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Aβ-carotene ketolase Gene NfcrtO from Subaerial Cyanobacteria Confers Drought Tolerance in Rice

Rice science 2024年第1期31卷 62-76页

作者： GAO Ningning YE Shuifeng ZHANG Yu ZHOU Liguo MA Xiaosong YU Hanxi LI Tianfei HAN Jing LIU Zaochang LUO Lijun College of Plant Science and Technology Huazhong Agricultural UniversityWuhan 430070China Shanghai Agrobiological Gene Center Shanghai 201106China College of Life Sciences Shangrao Normal UniversityShangrao 334001China Institute for Agri-food Standards and Testing Technology Shanghai Academy of Agricultural SciencesShanghai 201403China

Nostoc flagelliforme is a terrestrial cyanobacterium that can resist many types of stressors,including drought,ultraviolet radiation,and extreme *** this study,we identified the drought tolerance gene NfcrtO,which encodes aβ-carotene ketolase,through screening the transcriptome of *** under water loss *** expression of NfcrtO under 0.6 mol/L sorbitol or under 0.3 mol/L NaCl stress significantly increased the growth rate of Escherichia *** NfcrtO was heterologously expressed in rice,the seedling height and root length of NfcrtO-overexpressing rice plants were significantly higher than those of the wild type(WT)plants grown on½Murashige and Skoog solid medium with 120 mmol/L mannitol at the seedling *** analysis revealed that NfcrtO was involved in osmotic stress,antioxidant,and other stress-related ***,the survival rate of the NfcrtO-overexpression lines was significantly higher than that of the WT line under both hydroponic stress(24%PEG and 100 mmol/L H_(2)O_(2))and soil drought treatment at the seedling *** traits,including the activity levels of superoxide dismutase,peroxidase,catalase,total antioxidant capacity,and the contents of proline,trehalose,and soluble sugar,were significantly improved in the NfcrtO-overexpression lines relative to those in the WT line under 20%PEG ***,when water was withheld at the booting stage,the grain yield per plant of NfcrtO-overexpression lines was significantly higher than that of the WT *** two-hybrid analysis identified interactions between NfcrtO and Dna J protein,E3 ubiquitin-protein ligase,and pyrophosphate-energized vacuolar membrane proton ***,heterologous expression of NfcrtO in rice could significantly improve the tolerance of rice to osmotic stress,potentially facilitating the development of new rice varieties.

关键词： antioxidant enzyme β-carotene ketolase drought resistance Nostoc flagelliforme osmotic stress rice transcriptome analysis

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Engineering CrtW and CrtZ for improving biosynthesis of astaxanthin in Escherichia coli

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Chinese Journal of Natural Medicines 2020年第9期18卷 666-676页

作者： LI Di LI Yang XU Jiao-Yang LI Qing-Yan TANG Jin-Lei JIA Shi-Ru BI Chang-Hao DAI Zhu-Bo ZHU Xin-Na ZHANG Xue-Li College of Biotechnology Tianjin University of Science and TechnologyTianjin 310018China Tianjin Institute of Industrial Biotechnology Chinese of Academy of SciencesTianjin 300308China Key Laboratory of Systems Microbial Biotechnology.Chinese Academy of Sciences Tianjin 300308China University of Chinese Academy of Sciences.Beijing 100071 China National Technology Innovation Center of Synthetic Biology Tianjin 300308China

This study engineered β-carotene ketolase CrtW and β-carotene hydroxylase CrtZ to improve biosynthesis of astaxanthin in Escherichia coli. Firstly, crtW was randomly mutated to increase CrtW activities on conversion from β-carotene to astaxanthin. A crtW* mutant with A6 T, T105 A and L239 M mutations has improved 5.35-fold astaxanthin production compared with the wild-type control. Secondly, the expression levels of crtW* and crtZ on chromosomal were balanced by simultaneous modulation RBS regions of their genes using RBS library. The strain RBS54 selected from RBS library, directed the pathway exclusively towards the desired product astaxanthin as predominant carotenoid(99%). Lastly, the number of chromosomal copies of the balanced crtW*-crtZ cassette from RBS54 was increased using a Cre-loxP based technique, and a strain with 30 copies of the crtW*-crtZ cassette was selected. This final strain DL-A008 had a 9.8-fold increase of astaxanthin production compared with the wild-type control. Fed-batch fermentation showed that DL-A008 produced astaxanthin as predominant carotenoid(99%) with a specific titer of 0.88 g·L^(-1) without addition of inducer. In conclusion, through constructing crtW mutation, balancing the expression levels between crtW* and crtZ, and increasing the copy number of the balanced crtW*-crtZ cassette, the activities of β-carotene ketolase and β-carotene hydroxylase were improved for conversion of β-carotene to astaxanthin with higher efficiency. The series of conventional and novel metabolic engineering strategies were designed and applied to construct the astaxanthin hetero-producer strain of E. coli, possibly offering a general approach for the construction of stable hetero-producer strains for other natural products.

关键词： Astaxanthin RBS library Metabolic engineering β-carotene ketolase Cre-loxP Escherichia coli

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Expression of bkt and bch genes from Haematococcus pluvialis in transgenic Chlamydomonas

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Science China(Life Sciences) 2014年第10期57卷 1028-1033页

作者： ZHENG KaiJing WANG ChaoGang XIAO Ming CHEN Jun LI JianCheng HU ZhangLi Shenzhen Key Laboratory of Marine Bioresource & Eco-environmental Science Shenzhen Engineering Laboratory for Marine Algal Biotechnology College of Life Science Shenzhen University

β-carotene ketolase and β-carotene hydroxylase encoded by bkt and bch, respectively, are key enzymes required for astaxanthin biosynthesis in Haematococcu pluvialis 34-1n. Two expression vectors containing cDNA sequences of bkt and bch were constructed and co-transformed into cell-wall-deficient Chlamydomonas reinhardtii CC-849. Transgenic algae were screened on TAP agar plates containing 10 gg mL 1 Zeomycin. PCR-Southern analysis showed that bkt and bch were integrated into the genomes of C. reinhardtii. Transcripts of bkt and bch were further confirmed by RT-PCR-Southern analysis. Compared with the wild type, transgenic algae produced 29.04% and 30.27% more carotenoids and xanthophylls, respectively. Moreover, the transgenic algae could accumulate 34% more astaxanthin than wild type. These results indicate that foreign bkt and bch genes were successfully translated into β-carotene ketolase and β-carotene hydroxylase, which were responsible for catalyzing the biosynthesis of astaxanthin in transgenic algae.

关键词： Chlamydomonas β-carotene ketolase β-carotene hydroxylase carotenoids xanthophylls astaxanthin

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