Part:BBa_K3629023
2-isopropylmalate synthase (LEU4) overexpression construct with Gibson homology
2-isopropylmalate synthase (LEU4) expression construct with Gibson homology, TEF intronic promoter (BBa_K3629001)double XPR2 terminator(BBa_K3629004),Yarrowia lipolytica LEU4 coding sequence (BBa_K3629019) and reverse gPCR primer binding site for Yarrowia lipolytica.
Usage and Biology
This construct can be used to create a Yarrowia lipolytica leucine overproducing strain. One of the key enzymes, LEU4, of the leucine biosynthesis pathway is overexpressed using a strong constitutive promoter.
2-isopropylmalate synthase (LEU4) (BBa_K3629019) is the first enzyme involved in the biosynthesis of leucine. This is a very important enzyme in the pathway as it serves as a regulatory point in the biosynthesis pathway (1). The protein has a leucine binding domain which allows for downregulation of the pathway through negative-feedback inhibition of the enzyme. Expression of this enzyme especially on a strong promoter could be used to upregulate leucine synthesis and produce a leucine overproducing strain.
Expression of LEU4 is under the TEF intronic promoter (BBa_K3629001) which is the natural promoter found in the wild-type Yarrowia lipolytica for the Translation Elongation Factor 1 (TEF1) gene; it is a strong constitutive promoter. It also contains the first intron of the gene for stronger expression which helps with the overexpression of LEU4 (2). XPR2 (BBa_K3629004) was used in the construct which is the terminator sequence from the Yarrowia lipolytica alkaline extracellular protease XRP2 gene (3).
Design
The construct is flanked with two Gibson homology sequences. Upon digestion with BbsI, the construct can be connected to the nourseothricin resistance expression construct (BBa_K3629015) on the 5' side and to either the mCherry (BBa_K3629025) or mCitrine (BBa_K3629026) expression construct on the 3' side. This allows for both a selection marker and a fluorescence protein to be easily added to the construct in one Gibson reaction.
The construct was designed to be ultimately integrated into the genome of Yarrowia lipolytica. Therefore, a pair of gPCR primers can be used to help determine the location of the integration of the construct in the genome. The forward gPCR primer binding site is found in the nourseothricin resistance expression construct (BBa_K3629015) while the reverse gPCR primer binding site is located at the end of this construct (LEU4 Overexpression construct).
An extra XPR2 terminator (BBa_K3629004) was included at the beginning of the construct, right before the promoter in case the construct is integrated into the coding sequence of a gene in the genome. This would stop the expression of that gene which might have otherwise interfered with the expression of the LEU4 gene.
LEU4 coding sequence was also codon-optimized for expression and function in Yarrowia lipolytica.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 339
Illegal EcoRI site found at 2875
Illegal SpeI site found at 569
Illegal PstI site found at 534
Illegal PstI site found at 986
Illegal PstI site found at 2144 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 339
Illegal EcoRI site found at 2875
Illegal NheI site found at 91
Illegal SpeI site found at 569
Illegal PstI site found at 534
Illegal PstI site found at 986
Illegal PstI site found at 2144 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 339
Illegal EcoRI site found at 2875
Illegal BglII site found at 1629
Illegal XhoI site found at 1329
Illegal XhoI site found at 1878
Illegal XhoI site found at 2136 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 339
Illegal EcoRI site found at 2875
Illegal SpeI site found at 569
Illegal PstI site found at 534
Illegal PstI site found at 986
Illegal PstI site found at 2144 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 339
Illegal EcoRI site found at 2875
Illegal SpeI site found at 569
Illegal PstI site found at 534
Illegal PstI site found at 986
Illegal PstI site found at 2144 - 1000COMPATIBLE WITH RFC[1000]
References
1. KEGG PATHWAY: Valine, leucine and isoleucine biosynthesis. (n.d.). Retrieved October 26, 2020, from https://www.genome.jp/kegg-bin/show_pathway?yli00290
2. Blazeck, J., Liu, L., Redden, H., & Alper, H. (2011). Tuning gene expression in Yarrowia lipolytica by a hybrid promoter approach. Applied and environmental microbiology, 77(22), 7905–7914. https://doi.org/10.1128/AEM.05763-11
3. Czajka, Jeffrey J, Nathenson, Justin A, Benites, Veronica T, Baidoo, Edward E. K, Cheng, Qianshun, Wang, Yechun, & Tang, Yinjie J. (2018). Engineering the oleaginous yeast Yarrowia lipolytica to produce the aroma compound β-ionone. Microbial Cell Factories, 17(1), 136–136. https://doi.org/10.1186/s12934-018-0984-x
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