Part:BBa_K1462000
CoxVI
Characterised by GreatBay_SCIE2023
In order to increase the production of isoamyl acetate, we are determined to increase the accumulation of isopentanol by overexpression of ARO10 and ADH7. (Fig.1A) The overexpression of ATF1 enables the conversion from the alcohol to isoamyl acetate. The endogenous DNA strands were extracted and amplified from CEN.PK2-1C genome and integrated simultaneously to site 106 of CEN.PK2-1C using lithium acetate transformation, in which CRISPR-Cas9 is utilized to cut the site and the donor DNA strands, which were our coding sequences, would be inserted through homologous recombination. (Fig.1B.1) The construction was then verified by conducting yeast colony PCR followed by gel electrophoresis, which shows the target strands were integrated into the genome successfully (Fig.1C). The constructed strain was named SCIE L1. The fermentation was carried out and lasted for 48 hours using YPD+2% glucose medium and dodecane as solvent. We collected the product, and detected by GC-MS analysis. The result of the analysis demonstrates that we have successfully produced isoamyl acetate through our engineered S. cerevisiae using isoamyl acetate as the control. (Fig.2)
Fig.1 Expression of isoamyl acetate in S. cerevisiae. (A) Genetic pathway of producing isoamyl acetate. LEU4^S547∆, encoding for 2-isopropylmalate synthase (2-IPMS), in which the codon of the 547th amino acid S is deleted. LEU1, encoding isopropylmalate isomerase. LEU2, encoding 3-IPM dehydrogenase. ARO10, encoding 2-oxoacid decarboxylase. ADH7, encoding for alcohol dehydrogenase 7. ATF1, encoding for alcohol acetyl-coenzyme A (acetyl-CoA) transferase (AATase) (B) Genetic circuit construction for producing isoamyl acetate. ARO10, ADH7, and ATF1 are transformed into site 106 through homologous recombination using 106 upstream (106 US) and 106 downstream (106 DS). CDC9(first 47 amino acids)-LEU1, COX6(first 41 amino acids)-LEU2, COX4(first 26 amino acids)-LEU4S547∆ are transformed into site His3 through homologous recombination using His3 upstream (His3 US) and His3 downstream (His3 DS) (C) Gel electrophoresis analysis of integrated sequence ARO10-ADH7-ATF1 and CDC9-LEU1-COX6-LEU2-COX4-LEU4S547∆
Fig.2 GC-MS analysis of the product isoamyl acetate of SCIE L1 and SCIE L2 fermentation, using Standard isoamyl acetaet as control
For further improvement in the production of isoamyl acetate, it is necessary to enhance the supply of precursor 2-ketoisocaproate (KIC) in our modified SCIE L1. This can be achieved by overexpression of endogenous LEU4, LEU2 and LEU1 (Fig.3A). Since the presence of leucine can result in a decrease in the activity of LEU4, the 547th amino acid of the LEU4 gene is deleted (LEU4S547∆) to diminish its sensitivity to leucine while maintaining its function. Furthermore, to maximize the supply of KIC and minimize the formation of by-products, LEU4S547∆, LEU2 and LEU1 were targeted and overexpressed in mitochondria, reaching a higher regional enzyme concentration. It can be achieved by appending the first 26, 47, 41 amino acids from COX4, CDC9, COX6 to the N-terminus of LEU4S547∆, LEU2, and LEU1 coding sequences, respectively. The strands are simultaneously integrated into SCIE L1 at site His3 (Fig.3B.2). Yeast colony PCR and gel electrophoresis were carried out, and it successfully proved that the DNA strands have been integrated into the genome (Fig.3C). The constructed strain was named SCIE L2. We conducted fermentation of the yeast and tested our product through GC-MS using isoamyl acetate as the control. The results show an increase in the overall expression level of isoamyl acetate (Fig.4).
According to GC-MS analysis, the production of isoamyl acetate of SCIE L1 is 45.2, and SCIE L2 is 55.2 mg/L (Fig.5), which shows an increase in the yield.
Fig.5 Testing the production of isoamyl acetate (A) standard curve of isoamyl acetate (B) the production of isoamyl acetate of SCIE L1 and SCIE L2.
CoxVI is short for the subunit VI of the yeast cytochrome c oxidase, a N-terminal signal peptide to mitochondrial matrix.
Most mitochondrial proteins are synthesized in the cytosol as larger precursors carrying mitochondria targeting signals. In this pathway, the precursor is bound by cytosolic chaperones and then delivered to a set of receptors on the outer surface of mitochondria. Then, the polypeptide chain is passed through the TOM complex in the outer membrane and the TIM23 complex in the inner membrane. Insertion into the inner membrane is driven electrophoretically by the electrochemical potential across the membrane. Finally, the precursor is pulled completely across the membrane into the matrix by an ATP-powered translocation motor attached to the inner side of the TIM23complex. After that, the protein will be refolded by mitochondrial chaperone, and the mitochondria targeting signal will be cleaved.(Fig.3)
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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