Yutaka Yano

Production of docosahexaenoic acid by marine bacteria isolated from deep sea fish.

Five bacterial strains isolated from the intestine of deep sea fish were shown to produce docosahexaenoic acid (22∶6n−3; DHA) at a level of 6.4 to 11.6% of total fatty acids when incubated in DHA-free medium. In all of the strains examined, other polyunsaturated fatty acids were barely detectable, except for eicosapentaenoic acid (20∶5n−3). A typical strain, such as T3615, produced DHA at a concentration of about 0.8 mg/L within six days of aerobic incubation at 5°C and under atmospheric pressure. The T3615 strain, belonging to the genusVibrio, is rod-shaped, Gram-negative, motile and facultatively anaerobic.

pfaB products determine the molecular species produced in bacterial polyunsaturated fatty acid biosynthesis

When pDHA4, a vector carrying all five pfaA-pfaE genes responsible for docosahexaenoic acid (DHA; 22:6) biosynthesis in Moritella marina MP-1, was coexpressed in Escherichia coli with the individual pfaA-pfaD genes for eicosapentaenoic acid (EPA; 20:5) biosynthesis from Shewanella pneumatophori SCRC-2738, both polyunsaturated fatty acids were synthesized only in the recombinant carrying pfaB for EPA synthesis. Escherichia coli coexpressing a deleted construct comprising pfaA, pfaC, pfaD and pfaE for EPA and pfaB for DHA produced EPA and DHA. Both EPA and DHA were detected in bacteria that inherently contained pfa genes for DHA. These results suggest that PfaB is the key enzyme determining the final product in EPA or DHA biosynthesis.

Possible Biosynthetic Pathways for all cis-3,6,9,12,15,19,22,25,28-Hentriacontanonaene in Bacteria

A very long chain polyunsaturated hydrocarbon, hentriacontanonaene (C31:9), was detected in an eicosapentaenoic acid (EPA)-producing marine bacterium, which was isolated from the mid-latitude seashore of Hokkaido, Japan, and was tentatively identified as mesophilic Shewanella sp. strain osh08 from 16S rRNA gene sequencing. The geometry and position of the double bonds in this compound were determined physicochemically to be all cis at positions 3, 6, 9, 12, 15, 19, 22, 25, and 28. Although C31:9 was detected in all of the seven EPA- or/and docosahexaenoic acid-producing bacteria tested, an EPA-deficient mutant (strain IK-1Δ8) of one of these bacteria had no C31:9. Strain IK-1Δ8 had defects in the pfaD gene, one of the five pfa genes responsible for the biosynthesis of EPA. Although Escherichiacoli DH5α does not produce EPA or DHA inherently, cells transformed with the pfa genes responsible for the biosynthesis of EPA and DHA produced EPA and DHA, respectively, but not C31:9. These results suggest that the Pfa protein complex is involved in the biosynthesis of C31:9 and that pfa genes must not be the only genes responsible for the formation of C31:9. In this report, we determined for the first time the molecular structure of the C31:9 and discuss the possible biosynthetic pathways of this compound.

Genes and Pathways Involved in Biosynthesis of Eicosapentaenoic and Docosahexaenoic Acids in Bacteria

Some eubacteria produce long chain polyunsaturated fatty acids (PUFAs) such as eicosapentaenoic acid (EPA) and/or docosahexaenoic acid (DHA) by the mechanism differing from the combination of elongation and oxygen-dependent desaturation of fatty acids. Metz et al. (2001) proposed a possible EPA biosynthetic pathway of bacteria, where EPA can be produced by polyketide synthase (PKS)-like enzyme encoded by the EPA gene cluster. Accordingly, a trans double bond at Δ2 of acyl intermediates is isomerized to cis configuration either at Δ3 or at Δ2, when it is not reduced to the C-C bond.