Two acetyl-CoA synthetase isoenzymes are encoded by distinct genes in marine yeast Rhodosporidium diobovatum
Two genes encoding two acetyl-CoA synthetase (ACS) isoenzymes have been identified in the marine yeast Rhodosporidium diobovatum MCCC 2A00023.
ACS1 encoded a polypeptide with a sequence of 578 amino acid residues, a predicted molecular weight of 63.73 kDa, and pI of 8.14, while the ACS2 encoded a polypeptide containing 676 amino acid residues with a deduced molecular mass of 75.61 kDa and a pI of 5.95. Biological activity of Acs1p and Acs2p was confirmed by heterologous expression in Escherichia coli. A 1.5-kb DNA fragment of the ACS1 gene and a 2.7-kb DNA fragment of the ACS2 gene were deleted using the RNA guide CRISPR-Cas9 system. The strain lacking ACS1 was unable to grow on acetate and ethanol media, while the ACS2 deletant was unable to grow on glucose medium. ACS1-ACS2 double mutants of R. diobovatum were non-viable.
ACS isoenzymes are essential to the yeast metabolism, and other sources of ACSs cannot compensate for the lack of ACSs encoded by the two genes.
KeywordsAcetyl-CoA synthetase CRISPR-Cas9 Isoenzymes Rhodosporidium diobovatum
This work was supported by the Science Research Plan of Hebei Higher Schools (No. Z2010225) and open fund of Key laboratory (No. 3333112).
Supplementary Table 1—Primer sequences used in this study.
Supplementary Fig. 1—(a) Comparison of the nucleotide sequences of R. diobovatum MCCC 2A00023 ACS1 genomic DNA and cDNA. (b) Comparison of the nucleotide sequences of R. diobovatum MCCC 2A00023 ACS2 genomic DNA and cDNA.
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