Team:Marburg/Production

SynDustry Fuse. Produce. Use.

The Production Line

Description
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After establishing a system that is suitable to introduce an endosymbiont into one of the most commonly used eukaryotic organisms in both laboratories and industry, Saccharomyces cerevisiae, we wanted to take this system to a next level. Within Syndustry, we aimed to use different organisms according to their natural abilities and specialties to overcome bottlenecks in order to improve biotechnological productions as well as enable for productions that could not be established yet at all.

The biotechnological industry is capable of generating a lot of different highly valuable products that are useful in various ways. To show the potential of Syndustry we have chosen to implement a model system for a production line within the cell. The focus was kept on the level of terpenoids, which opens up a tremendous amount of different valuable chemicals that can be expressed in a biological system. Terpenoids usually derive from two different pathways (Fig. 1): The mevalonate pathway, mostly used in eukaryotes, and the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway also known as deoxyxylose 5-phosphate (DXP) pathway, mostly used in prokaryotes. Both pathways synthesize the terpenoid precursors isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) [1]. Terpenoids are hydrocarbons and classified by the number of isoprene units that form the structure.

Figure 1
Figure 1. Overview of the two given pathways for the formation of isoprenoids. The mevalonate pathway and the DXP pathway lead to IPP and DMAPP as direct starting points for the synthesis of the several terpenoid classes. The production line was designed to synthesize limonene as a monoterpenoid from GPP. [2]

The production line within the cell will start with a heterologous, engineered mevalonate-pathway for terpenoid production in E. coli to produce (-)-S-Limonene (Fig. 2) [3]. Limonene is a highly valuable chemical, which can find applications in the fragrance and food industry, in jet- and rocketfuels as well as in several other products. This variability makes this chemical a suitable intermediate for Syndustry. In the production line of Syndustry, the limonene gets pumped out of the endosymbiont into the yeast cell by a limonene efflux pump. That should increase the limonene yields in the host and with that the efficiency of the general production process. Beneath that, the efflux pump should also increase the resistance of E. coli towards the highly lipophilic limonene [4].

Figure 1
Figure 2. Construct that has been used for the limonene synthesis. This construct introduces a heterologous mevalonate pathway into E. coli and additionally, makes it possible to produce GPP and Limonene. The different origins of the several proteins that are encoded on the plasmid are shown in different colors. [3, modified]

With limonene transported into yeast we focused on producing perillyl alcohol, which is a promising anti-cancer drug [5][6]. For the production of perillyl alcohol the limonene has to be hydroxylated by a cytochrome P450. The expression of cytochrome P450 in prokaryotes was experienced to be troubling [7]. That means the expression of the cytochrome P450 in a eukaryotic host could lead to a more efficient production. Furthermore, the produced perillyl alcohol is pumped out into the medium by the monoterpenoid efflux pump GcABC-G1 from Grosmannia clavigera [8]. With that step the production line within the cell is complete and an extraction of perillyl alcohol from the medium is possible via a commercially available resin that grants a constant workflow. Furthermore, an additional point mutation in the Tcb3 gene, which leads to a truncated version of the protein and with that to a change in cell wall composition was introduced [9]. Together with the efflux pump the point mutation enables a higher resistance towards the lipophilic monoterpenoids.

Figure 1
Figure 3. Overview of the production line. The design shows the simple workflow that combines the strengths of two organisms towards a specialization of each one to generate increased productivity.

This system is highly variable as limonene is the basic component for several terpenoid derived products. The production step in the host could be changed to produce different monoterpenoids, which could still be exported into the medium by the efflux pump.

Literature

  1. [1] Pérez-Gil, Jordi, and Manuel Rodríguez-Concepción. "Metabolic plasticity for isoprenoid biosynthesis in bacteria." Biochemical Journal 452.1 (2013): 19-25.
  2. [2] Wheeldon, Ian. "Microbial production of isoprenoids enabled by synthetic biology." Synthetic biology applications in industrial microbiology 94 (2014): 100.
  3. [3] Alonso-Gutierrez, Jorge, et al. "Metabolic engineering of Escherichia coli for limonene and perillyl alcohol production." Metabolic engineering 19 (2013): 33-41.
  4. [4] Dunlop, Mary J., et al. "Engineering microbial biofuel tolerance and export using efflux pumps." Molecular systems biology 7.1 (2011): 487.
  5. [5] Stark, M. Jennifer, et al. "Chemotherapy of pancreatic cancer with the monoterpene perillyl alcohol." Cancer letters 96.1 (1995): 15-21.
  6. [6] Yeruva, Laxmi, et al. "Perillyl alcohol and perillic acid induced cell cycle arrest and apoptosis in non small cell lung cancer cells." Cancer letters 257.2 (2007): 216-226.
  7. [7] Chang, Michelle CY, et al. "Engineering Escherichia coli for production of functionalized terpenoids using plant P450s." Nature chemical biology 3.5 (2007): 274-277.
  8. [8] Wang, Ye, et al. "A specialized ABC efflux transporter GcABC‐G1 confers monoterpene resistance to Grosmannia clavigera, a bark beetle‐associated fungal pathogen of pine trees." New Phytologist 197.3 (2013): 886-898.
  9. [9] Brennan, Timothy CR, et al. "Evolutionary engineering improves tolerance for replacement jet fuels in Saccharomyces cerevisiae." Applied and environmental microbiology 81.10 (2015): 3316-3325.