An effective technique for enrichment and isolation of Candida cloacae mutants defective in alkane catabolism

Techniques are described which allow mutated populations of Candida cloacae to be enriched efficiently (up to 167-fold in one round of enrichment) for mutants deficient in the alkane degradation pathway (Alk-). Such mutants, as well as being of scientific importance in studies of the degradation pathway, are also of commercial interest because several of the degradative intermediates are of value to the chemical industry. The Alk- mutants were readily isolated by their inability to grow on agar plates supplied with hexadecane as sole carbon source. A total of 288 Alk- mutants were isolated from, effectively, 4 x 10(6) mutagen-treated cells. They were further characterized by replica-plating using palmitic acid (PA) or acetate (Ac) as sole carbon source. Preliminary screening studies showed that of the 84 Alk- PA- Ac+ mutants, most could accumulate dicarboxylic acids from hexadecane and palmitic acid and at least one mutant also produced 3-hydroxyhexadecanedioic acid. Of the 80 mutants characterized as Alk- PA+, 16 produced small amounts of hexadecanol.