Team:Marburg/Production/Results

SynDustry Fuse. Produce. Use.

The Production Line

« Description
Results
Outlook »

The production of (-)-(S)-limonene should be accomplished by transforming the plasmids pJBEI-6410 (Fig. 1) and pBbA5k-EPL95. We got positive transformations but could not further verify the production of limonene. One further optimization step regarding the limonene production would be the reduction of metabolic burden by establishing a one plasmid-based system.

Figure 1
Figure 1. Plasmid map for pJBEI-6410. This plasmid has been optimized by Alonso-Gutierrez et al. for the synthesis of (-)-(S)-limonene. Proteins responsible for the synthesis expressed on this plasmid express a heterologous mevalonate pathway for isoprenoid synthesis and further truncated, codon optimized geranylpyrophosphate synthase and limonene synthase for the further limonene synthesis.

In order to increase the possibility of high yields using this novel system it was necessary to codon optimize the genes of the Cytochrome P450 enzyme complex and the GcABC-G1 efflux pump for S. cerevisiae. Therefore we codon optimized the genes and ordered them without biobrick cut sites from idtDNA. The GcABC-G1 efflux pump does not suit the gblock standards as the gene itself with a length of 4377 bp is too long for just one biobrick. Because of this limitation we decided to split the gene into three fragments with 50 bp overhangs to the next fragment. The assembly of fragments was done via modified PCR with primers that bound to the outer fragments. The overhangs at the end of the gene fragments served as links for the amplification. To increase efficiency we used longer annealing times and more cycles. We have experienced reliable results, however sometimes unspecific fragments could be detected (Fig. 2).

Figure 1
Figure 2. Gel image of the PCR assembly of GcABC-G1 fragments. The marked bands show the amplification of the 4377bp GcABC-G1 gene.

As highest yields of perillyl alcohol would require a similar expression of the cytochrome P450 subunits, the ordered parts were designed with T2A-sequences from the Thosea asigna virus which allow the expression of up to three individual proteins from a polycistronic construct [1]. For the design of the T2A-sequences it was crucial to exclude the possibility of unwanted homologous recombination. Additionally, one of the T2A-sequences served as an overhang for another PCR assembly with two fragments. The PCR assembly with just two fragments worked better in comparison to the assembly of three fragments (Fig. 3).

Figure 1
Figure 3. Gel image of the pcr assembly of the two Cytochrome P450 gene fragments. The marked bands show the assembled and amplified fragments of the 2970bp Cytochrome P450 sequence with additional T2A-sequences.
Figure 1
Figure 4. Integration cassette with the Cytochrome P450 subunits and GcABC-G1. The desired integration cassette constists of the LEU2 homologies, GPD promoter and terminator for the Cytochrome P450 proteins alkane hydroxylase, ferredoxin reductase and ferredoxin linked by T2A-sequences. The GcABC-G1 should be flanked by a cyc1 promoter and an ADH1 terminator.

The chosen backbone was an integration backbone, which is capable of integrating the sequence into the LEU2 locus on the yeast genome (Fig. 4).

Cytochrome P450 was amplified with overhangs to clone it into this backbone in between the integrated GPD promoter and terminator to ensure a constant and high expression of the cytochrome P450 complex. Furthermore we planned to insert the GcABC-G1 with a Cyc1 promoter and an ADH1 terminator behind the GPD terminator. This would secure a suitable expression of the GcABC-G1 efflux pump to not affect the natural fitness of the yeast. The integration of the cassette would require to be cut out via digestion with the restriction enzyme PmeI.

Avoiding another plasmid based expression system could further increase the yield of perillyl alcohol. The next planned step was to extract the produced perillyl alcohol directly from the media using the commercially available Amberlite IRA 410 Cl resin.

Additional steps would have been a point mutation of Tcb3 with a hygromycin cassette. After that the limonene resistance of those cultures compared to WT cultures could have been measured by colony forming units and growth curves.

Literature

  1. [1] Beekwilder, Jules, et al. "Polycistronic expression of a β-carotene biosynthetic pathway in Saccharomyces cerevisiae coupled to β-ionone production." Journal of biotechnology 192 (2014): 383-392.