems (including hexane, ethanol, methanol, chloroform, and butanol). Further, derivatization of FAs tends to rely on heavy metal catalysts. The current preferred technology for FA production is continuous high-pressure hydrolysis of fats and oils with superheated steam and has been in continuous use since the early 20th century. The process is usually carried out at around 250 °C at 3000–5000 kN/m2 pressure with sulfide or noble metal catalyst. This is a costly process due to the high energy input requirements in combination with the corrosive nature of the acids produced at these temperatures and pressures (Anneken et al., 2000; Guczi & Erdôhelyi, 2012). Methods using lipase enzymes as catalysts at mild conditions became available in the 1980s and are well suited for small-scale production of FAs, but are challenging for large-scale production due to problems of catalyst recovery and the low rate of attaining equilibrium in the reaction vessels (Daiha et al., 2015; Gandhi, 1997; Kubička, 2008). Another challenge in the production of FAs (which plant oils happen to share with petroleum) is the wide range of products made by the processes involved, with as many as 8-10 products being made at the same time with most of them sold into different markets, with different dynamics. While possibly supplying added value, this makes it difficult to balance the supply and the demand for all, or even most of the products by the frequently changing market demands. Given the tremendous industrial importance of oleochemicals, there is a need to decrease the world’s reliance on oleochemicals sourced from plant oil by finding alternative manufacturing methods for these types of compounds.

  The future of oleochemicals from cell factories


The development of cell factories for the sustainable manufacturing of oleochemical is an enticing prospect. Using cell factories, it may be possible to produce a narrower range of FAs and oleochemicals from unspecific biomass as the feedstock, rather than unsustainable plant- or petroleum materials. Cell factories have the potential to address many of the challenges associated with plant-based supply chains. Cultivation of many microorganisms occurs over days and in closed environmental systems such as bioreactors, whereas plant production is often annually and regionally limited, and potentially allows more localized production, limiting the need for extensive transportation to maintain supply chains. In comparison with oleochemicals based on plant oils, cell factories allow greater consistency in product composition and purity profiles. Where plant-based production results in a mix of different acyl chain lengths (Carlsson, 2009), cell factories can be made compatible with renewable plant-based materials that can be grown on land not suitable for the cultivation of food crops. As previously noted, the use of edible oil in biochemical production has an influence on the global imbalance to the market demand and the food supply by their high prices, the reduction of food sources and the growth of commercial plant capacities. Thus, focus should be shifted to non-edible resources, which are not used in the human nutrition and could be grown in barren lands (Bozell & Petersen, 2010).

While the low abundance or yield of oleochemicals in wild microorganisms have rendered their isolation from such sources in many cases non-economically viable on an industrial scale, significant efforts have been put into finding genetic- and metabolic engineering strategies to increase production yields (Cravens et al., 2019; Ko et al., 2020; Nielsen & Keasling, 2016; T. Yu et al., 2018). The challenge is not only to design cell factories that can produce high yields, rates, and product titers of oleochemicals, but also to shorten the development time of each metabolic- and biosynthesis engineering design cycle, allowing cell factories to compete effectively with current petroleum-, and plant-based manufacturing processes as new markets develop.

The metabolism of natural producers with specific FA compositions are usually poorly understood, and they often lack immediate available tools for genetic modification available for model organism such as Saccharomyces cerevisiae and Escherichia coli. This makes them more difficult targets for engineering. Consequently, many new technologies depend on these well-known organisms by modifying existing biosynthesis pathways or introducing new ones (Calero & Nikel, 2019; Navarrete et al., 2020), showing great success in producing many highly-valuable target compounds such as complex opioids and vitamins (Galanie et al., 2015; Sun et al., 2019). Even as methods to produce and extract oleochemicals from microorganisms have been extensively explored (Marella et al., 2018; A.-Q. Yu et al., 2014) much of the current research has been focused on metabolic engineering to yield high-producing strains of one, or a few, number of structurally simple, low-molecular weight products such as such as aromatics, amines, terpenoids, terpenes, and esters (Calero & Nikel, 2019; Navarrete et al., 2020; Nielsen & Keasling, 2016). Although some natural products can be chemically synthesized - for example tunable synthesis of oleochemicals has been attempted using metal catalysts (Gollwitzer et al., 2017) - the complex structures of many of these compounds makes chemical synthesis either difficult or commercially infeasible (De Luca et al., 2012). The nature of biosynthesis in cell factories provide a promising route to generate a vast library of oleochemicals, even those difficult to extract, or not found in nature (Biermann et al., 2011; Nikolau et al., 2008).


OUR PROJECT

Inspired by previous research from Fernandez‐Moya and colleagues in 2015 and the work conducted in collaboration with the Siewers Lab concerning yeast metabolism (Marella et al., 2018; Zhou et al., 2014, 2016), we decided to design a system that would allow us to regulate the chain lengths of FAs produced by S. cerevisiae (a large group that includes both laboratory and industrial strains). The decision to use S. cerevisiae as a chassis instead of other well studied model organisms such as E. coli was based on its proven record as a reliable platform for industrial large-scale production of various compounds due to its robustness, genetic malleability, and tolerance towards harsh fermentation conditions (Parapouli et al., 2020), and some of the highest production titres of FAs in microbial fermentation have been reported for this organism (Fernandez-Moya & Da Silva, 2017; T. Yu et al., 2018). Being a model organism, substantial knowledge has been accumulated concerning the metabolism, genetics, and physiology of S. cerevisiae, with several well-established tools for genome engineering and fermentation technologies (Besada-Lombana et al., 2018; Lian, HamediRad, et al., 2018; Lian, Mishra, et al., 2018; Nevoigt, 2008). Working in a yeast lab with d irect access to knowledgeable experts who had worked with S. cerevisiae also played a part in our decision. To further explore the potential of this kind of system, we wanted to make it programmable by using a combination of different signaling agents.

  Replacement of the native FA acid synthase system allows the chain lengths to be regulated


Fernandez‐Moya and colleagues in 2015 showed that the functional replacement of the S. cerevisiae FA synthase (FAS) with a bacterial type II system (eFAS) from E. coli allows the FA product profiles to be controlled by regulating the expression of different thioesterase (TE) enzymes.

S. cerevisiae produces cytosolic FAs de novo using a large barrel-shaped protein complex (2.6 MDa), consisting of two main subunits (α- and β) expressed by separate genes (FAS1 and FAS2, respectively). The process is iterative, and the reactions are catalyzed using acetyl coenzyme A as the initial active donor of two carbon atoms and extended two carbons at a time by the addition of an acetyl group from malonyl coenzyme A. Throughout this process the growing FA remains tethered to an acyl carrier protein (ACP) (Sztain et al., 2019). When the final length is achieved, FA synthesis is terminated by the TEs hydrolyzing a thioester bond, releasing a fully formed FA. The complexity and rigidity of native S. cerevisiae FAS makes it a challenging enzyme to target for protein engineering efforts. In comparison, the bacterial type II FAS consists of eight dissociated and mono-functional enzymes expressed from separate genes that can be manipulated individually and have been extensively reviewed (Chan & Vogel, 2010; Dodge et al., 2019; White et al., 2005).

The sequence for all eFAS genes (including the tesA) used in our project was retrieved from the NCBI database and originate from E. coli strain K-12 MG1655 (NCBI:txid511145). All genes were sequence optimized for yeast using Integrated DNA Technologies online codon optimization tool.
Listing the nine genes, proteins and reactions required to produce FA in S. cerevisiae using the eFAS system from E. coli. Retrieved from Fernandez‐Moya et al., 2015 and White et al., 2005.

*Two enzymes FabB and FabF catalyze the condensation of malonyl-CoA with the fatty acyl-ACP growing chain. Fernandez‐Moya et al., 2015 expressed only FabB since expression of FabF in active form had proven to be challenging.
**There are two genes in E. coli that encode β-hydroxyacyl-ACP dehydrases (fabA and fabZ). Fernandez‐Moya et al., 2015 used fabZ due to its broad substrate specificity.
Gene Protein Substrate(s) Product(s)
acpP Acyl Carrier Protein Apo-ACP + 4'-Phosphopantetheine Group ACPSH
acpS ACP Synthase AcpS activates apo-ACP (encoded by acpP), transferring to it the 4’- phosphopantetheine from coenzyme A, resulting in holo-ACP
fabB* β-Ketoacyl-ACP Synthase I (1) (n)Acyl-ACP + Malonyl-ACP (n+2)β-Ketoacyl-ACP
(2) Acetoacetyl-ACP + Malonyl-ACP β-Ketobutyryl-ACP + CO2 + ACPSH
fabG β-Ketoacyl-ACP Reductase β-Ketoacyl-ACP + NADPH + H+ β-Hydroxyacyl-ACP + NADP+
fabZ** β-Hydroxyacyl dehydratase β-Hydroxyacyl-ACP Anoyl-ACP + H2O
fabI Enoyl-ACP Reductase Anoyl-ACP + NADH + H+ Acyl-ACP + NAD+
fabD Malonyl-CoA:ACP Transacylase Transfers a malonyl group from coenzyme A to ACP, providing the primary carbon source for the formation of the fatty acid chain.
fabH β-Ketoacyl-ACP Synthase III Start to initiate cycles of elongation by condensing acetyl-CoA with malonyl-ACP to form acetoacetyl-ACP.
tesA Thioesterase I Acyl-ACP Free fatty acid + ACPSH
Showing a comparison between (A) FAS (B) eFAS FA acid synthesis. In FAS, the catalytic domains are located on the interconnected α-, and β-chains. In eFAS, the similarly performing enzymes are distinct and monofunctional (marked in blue). In both systems, FA synthesis in initiated by acetyl coenzyme A, the starting point for a new chain capped by a coenzyme A cofactor. Malonyl coenzyme A is then iteratively fed into the reaction cycle to continue building the chain by two extra carbon atoms at a time. The process stops when the terminal coenzyme A unit is cleaved off by a TE. Most TEs target certain ranges of chain lengths. The overall FA profile that is produced by the cell is then dependent on which TEs are expressed. Image and text modified from Zhou et al., 2014 distributed under the terms of the Creative Commons Attribution License (CC BY).
Figure: showing the structure of the FAS complex (PDB 6TA1) and nine enzymes involved in the eFAS (PDB 1T8K, 5VBX, 1FJ4, 2G2Y, 1I01, 1HNK, 5CFZ, 6N3P, and 5TIC). Visualising complexes of ACP with its enzymatic partners has been a challenging research effort as many complexes are short-lived. Dodge et al., 2019 overcame this problem by employing cross-linking probes in combination with protein fusion methods to covalently trap E. coli FabZ in functional association with ACP to solve the crystal structure of the resulting complex (ACP=FabZ).

  Expression of different thioesterases is key for the formation of FA with specific chain lengths


TEs (or thiolester hydrolases) (E.C.3.1.2) are enzymes which belong to the esterase family. Enzymes in this group exhibit esterase activity (splitting of an ester into acid and alcohol, in the presence of water) specifically at a thiol group. TEs tend to have strong sequence similarity, tertiary structures, and they share general mechanisms, as well as catalytic residues. TEs tend to be promiscuous and have been shown to display specificity for a broad range of different chain lengths, but some are more specific than others (Cantu et al., 2010; Jing et al., 2011).

When designing our system, we decided to use TesA (E. coli), as a part of the native eFAS system, and two heterologous thioesterases (FatB and TesBT), with substrate specificity for different target chain lengths. TesA is located in the periplasm of E. coli and has been shown to exhibits substrate preference for C14 (Steen et al., 2010), although other studies have reported a preference for C16 and C18 (Cho & Cronan Jr, 1993). FatB (Ricinus communis) overexpressed in E. coli was shown to lead to the accumulation of C14 and C16 at levels about 40 % and 55 %, respectively (Zhang et al., 2011). TesBT (Bacteroides thetaiotaomicron VPI-5482) expressed in E. coli has been shown to have a preference for C6 and C8, and significantly increased the yield of butyric acid (C4) (Jawed et al., 2016). Unfortunately, we managed to find very little data on these proteins when expressed in yeast. The overlap in substrate specificity between TesA and TesBT reported in the literature was deemed to not pose a problem for our design, as a proof-of-concept needed to show a tuneable shift between short-, medium-, and long FA production profiles in the final strain, not a preference for a very specific number of carbons. We expect more narrow ranges will be accomplished by future protein engineering efforts (Zhu et al., 2020).

The amino acid sequences for FatB and TesBT was retrieved from the NCBI database (ref. NM_001323748.1 and AAO77182.1, respectively) and converted to a DNA sequence using the Sequence Manipulation Suite. We optimized all gene sequences for S. cerevisiae using the IDT Codon Optimization tool.

  A combination of different chemical induction systems facilitates tunable FAs profiles


Regulation of gene expression is crucial in synthetic biology research and applications and can be accomplished by means of inducible promotor systems. Most induction agents are transcription-factor based, meaning they bind to proteins that either directly target a particular promoter, or an activation and/or repression domain that regulate the transcription. Inducible gene expression systems are sometimes favored over systems that facilitate a stable expression due to being reversible, and thus more flexible to use. Promoters have been identified that respond to many different types of stimuli, including small-molecules, metal ions, light, and temperature. Chemical approaches to control gene expression have extensively been explored in literature both concerning synthetic biology (Ford & Silver, 2015; Hanczyc, 2020) and medical science, as the ability to switch genes ‘on’ and ‘off’ is of great importance for the developing field of gene therapy (Goverdhana et al., 2005; Kallunki et al., 2019).

In our system, tunable gene expression is accomplished using a combination three chemical induction systems based on copper ions (Etcheverry, 1990; Labbé & Thiele, 1999), tetracycline (Gossen et al., 1995; Gossen & Bujard, 1992), and estradiol (Kumar et al., 1986; Pratt, 1990). Each system is carried on a separate plasmid and linked to the expression of the TEs by placing the genes downstream of the regulatory elements and promotors belonging to each system. Apart from the CUP1 promotor, the tetracycline- and estradiol systems utilized the Scpho5- and GAL1 promotors, respectively. Scpho5 and GAL1 are naturally phosphate- and galactose-sensitive promoters that have been frequently used and engineered to control gene expression in S. cerevisiae (Da Silva & Srikrishnan, 2012).

To find optimal concentration ranges, we benchmarked each system individually, in pairs, and as combination of all three systems, by replacing the TE genes with different fluorescent proteins (GFP, RFP and BFP, respectively).

Many synthetic induction systems are hybrid constructs, utilizing parts from both eukaryotic- and prokaryotic organisms, and often require the combined action of many different proteins to work. When this has been the case, as with the tetracycline- and the estradiol-based systems, these proteins were carried on the same plasmids as the TEs and constitutively expressed.

  The CUP1, TET and Estradiole induction systems


The copper-based induction system (CUP1) is native to yeast where it responds to copper ions by producing metallothioneins and help to protect the cell from toxic levels. Yeast naturally expresses Ace1, a copper-dependent transcription factor (Gralla et al., 1991). The transcription factor is activated by binding to copper ions, and in turn activates the CUP1 promoter containing four metal regulatory elements. By inserting the CUP1 promoter upstream from a desired gene we can use the same system to stimulate expression. Since the system is native to yeast, no additional associated proteins need be included to make it work.

In the tetracycline-based induction system (TET), gene expression is induced by the presence, or absence, of the antibiotic tetracyline (or one of its derivatives such as doxycycline). There are two commonly used variants of this system, TetON and TetOFF. The TetON system is based on a reverse tetracycline-controlled transactivator (rtTA) composed of the TetR repressor binding protein from the tetracycline resistance operon of E. coli transposon Tn10. In the original version of the system, TetR is fused to the strong transactivating domain of VP16 (alternatively referred to as vmw65 or α-TIF in the literature) from Herpes simplex virus (Hirai et al., 2010). TetON is a variation of TetOFF in which four amino acids have been modified in the TetR DNA binding moiety which alters its binding characteristics to the promotors. The difference between the two systems is not whether the transactivator turns a gene on or off (as both proteins activate expression). Rather, TetON activates expression in the presence of tetracycline, while and TetOFF activates expression in the absence of tetracyline. The promotor contains Tet operator (TetO) sequences derived from the human cytomegalovirus immediate-early promoter and multiple TetO repeats can be placed in sequence to increases induction efficiency.

In our project we replaced VP16 with the yeast Gal4 activation domain (Traven et al., 2006), and rtTA is linked to a nuclear localization signal sequence. We also utilized seven TetO sequence repeats in the Scpho5 promoter.
Figure: Showing the similarities in the chemical structure of tetracycline (left) and doxycycline (right). The differences are highlighted in red.
The estradiol-inducible system (also given to us by the Tom Ellis lab) induced by the mammalian hormone β-estradiol. Similar to TET, several parts are required in order to make this system function properly. An estradiol ligand recognizing domain (ER) alters conformation upon binding to estradiol. This leads to the exposure of a nuclear localization signal, and thus to a transfer of the transcription factor from the cytosol to the nucleus. ER has further been fused to a human zinc finger protein native to neurons (Zif268). Zif268 recognizes a short DNA sequence known as Z3BS. ER-Zif268 is further fused with VP16 to ensure strong activation, and multiple repeats of Z3BS are used to increasing binding efficiency.

The variant of this system we decided to use for our project is an improvement of the original system more optimized for induction in yeast. In the original system, ER was fused with the yeast Gal4 binding domain, making it bind to the endogenous GAL promoters and thereby unwanted influenced the cells metabolism. As other Gal4 regulators could also bind to the promoter, this system could not be used in media containing galactose, unless GAL4 was knocked out. By substituting the Gal4 binding domain with Zif268, the variant system allows tightly regulated and growth condition-independent transcription (McIsaac et al., 2011, 2013, 2014; Ottoz et al., 2014).
The chemical structure of the mammalian hormone β-estradiol.
While not optimal for large-scale manufacturing processes, these induction systems were chosen for their availability and being well-characterized in the current literature to provide a basis for a proof-of-concept of our design. The use of small-molecule inducers is mainly limited to the diffusion of the agent in the growth media, the sensitivity of the system and the cost of the compounds used. Background expression (‘leakiness’) is common for many types of induction systems. In addition, specific responses by the chassis used add an extra level of complexity when trying to design and optimize the induction system, especially when several systems are combined and made to work in parallel as on our project (Da Silva & Srikrishnan, 2012).

With our project we aim to illustrate that the use of multiple signals can allow programmability and fine-tuning of gene expression involved in biosynthesis pathways, and that there is a potential for using this kind of systems as platform technologies for large-scale manufacturing purposes.


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