Mutants and intersexual heterokaryons of Blakeslea trispora for production of beta-carotene and lycopene

Abstract

The industrial production of beta-carotene with the zygomycete Blakeslea trispora involves the joint cultivation of mycelia of opposite sex in the presence of beta-ionone and other chemical activators. We have obtained improved strains by mutation and heterokaryosis. We chose wild strains on the basis of their growth and carotene content in single and mated cultures. Following exposure of their spores to N-methyl-N'-nitro-N-nitrosoguanidine, we obtained high-carotene mutants, which were more productive than their parents but similar to them in having beta-carotene as the main product. Further increases in carotene content were obtained after a new round of mutagenesis in one of the mutants. The production was shifted to lycopene in cultures incubated in the presence of nicotine and in lycopene-rich mutants derived from the wild strains. The highest production levels were achieved in intersexual heterokaryons, which contained mutant nuclei of opposite sex. These contained up to 39 mg of beta-carotene or 15 mg of lycopene per g (dry mass) under standard laboratory conditions in which the original wild strains contained about 0.3 mg of beta-carotene per g (dry mass). Beta-ionone did not increase the carotene content of these strains. Not all wild strains lent themselves to these improvements, either because they produced few mutants or because they did not increase their carotene production in mated cultures.

FIG. 1.

Cumulative distribution of the number of nuclei per spore in the wild-type strains F921 (○), F986 (•), and F989 (▵) as determined by fluorescence microscopy of DAPI-stained samples.

FIG. 2.

Colored carotenes in the wild-type strains, mutant strains, and intersexual heterokaryons. High-pressure liquid chromatography analyses of mycelia grown for 5 days on minimal agar were performed. The three homogeneous coordinates represent the proportions of lycopene, γ-carotene, and β-carotene in the sum of the three compounds. Values are the means of two to five determinations. The mean relative error in 45 determinations was 2.2%. (Left) Wild and mutant strains as indicated. (Right) Intersexual heterokaryons.

FIG. 3.

Predicted genotype distributions in the spores produced by heterokaryotic mycelia of the wild-type strains F921, F986, and F989. The two kinds of nuclei in the heterokaryons are called A and B, and the spores can be heterokaryons, homokaryons with A nuclei (○), or homokaryons with B nuclei (•).