Team:Tartu TUIT/Project

Project: Yeasthylene
The demand on ethylene has only been increasing during the last decade. It is used as an essential building block in many chemical compounds.The main aim of our project is to find an alternative and biological way of producing ethylene. That is why we have decided to genetically engineer yeast cells to produce ethylene from sucrose.
Scientific Background
The focus of our project is to produce ethylene from sucrose (molasses) by using two different yeast strains. Both strains have dissimilar roles in the ethylene production and would be dependent on each other, in order to provide a balanced growth. 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 as 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). Strain A is unable to break sucrose while strain B will be in charge of expressing the BioBrick for the SUC2 invertase, which will be secreted into the medium and convert it into glucose and fructose (Equation 1). The hexoses will be used for biomass and ethanol production by strain A. Strain B is not able to transport hexoses, therefore, it will use the ethanol for growth and ethylene production (Figure 2). Thus, the growth of strain B is directly conditioned by the amount of ethanol secreted into the medium by strain A, while the growth and ethanol production of the latter is directly proportional to 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 strain 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 derived from the CEN.PK 2-1C strain, it has a SUC2 gene integrated into the genome, the expression of which 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 (strain 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 acquired. 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 and literature usually describes the insertion of the ethylene forming enzyme (EFE) from the plant pathogenic bacterium, Pseudomonas syringae, for the expression in yeast. The EFE 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 strain A will diffuse into strain B and be converted into acetyl-CoA (Eqs. 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)

[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.