Presentation Information
[P02-295]Production of pure all-trans retinal from agricultural byproducts by metabolically engineered Corynebacterium glutamicum
○Shungo Yamada1, Wenhui Hao1, Yoko Hirono2, Kiyotaka Y Hara2, Yota Tsuge1 (1. Kanazawa University (Japan), 2. University of Shizuoka (Japan))
Keywords:
All-trans retinal,Corynebacterium glutamicum,Molasses,Two-phase cultivation
[Purpose] Retinoids, including retinal, retinol, and retinoic acid, comprise vitamin A and its biologically active metabolites, which are widely used in pharmaceutical, healthcare, and cosmetic applications. Among these, retinal has attracted increasing attention in skincare applications because of its favorable combination of high biological efficacy with low skin irritation, as well as its potent antibacterial activity. However, microbial production of retinal generally yields mixtures with other retinoids and require expensive nutrient media, vitamins, and refined sugars. Corynebacterium glutamicum, a natural producer of the C50 carotenoid decaprenoxanthin, has been used to produce other carotenoids such as lycopene and β-carotene. However, C. glutamicum has not yet been used to produce retinoids, which are synthesized from these carotenoids as precursors. In this study, we investigated the production of retinal from agricultural byproducts using metabolically engineered C. glutamicum.
[Methods] A lycopene producer, serving as a retinal precursor producer, was constructed by blocking the downstream pathway and enhancing carotenoid biosynthesis. By introducing the crtY gene encoding lycopene cyclase from Pantoea ananatis, lycopene was efficiently converted into β-carotene. The blh gene, encoding β-carotene 15,15′-dioxygenase, which cleaves β-carotene into two molecules of retinal, from uncultured marine bacterium 66A03 was introduced to construct a retinal producer. The strain was cultivated in shake flasks using a synthetic medium with sucrose as a carbon source. Since retinal is fat-soluble and easily oxidized, a two-phase cultivation system using n-dodecane and butylated hydroxytoluene was employed for in-situ extraction and stabilization. The optimal timing for n-dodecane addition for retinal production was investigated from 0 to 48 h, as well as the optimal temperature from 15 to 33 ℃.
[Results] The retinal concentration and retinal/β-carotene ratio were the highest when n-dodecane was added at 24 h. The optimal temperature for retinal production was identified as 21 ℃, yielding 32.4 mg/L retinal (calculated based on the volume of the aqueous phase), which was 2.6-fold higher than that at the optimal growth temperature of 30 ℃. Using molasse as the carbon and vitamin source, 30.7 mg/L retinal was produced. Scale-up cultivation in a 2.5-L jar fermenter produced 104.9 mg/L retinal (2,099 mg/L in n-dodecane phase) from molasses. Unlike the other studies on microbial production of retinal, no other retinoids including retinol and retinoic acid were formed. These results showed that pure all-trans retinal was produced from molasses using metabolically engineered C. glutamicum.
[Conclusion] This study demonstrates a cost-effective and sustainable bio-based strategy for producing pure retinal from renewable resources, paving the way for its industrial bio-based manufacture.
[Methods] A lycopene producer, serving as a retinal precursor producer, was constructed by blocking the downstream pathway and enhancing carotenoid biosynthesis. By introducing the crtY gene encoding lycopene cyclase from Pantoea ananatis, lycopene was efficiently converted into β-carotene. The blh gene, encoding β-carotene 15,15′-dioxygenase, which cleaves β-carotene into two molecules of retinal, from uncultured marine bacterium 66A03 was introduced to construct a retinal producer. The strain was cultivated in shake flasks using a synthetic medium with sucrose as a carbon source. Since retinal is fat-soluble and easily oxidized, a two-phase cultivation system using n-dodecane and butylated hydroxytoluene was employed for in-situ extraction and stabilization. The optimal timing for n-dodecane addition for retinal production was investigated from 0 to 48 h, as well as the optimal temperature from 15 to 33 ℃.
[Results] The retinal concentration and retinal/β-carotene ratio were the highest when n-dodecane was added at 24 h. The optimal temperature for retinal production was identified as 21 ℃, yielding 32.4 mg/L retinal (calculated based on the volume of the aqueous phase), which was 2.6-fold higher than that at the optimal growth temperature of 30 ℃. Using molasse as the carbon and vitamin source, 30.7 mg/L retinal was produced. Scale-up cultivation in a 2.5-L jar fermenter produced 104.9 mg/L retinal (2,099 mg/L in n-dodecane phase) from molasses. Unlike the other studies on microbial production of retinal, no other retinoids including retinol and retinoic acid were formed. These results showed that pure all-trans retinal was produced from molasses using metabolically engineered C. glutamicum.
[Conclusion] This study demonstrates a cost-effective and sustainable bio-based strategy for producing pure retinal from renewable resources, paving the way for its industrial bio-based manufacture.
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