Difference between revisions of "Team:Tartu TUIT/Project"

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<h1> Project Description
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<h1> Yeasthylene
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    {{Tartu_TUIT_project}}
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Nowadays society is deeply dependent on non-renewable energy sources, such as oil and gas. These sources are mainly used as fuels, intended to meet the energy and electricity demands of today’s world. However, a wide range of other important chemical compounds are produced from petroleum, most notably hydrocarbon monomers such as ethylene (C2H4). The demand on ethylene has only been increasing during the last decade; the average annual growth from 2007 to 2014 was 2.2%, whereas it is predicted that the growth would increase to 3.6% for the years 2014-2020. This high demand is mainly due to the fact that ethylene is rather multipurposed, and it is used as an essential building block in many chemical compounds. Its most commonly used polymer product, polyethylene, is a main compound in many plastic materials.The main aim of our project is to find an alternative and biological way of producing ethylene.
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In this project, two yeast subpopulations with completely different roles will be genetically modified to produce ethylene from sucrose. The focus of the project is set to make those subpopulations dependent on each-other and to provide a balanced growth. The subpopulation approach represents a more efficient method than cloning the whole pathway into one population, especially when longer heterologous pathways will be used in the future to produce more complex chemicals. Energy gain due to lower metabolic burden and balanced co-factor metabolism will result in higher production rates.
 
  
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We will make both of our subpopulations unable to metabolise sucrose. However, in the second subpopulation the suc2 gene, responsible for conversion of sucrose to glucose and fructose, will be overexpressed. The protein will be automatically secreted into the sucrose media where it will convert sucrose into glucose and fructose. Our second subpopulation will not be able to metabolise glucose or fructose. In this way, all the glucose or fructose will be used up by the first subpopulation. The first subpopulation will naturally convert glucose and fructose into ethanol. Since the second subpopulation is not able to metabolise sucrose, glucose or fructose it will use ethanol as it only carbon source. This ethanol will enter the TCA cycle and will eventually be converted into ethylene through EFE enzyme which will be introduced to the second subpopulation.
 
  
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        Scientific Background
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           The focus of our project is to produce ethylene from sucrose (molasses) by using two different yeast strains. Both strains would be dependent on each other, in order to provide a balanced growth, and have dissimilar roles in the ethylene production. By using co-cultured strains we expect to lower the metabolic burden and, therefore, increase our yields.  This approach could represent a more efficient method than cloning long heterologous pathways into one population for the involved in the production of complex chemicals. Also, it could also decrease the time needed for metabolic engineering, along with lowering a chance of redox unbalance in the cell.
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          Our strategy to obtain balanced growth between the strains was through substrate feeding (Figure 1). As sucrose is our substrate, the strain A is unable to break it while strain B will be in charge of expressing our biobrick for the secreted suc2 invertase, which will convert it into glucose and fructose (Equation 1. Those hexoses will used as carbon and energy source by A, which will convert them into ethanol. Strain B is not able to transport hexoses, therefore it needs another carbon source for growth and energy: ethanol (Figure 2). Thus, the growth of the B strain is directly conditioned by the amount of ethanol secreted into the media by A strain, while the growth and ethanol production of the later is directly proportional by the sucrose breakage by the suc2 biobrick expressed by strain B.
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          C12H22O11 + H2O + invertase → 2 C6H12O6 (fructose and glucose)  (Eq. 1)
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            Figure 1. Scheme of the strains A and B used for ethylene production
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          For B we used EBY.VW4000[1] strain, which was kindly donated by Prof. Eckhard Boles’ research group from Heinrich-Heine-Universität. This strain had around 20 hexose transporters knocked out: his3-Δ1, hxt17Δ,  hxt13Δ::loxP, hxt15Δ::loxP, hxt16Δ::loxP, hxt14Δ::loxP, hxt12Δ::loxP, hxt9Δ::loxP, hxt11Δ::loxP, hxt10Δ::loxP,  hxt8Δ::loxP, hxt514Δ::loxP, hxt2Δ::loxP, hxt367Δ::loxP ,gal2Δ,  stl1Δ::loxP ,agt1Δ::loxP ,ydl247wΔ::loxP, yjr160cΔ::loxP. As this strain is derivate from the CEN.PK 2-1C strain it has a SUC2 gene integrated into the genome, which expressions is highly dependent on the glucose concentration in the medium: induced by low glucose concentration, but repressed by high concentrations of the same sugar[2].
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            Figure 2. Sucrose break by suc2 biobrick (from strain B) into glucose and fructose and ethanol production by strain A
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          IMU051[3] strain (referred to as a strain number A) has been deleted for the following genes: malΔ(mal11-mal12::loxP, mal21-mal22::loxP, mal31-32::loxP), mphΔ(mph2/3::loxP, mph2/3::loxP-hphNT1-loxP), suc2Δ. The strain showed no growth in a sucrose media even after a 10 day incubation period. [3] This strain has been kindly provided by Prof. Antonius J.A. van Maris’ research group from the University of Delft.
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These two sugars (glucose and fructose) will be consumed by IMU051 strain (Figures 2) and, after a series of steps, converted into ethanol (C2H5OH). Fructose and glucose are broken down into two pyruvate molecules in a process known as glycolysis[4]
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C6H12O6 (fructose/glucose) + 2 ADP + 2 Pi + 2 NAD+ → 2 CH3COCOO− + 2 ATP + 2 NADH + 2 H2O + 2 H+
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Pyruvate molecule is converted into ethanol and CO2, during a 2 step process:
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1. CH3COCOO− + H+ → CH3CHO + CO2
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The second step is catalysed by ADH1(alcohol dehydrogenase)[5]
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2. CH3CHO + NADH+H+ → C2H5OH + NAD+
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Ethanol, being an uncharged polar molecule, will cross the cellular membrane by passive diffusion; a process in which active transporter proteins are required. As the movement is performed from the area of high to the area of low concentration of the molecule, no metabolic energy will be expended.[6]
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          S. cerevisiae is not a natural ethylene producer organism and literature usually describes the insertion of the ethylene forming enzyme (EFE) from the plant pathogenic bacterium, Pseudomonas syringae for expression in yeast. The efe enzyme catalyzes the formation of ethylene along with carbon dioxide and succinate from 2-oxoglutarate, arginine and oxygen [7,8]. We expect that the ethanol produced from population A will difuse into strain B and be converted into acetil-CoA (Eq 2-4). This molecule will  enter the TCA cycle and the intermediate 2-oxoglutarate (alpha-keto glutaric acid) will be converted into ethylene by the EFE biobrick (Eq. 5) transformed into strain B (Figure 3).
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          C2H6O(Ethanol) + NAD+ →C2H4O(Acetaldehyde) + NADH + H+  (Eq. 2)
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          C2H4O(Acetaldehyde) + NAD+ + H2O → C2H4O2(acetic acid) + NADH + H+  (Eq. 3)
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          C2H4O2(acetic acid) + CoA + ATP → Acetyl-CoA + AMP + PPi  (Eq. 4)
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          3 2−oxoglutarate + 3 O2 + 1 Arginine→2 Ethylene + 1 Succinate + 1 Guanidine + 1 (S)−1−pyrroline−5−carboxylate (P5C) + 7CO2 + 3H2O (Eq. 5)
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          References:
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        <div class="col-xs-12 project-background-references project-background-text">
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          [1] - R. Wieczorke, S. Krampe, T. Weierstall, K. Freidel, C. P. Hollenberg, E.Boles: Concurrent knock-out of at least 20 transporter genes is required to block uptake of hexoses in Saccharomyces cerevisiae. FEBS Letters 1999, 464: 123-128
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<br/></br/>
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          [2] - Mohandesi, N., Siadat, S.O.R., Haghbeen, K. and Hesampour, A., 2016. Cloning and expression of Saccharomyces cerevisiae SUC2. 3 Biotech, 6(2), pp.1-10.
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<br/></br/>
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          [3] - Marques, W.L., Mans, R., Marella, E.R., Cordeiro, R.L., van den Broek, M., Daran, J.M.G., Pronk, J.T., Gombert, A.K. and van Maris, A.J., 2017. Elimination of sucrose transport and hydrolysis in Saccharomyces cerevisiae: a platform strain for engineering sucrose metabolism. FEMS yeast research, 17(1).
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<br/></br/>
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          [4] - Stryer, Lubert (1975). Biochemistry. W. H. Freeman and Company.
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<br/></br/>
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          [5] - Raj, S.B., Ramaswamy, S. and Plapp, B.V., 2014. Yeast alcohol dehydrogenase structure and catalysis. Biochemistry, 53(36), p.5791.
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<br/></br/>
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          [6] - Lodish, H., Berk, A., Zipursky, S.L., Matsudaira, P., Baltimore, D. and Darnell, J., 2000. Section 15.1, Diffusion of small molecules across phospholipid bilayers. Molecular Cell Biology.
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<br/></br/>
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          [7] Pirkov, I., Albers, E., Norbeck, J.,  Larsson, C. 2008. Ethylene production by metabolic engineering of the yeast Saccharomyces cerevisiae. Metabolic Engineering, 10(5), 276–280.
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<br/></br/>
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          [8] Johansson, N., Quehl, P., Norbeck, J.,  Larsson, C. 2013. Identification of factors for improved ethylene production via the ethylene forming enzyme in chemostat cultures of Saccharomyces cerevisiae. Microbial Cell Factories, 12(1), 89.
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Scientific Background
The focus of our project is to produce ethylene from sucrose (molasses) by using two different yeast strains. Both strains would be dependent on each other, in order to provide a balanced growth, and have dissimilar roles in the ethylene production. By using co-cultured strains we expect to lower the metabolic burden and, therefore, increase our yields. This approach could represent a more efficient method than cloning long heterologous pathways into one population for the involved in the production of complex chemicals. Also, it could also decrease the time needed for metabolic engineering, along with lowering a chance of redox unbalance in the cell.
Our strategy to obtain balanced growth between the strains was through substrate feeding (Figure 1). As sucrose is our substrate, the strain A is unable to break it while strain B will be in charge of expressing our biobrick for the secreted suc2 invertase, which will convert it into glucose and fructose (Equation 1. Those hexoses will used as carbon and energy source by A, which will convert them into ethanol. Strain B is not able to transport hexoses, therefore it needs another carbon source for growth and energy: ethanol (Figure 2). Thus, the growth of the B strain is directly conditioned by the amount of ethanol secreted into the media by A strain, while the growth and ethanol production of the later is directly proportional by the sucrose breakage by the suc2 biobrick expressed by strain B.
C12H22O11 + H2O + invertase → 2 C6H12O6 (fructose and glucose) (Eq. 1)
Figure 1. Scheme of the strains A and B used for ethylene production
For B we used EBY.VW4000[1] strain, which was kindly donated by Prof. Eckhard Boles’ research group from Heinrich-Heine-Universität. This strain had around 20 hexose transporters knocked out: his3-Δ1, hxt17Δ,  hxt13Δ::loxP, hxt15Δ::loxP, hxt16Δ::loxP, hxt14Δ::loxP, hxt12Δ::loxP, hxt9Δ::loxP, hxt11Δ::loxP, hxt10Δ::loxP, hxt8Δ::loxP, hxt514Δ::loxP, hxt2Δ::loxP, hxt367Δ::loxP ,gal2Δ,  stl1Δ::loxP ,agt1Δ::loxP ,ydl247wΔ::loxP, yjr160cΔ::loxP. As this strain is derivate from the CEN.PK 2-1C strain it has a SUC2 gene integrated into the genome, which expressions is highly dependent on the glucose concentration in the medium: induced by low glucose concentration, but repressed by high concentrations of the same sugar[2].
Figure 2. Sucrose break by suc2 biobrick (from strain B) into glucose and fructose and ethanol production by strain A
IMU051[3] strain (referred to as a strain number A) has been deleted for the following genes: malΔ(mal11-mal12::loxP, mal21-mal22::loxP, mal31-32::loxP), mphΔ(mph2/3::loxP, mph2/3::loxP-hphNT1-loxP), suc2Δ. The strain showed no growth in a sucrose media even after a 10 day incubation period. [3] This strain has been kindly provided by Prof. Antonius J.A. van Maris’ research group from the University of Delft. These two sugars (glucose and fructose) will be consumed by IMU051 strain (Figures 2) and, after a series of steps, converted into ethanol (C2H5OH). Fructose and glucose are broken down into two pyruvate molecules in a process known as glycolysis[4]

C6H12O6 (fructose/glucose) + 2 ADP + 2 Pi + 2 NAD+ → 2 CH3COCOO− + 2 ATP + 2 NADH + 2 H2O + 2 H+

Pyruvate molecule is converted into ethanol and CO2, during a 2 step process: 1. CH3COCOO− + H+ → CH3CHO + CO2

The second step is catalysed by ADH1(alcohol dehydrogenase)[5]

2. CH3CHO + NADH+H+ → C2H5OH + NAD+

Ethanol, being an uncharged polar molecule, will cross the cellular membrane by passive diffusion; a process in which active transporter proteins are required. As the movement is performed from the area of high to the area of low concentration of the molecule, no metabolic energy will be expended.[6]
S. cerevisiae is not a natural ethylene producer organism and literature usually describes the insertion of the ethylene forming enzyme (EFE) from the plant pathogenic bacterium, Pseudomonas syringae for expression in yeast. The efe enzyme catalyzes the formation of ethylene along with carbon dioxide and succinate from 2-oxoglutarate, arginine and oxygen [7,8]. We expect that the ethanol produced from population A will difuse into strain B and be converted into acetil-CoA (Eq 2-4). This molecule will enter the TCA cycle and the intermediate 2-oxoglutarate (alpha-keto glutaric acid) will be converted into ethylene by the EFE biobrick (Eq. 5) transformed into strain B (Figure 3).

C2H6O(Ethanol) + NAD+ →C2H4O(Acetaldehyde) + NADH + H+ (Eq. 2)

C2H4O(Acetaldehyde) + NAD+ + H2O → C2H4O2(acetic acid) + NADH + H+ (Eq. 3)

C2H4O2(acetic acid) + CoA + ATP → Acetyl-CoA + AMP + PPi (Eq. 4)

3 2−oxoglutarate + 3 O2 + 1 Arginine→2 Ethylene + 1 Succinate + 1 Guanidine + 1 (S)−1−pyrroline−5−carboxylate (P5C) + 7CO2 + 3H2O (Eq. 5)
References:
[1] - R. Wieczorke, S. Krampe, T. Weierstall, K. Freidel, C. P. Hollenberg, E.Boles: Concurrent knock-out of at least 20 transporter genes is required to block uptake of hexoses in Saccharomyces cerevisiae. FEBS Letters 1999, 464: 123-128

[2] - Mohandesi, N., Siadat, S.O.R., Haghbeen, K. and Hesampour, A., 2016. Cloning and expression of Saccharomyces cerevisiae SUC2. 3 Biotech, 6(2), pp.1-10.

[3] - Marques, W.L., Mans, R., Marella, E.R., Cordeiro, R.L., van den Broek, M., Daran, J.M.G., Pronk, J.T., Gombert, A.K. and van Maris, A.J., 2017. Elimination of sucrose transport and hydrolysis in Saccharomyces cerevisiae: a platform strain for engineering sucrose metabolism. FEMS yeast research, 17(1).

[4] - Stryer, Lubert (1975). Biochemistry. W. H. Freeman and Company.

[5] - Raj, S.B., Ramaswamy, S. and Plapp, B.V., 2014. Yeast alcohol dehydrogenase structure and catalysis. Biochemistry, 53(36), p.5791.

[6] - Lodish, H., Berk, A., Zipursky, S.L., Matsudaira, P., Baltimore, D. and Darnell, J., 2000. Section 15.1, Diffusion of small molecules across phospholipid bilayers. Molecular Cell Biology.

[7] Pirkov, I., Albers, E., Norbeck, J., Larsson, C. 2008. Ethylene production by metabolic engineering of the yeast Saccharomyces cerevisiae. Metabolic Engineering, 10(5), 276–280.

[8] Johansson, N., Quehl, P., Norbeck, J., Larsson, C. 2013. Identification of factors for improved ethylene production via the ethylene forming enzyme in chemostat cultures of Saccharomyces cerevisiae. Microbial Cell Factories, 12(1), 89.

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