Efficient production of retinol in Yarrowia lipolytica by increasing stability using antioxidant and detergent extraction |
| |
| Institution: | 1. Bio Research Institutes, CJ CheilJedang, Suwon, 16495, South Korea;2. School of Chemical and Biological Engineering, Institute of Chemical Processes, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul, 08826, South Korea;1. College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, 310014, China;2. MOE Key Lab of Bioinformatics, School of Life Sciences, Tsinghua-Peking Center for Life Sciences, Tsinghua University, Beijing, 100084, China;3. Center for Synthetic and Systems Biology, Tsinghua University, Beijing, 100084, China;4. MOE Key Laboratory for Industrial Biocatalysis, Dept Chemical Engineering, Tsinghua University, Beijing, 100084, China;5. Shandong Provincial Research Center for Bioinformatic Engineering and Technology, School of Life Sciences, Shandong University of Technology, Zibo, 255049, China;1. Department of Chemical and Biomolecular Engineering, The University of Tennessee, Knoxville, TN, USA;2. Center of Bioenergy Innovation, Oak Ridge National Laboratory, Oak Ridge, TN, USA;3. Chemical Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN, 37830, USA;4. Department of Biological Engineering, Konkuk University, Seoul, South Korea;1. School of Biotechnology, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China;2. College of Life and Environmental Sciences, Shanghai Normal University, 100 Guilin Rd, Shanghai 200234, China;3. Shanghai Laiyi Center for Biopharmaceutical R&D, 800 Dongchuan Road, Shanghai 200240, China;4. George Stevens Academy, 23 Union St, Blue Hill, ME 04614, USA;5. Shanghai Institute of Pharmaceutical Industry, 1320 West Beijing Road, Shanghai 200040, China;6. Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai 200032, China;7. Shanghai Research and Development Center of Industrial Biotechnology, 528 Ruiqing Road, Shanghai 201201, China;8. Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), 200 North Zhongshan Road, Nanjing 211816, China;1. Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China;2. National Technology Innovation Center of Synthetic Biology, Tianjin, 300308, China;3. Beijing Advanced Innovation Center for Soft Matter Science and Engineering, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing, 100029, China;4. Department of Biology and Biological Engineering, Chalmers University of Technology, SE 412 96, Gothenburg, Sweden |
| |
| Abstract: | The demand for bio-based retinol (vitamin A) is currently increasing, however its instability represents a major bottleneck in microbial production. Here, we developed an efficient method to selectively produce retinol in Yarrowia lipolytica. The β-carotene 15,15′-dioxygenase (BCO) cleaves β-carotene into retinal, which is reduced to retinol by retinol dehydrogenase (RDH). Therefore, to produce retinol, we first generated β-carotene-producing strain based on a high-lipid-producer via overexpressing genes including heterologous β-carotene biosynthetic genes, GGS1F43I mutant of endogenous geranylgeranyl pyrophosphate synthase isolated by directed evolution, and FAD1 encoding flavin adenine dinucleotide synthetase, while deleting several genes previously known to be beneficial for carotenoid production. To produce retinol, 11 copies of BCO gene from marine bacterium 66A03 (Mb.Blh) were integrated into the rDNA sites of the β-carotene overproducer. The resulting strain produced more retinol than retinal, suggesting strong endogenous promiscuous RDH activity in Y. lipolytica. The introduction of Mb.Blh led to a considerable reduction in β-carotene level, but less than 5% of the consumed β-carotene could be detected in the form of retinal or retinol, implying severe degradation of the produced retinoids. However, addition of the antioxidant butylated hydroxytoluene (BHT) led to a >20-fold increase in retinol production, suggesting oxidative damage is the main cause of intracellular retinol degradation. Overexpression of GSH2 encoding glutathione synthetase further improved retinol production. Raman imaging revealed co-localization of retinol with lipid droplets, and extraction of retinol using Tween 80 was effective in improving retinol production. By combining BHT treatment and extraction using Tween 80, the final strain CJ2104 produced 4.86 g/L retinol and 0.26 g/L retinal in fed-batch fermentation in a 5-L bioreactor, which is the highest retinol production titer ever reported. This study demonstrates that Y. lipolytica is a suitable host for the industrial production of bio-based retinol. |
| |
| Keywords: | Antioxidant β-Carotene Retinol Tween 80 Vitamin A |
| 本文献已被 ScienceDirect 等数据库收录！ |
|
