PART DESIGN

Genetic Constructs

In order to implement our biocontainment strategy in Y. lipolytica , we need to engineer two strains of yeast; one that is auxotrophic for tryptophan and overproduces leucine, and one that is auxotrophic for leucine and overproduces tryptophan.

Engineering amino acid overproduction:

To engineer a leucine overproducing and a tryptophan overproducing strain we designed two overexpression cassettes that obtained the following components:

2-isopropylmalate synthase (LEU4) BBa_K3629019 and Anthranilate synthase component 1 (TRP2) BBa_K3629020 are important regulatory enzymes involved in the biosynthesis pathway of leucine and tryptophan respectively. These enzymes prevent overproduction of the amino acids as they are feedback-inhibited by the end products of the pathway (leucine and tryptophan). Overexpression of these two enzymes could help remove the bottleneck effect of the feedback inhibition and allow for overproduction of the amino acid

herefore we designed two genetic constructs; one to overexprss LEU4 href="http://parts.igem.org/Part:BBa_K3629023">BBa_K3629023, and one to overxpress TRP2 href="http://parts.igem.org/Part:BBa_K3629024">BBa_K3629024. We used the stong intronic TEF promoter href="http://parts.igem.org/Part:BBa_K3629001">BBa_K3629001 in these constructs to help ensure that LEU4 and TRP2 are expressed at high levels. TEF promoter is one of the strongest native promoters of Y. lipolytica . We included the first intron of the TEF1 gene in the sequence of the promoter since the inclusion of the first intron has shown to allow for even stronger expression.

The mCherry reporter device href="http://parts.igem.org/Part:BBa_K3629025">BBa_K3629025 and the mCitrine reporter device href="http://parts.igem.org/Part:BBa_K3629025">BBa_K3629025 were included to help distinguish and quantify the leucine and tryptophan overproducing gene in the co-culture. Each strain will be engineered with one of the reporter devices and since each reporter device contains a fluorescent protein that fluoresces at a distinct emission wavelength, fluorescence spectroscopy can be used to distinguish and quantify the leucine and tryptophan overproducing strains in the co-culture.

Nourseothricin resistance expression construct href="http://parts.igem.org/Part:BBa_K3629012">BBa_K3629012, optimized for expression in Y. lipolytica , was also used in our cassettes as the selection marker.

Together, the LEU4/TRP2 overexpression constructs, the reporter devices (mCherry or mCitrine), and the Nourseothricin resistance expression construct made our overexpression cassette that could be used to engineer a leucine or a tryptophan overproducing Y. lipolytica . In order to allow for easy assembly of our cassettes, we make each of our genetic constructs compatible with Gibson assembly. Each part is flanked by a Gibson homology sequence that allows for all the parts to come together in a single Gibson reaction.

Engineering Auxotrophy

To introduce leucine auxotrophy, we plan to knock-out 3-isopropylmalate dehydrogenase (LEU2), a common gene targeted in Y. lipolytica leucine auxotrophs. Tryptophan auxotrophy, however, has not been introduced before inY. lipolytica . In order to make tryptophan auxotrophic strain of Y. lipolytica , we plan to target TRP1, a common gene targetted in S. cerevisiae tryptophan auxotrophs. Since the tryptophan biosynthesis pathway of S. cerevisiae and Y. lipolytica are almost identical, knockout of TRP1 should produce tryptophan auxotrophs in Y. lipolytica .

We wanted to engineer auxotrophy and amino acid overproduction in Y. lipolytica in one efficient step. Therefore, we included homology arms to upstream and downstream of the LEU2 or the TRP1 gene in our overexpression constructs. For example, our TRP2 overproducing cassette is flanked with homology arms to upstream and downstream of the LEU2. So that when the yeast is transformed with our cassette, through homologous recombination the TRP2 cassette would insert in the middle of the LEU2 gene, knocking out the gene. As a result, the engineered will become auxotrophic for leucine and overproducing for tryptophan in one engineering step.