Difference between revisions of "Part:BBa K1462000"

Revision as of 15:19, 12 October 2023

CoxVI Characterised by GB_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.3A) 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.3B.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.3C). 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.4)

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). In order to produce the odor of papaya oil, we engineered S.cerevisiae to produce alpha-pinene. The endogenous genes tHMG1 and IDi were overexpressed in the pathway to increase the supply of precursors isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) (Fig.6A). The strands were simultaneously integrated into CEN.PK2-1C at the His3 site (Fig.6B). The transformation was proven to be successful by colony PCR and gel electrophoresis (Fig.7A). The accuracy of the transformation was proved by the result of sequencing (Fig.7B.1). The modified strain was named SCIE L3. We carried out fermentation, and sent the product for GC-MS analysis using CEN.PK2-1C as the control (Fig.8).

To achieve a high expression level of alpha-pinene, it is indispensable to increase the supply of geranyl pyrophosphate (GPP) in our strain SCIE L3 and minimize the supply of farnesyl diphosphate (FPP) which would result in the production of by-products. Thus, ERG20 was overexpressed with the introduction of the mutations F96W and N127W (ERG20ww), mitigating the formation of FPP while maintaining its activity for producing GPP. The heterologous gene derived from Pinus taeda encoding for alpha-pinene synthase (PS) is required to convert GPP to alpha-pinene. In order to maximize the production of alpha-pinene, a N-terminus truncation of 48 amino acids was introduced (t48PS) (Fig.6A). In addition, we decided to express ERG20ww and t48PS in two possible ways. First, ERG20ww and t48PS were simultaneously transformed into SCIE L3 (Fig.6B.2). Second, we are determined to express ERG20ww linked to the t48PS through a flexible linker (FL) GGGGSGGGGS, which would increase the amount of GPP converted to alpha-pinene (Fig.6B.3). The result of the transformation was verified successfully by gel electrophoresis (Fig.7A) and sequencing (Fig.7B.2). The strain was named SCIE L4. Fermentation was conducted using the same procedure, and the product was collected and sent for GC-MS analysis using CEN.PK2-1C as the control (Fig.8). Although the result of the analysis was not alpha-pinene, the odor of our product resembled the smell of papaya.

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)

Fig.3: COX VI LEADING PEPTIDE MECHANISM

Sequence and Features