Shuliang Gao1, Yangyang Tong2, Li Zhu3, Mei Ge3, Yu Jiang4,5, Daijie Chen6,7, Sheng Yang4,5,8. 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. 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. 5. Shanghai Research and Development Center of Industrial Biotechnology, 528 Ruiqing Road, Shanghai, 201201, China. 6. School of Biotechnology, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, China. hccb001@163.com. 7. Shanghai Institute of Pharmaceutical Industry, 1320 West Beijing Road, Shanghai, 200040, China. hccb001@163.com. 8. Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), 200 North Zhongshan Road, Nanjing, 211816, China.
Abstract
OBJECTIVES: To obtain functional expression of a heterologous multifunctional carotene synthase containing phytoene synthase, phytoene dehydrogenase, and lycopene β-cyclase activities encoded by carS from Schizochytrium sp. in order to allow Yarrowia lipolytica to produce β-carotene. RESULTS: To increase the integration efficiency of a 3.8 kb carS under the control of P GPD promoter with a 2 kb selection marker, ura3, along with a geranylgeranyl diphosphate synthase (GGS1) expression cassette (~10 kb in total), was inserted into the Y. lipolytica chromosome, and the DNA assembler method was combined with double chromosomal deletions of ku70 and ku80. This method resulted in a 13.4-fold increase in integration efficiency compared with the original method, reaching 63% (10/16). The resulting recombinant Y. lipolytica produced 0.41 mg β-carotene per g dry cell weight, while the wild type did not produce any indicating the functionality of the multifunctional carotene synthase in Y. lipolytica. CONCLUSION: Expression of GGS1 and a multifunctional carotene synthase from Schizochytrium sp. in Y. lipolytica led to β-carotene production. DNA assembler efficiency was greatly increased by the deletion of ku70 and ku80, which resulted in decreased in vivo nonhomologous end-joining (NHEJ) in Y. lipolytica.
OBJECTIVES: To obtain functional expression of a heterologous multifunctional carotene synthase containing phytoene synthase, phytoene dehydrogenase, and lycopene β-cyclase activities encoded by carS from Schizochytrium sp. in order to allow Yarrowia lipolytica to produce β-carotene. RESULTS: To increase the integration efficiency of a 3.8 kb carS under the control of P GPD promoter with a 2 kb selection marker, ura3, along with a geranylgeranyl diphosphate synthase (GGS1) expression cassette (~10 kb in total), was inserted into the Y. lipolytica chromosome, and the DNA assembler method was combined with double chromosomal deletions of ku70 and ku80. This method resulted in a 13.4-fold increase in integration efficiency compared with the original method, reaching 63% (10/16). The resulting recombinant Y. lipolytica produced 0.41 mg β-carotene per g dry cell weight, while the wild type did not produce any indicating the functionality of the multifunctional carotene synthase in Y. lipolytica. CONCLUSION: Expression of GGS1 and a multifunctional carotene synthase from Schizochytrium sp. in Y. lipolytica led to β-carotene production. DNA assembler efficiency was greatly increased by the deletion of ku70 and ku80, which resulted in decreased in vivo nonhomologous end-joining (NHEJ) in Y. lipolytica.
Authors: Hery Rabeharindranto; Sara Castaño-Cerezo; Thomas Lautier; Luis F Garcia-Alles; Christian Treitz; Andreas Tholey; Gilles Truan Journal: Metab Eng Commun Date: 2019-01-18