Objectives Two genes encoding two acetyl-CoA synthetase (ACS) isoenzymes have been identified in the marine yeast Rhodosporidium diobovatum MCCC 2A00023. Results 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. Conclusions 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.