| Astaxanthin {dollar}(3,3spprime{dollar}-dihydroxy-{dollar}beta,beta,{dollar}-carotene-4,4{dollar}sp prime{dollar}-dione) is the principal carotenoid pigment of salmonids. Worldwide production of farm-raised salmon increased rapidly in the past decade, and more than 200,000 metric tons were raised in 1990. The yeast Phaffia rhodozyma has desirable properties as a biological source of pigment but its content of astaxanthin in wild strains is only 200-300 {dollar}mu{dollar}g/g yeast (0.02-0.03%). High-producers were obtained by strain improvement that produced 2000 to 3000 {dollar}mu{dollar}g carotenoid/g yeast. However, these were often unstable and further strain development was required. A 10,000-fold enrichment of carotenoid hyperproducing mutants (CHMs) was obtained by flow cytometry and cell sorting. 2-methyl-imidazole (MI) and triethylamine could be used for inducers or selectors of CHMs. A small-light colony on the plates containing MI (0.3g/l) produced more than 3500 {dollar}mu{dollar}g total carotenoid/g yeast ({dollar}>{dollar}0.30%) and 14 mg total carotenoid/l medium in shake flasks after 5 days growth. Measurement of carotenoid fluorescence in individual cells indicates that levels of 15,000 {dollar}mu{dollar}g/g can be obtained. Evidence is presented that P. rhodozyma contains 2 astaxanthin biosynthetic pathways: one involves di-cyclic intermediates (neurosporene {dollar}to{dollar} lycopene {dollar}to{dollar} {dollar}beta{dollar}-carotene {dollar}to{dollar} echinenone {dollar}to{dollar} 3-hydroxy-echinenone {dollar}to{dollar} phoenicoxanthin {dollar}to{dollar} trans-astaxanthin), and the other has mono-cyclic intermediates (neurosporene {dollar}to{dollar} {dollar}beta{dollar}-zeacarotene {dollar}to{dollar} torulene {dollar}to{dollar} 3-keto-torulene {dollar}to{dollar} 3-hydroxy-3{dollar}spprime{dollar},4{dollar}spprime{dollar}-didehydro-{dollar}beta,Psi{dollar}-carotene-4-one {dollar}to{dollar} 3,3{dollar}spprime{dollar}-dihydroxy-{dollar}beta,Psi{dollar}-carotene-4,4{dollar}spprime{dollar}-dione {dollar}to{dollar} cis- and trans-astaxanthin). The mono-cyclic pathway is activated in CHMs. Carotenoid distribution in P. rhodozyma was studied by laser confocal fluorescence microscopy. The carotenoids are located in lipid globules but not in endoplasmic reticulum or mitochondria. The physiology of astaxanthin biosynthesis was investigated. Antimycin + light increased carotenoid production up to two-fold in P. rhodozyma wild-type strain 67-385. Proline, aspartate, asparagine, glutamine and glutamate increased yeast growth but decreased carotenoid/g yeast. The supplementation ofthe 20 amino acids inhibited carotenoid accumulation during stationary phase. This study has demonstrated that carotenoid formation is a highly regulated process in P. rhodozyma and has provided methods of the isolation of carotenoid hyperproducing mutants of the yeast. |
