构建可合成非天然辅酶的圆红冬孢酵母工程菌<sup>*</sup>

doi:10.13523/j.cb.2201011

中国生物工程杂志

2022, Vol. 42

Issue (5): 58-68 DOI: 10.13523/j.cb.2201011

技术与方法

构建可合成非天然辅酶的圆红冬孢酵母工程菌^*

梁世玉^1,²,万里^1,²,郭潇佳¹,王雪颖¹,吕力婷¹,胡英菡^1,²,赵宗保^1,^**(

)

1 中国科学院大连化学物理研究所大连 116023
2 中国科学院大学北京 100049

Engineered Rhodosporidium toruloides Strains Capable of Biosynthesizing a Non-natural Cofactor

LIANG Shi-yu^1,²,WAN Li^1,²,GUO Xiao-jia¹,WANG Xue-ying¹,LV Li-ting¹,HU Ying-han^1,²,ZHAO Zong-bao^1,^**(

)

1 Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China
2 University of Chinese Academy of Sciences, Beijing 100049, China

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摘要：

烟酰胺腺嘌呤二核苷酸(nicotinamide adenine dinucleotide,NAD)及其还原态是生物体通用的氧化还原辅酶和重要小分子,参与胞内众多代谢反应,因此调控NAD水平不仅难以选择性作用于代谢途径,还常常产生意外的生物学效应。最近研究发现利用非天然辅酶烟酰胺胞嘧啶二核苷酸(nicotinamide cytosine dinucleotide,NCD),可构建正交的氧化还原催化体系,为调控胞内代谢提供了新机遇。为实现在产油酵母圆红冬孢酵母中建立NCD介导的氧化还原代谢,采用农杆菌介导转化方法,在基因组整合表达密码子优化的NCD合酶(NcdS)编码基因NCDS,获得系列有效表达NcdS的工程菌株。酶偶联法分析发现,工程菌细胞裂解液NcdS酶活达8.1×10^-3 U/OD_{600 nm}。通过高效液相色谱法(HPLC)和超高分辨率质谱检测,确定细胞裂解液可催化合成NCD。在培养基内补加5.0 mmol/L烟酰胺核糖后,工程菌胞内合成NCD达41.6 μmol/L。对工程菌进行发酵和油脂提取,发现胞内表达NCD合酶未导致细胞产油性能降低,后续可通过表达其他NCD偏好性酶,有望在圆红冬孢酵母中建立受NCD调控的油脂合成代谢体系。

关键词： 圆红冬孢酵母; 非天然辅酶; 烟酰胺胞嘧啶二核苷酸; 合成生物学; 氧化还原代谢

Abstract:

Nicotinamide adenine dinucleotide (NAD) and its reduced form are universal redox cofactors involved in many cellular reactions. Cellular NAD level disturbance routinely leads to unexpected biological effects, and thus it is difficult to regulate a redox pathway-of-interest by manipulating NAD level. Recently, a non-natural cofactor nicotinamide cytosine dinucleotide (NCD) was devised and orthogonal redox systems based on NCD were developed, providing a new strategy for regulating cellular metabolism. To achieve more efficient regulation of lipid metabolism of the oleaginous yeast Rhodosporidium toruloides, a codon-optimized gene NCDS encoding NCD synthetase (NcdS) was integrated into the genome of R. toruloides by an Agrobacterium-mediated transformation method. Engineered strains were found with proper expression of NcdS and enzyme activity of the cell lysates reached 8.1×10^-3 U/OD_{600 nm} based on a coupled colorimetric assay. Successful biosynthesis of NCD by the cell lysates was further verified by high-performance liquid chromatography and ultra-high-resolution mass spectrometry. Intracellular NCD levels up to 41.6 μmol/L were realized upon feeding 5.0 mmol/L of nicotinamide riboside to the media used for cell culture. Results also showed that the expression of NcdS had little detrimental effect on lipid production capacity. It is expected that lipid metabolism may be reconstructed and regulated by NCD upon further expression of other NCD-preferred enzymes in R. toruloides.

Key words: Rhodosporidium toruloides Non-natural cofactor Nicotinamide cytosine dinucleotide(NCD) Synthetic biology Redox metabolism

收稿日期: 2022-01-10 出版日期: 2022-06-17

ZTFLH:

Q819

基金资助: *国家重点研发计划(2021YFA0910600)

通讯作者: 赵宗保 E-mail: zhaozb@dicp.ac.cn

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引用本文:

梁世玉,万里,郭潇佳,王雪颖,吕力婷,胡英菡,赵宗保. 构建可合成非天然辅酶的圆红冬孢酵母工程菌^*[J]. 中国生物工程杂志, 2022, 42(5): 58-68.

LIANG Shi-yu,WAN Li,GUO Xiao-jia,WANG Xue-ying,LV Li-ting,HU Ying-han,ZHAO Zong-bao. Engineered Rhodosporidium toruloides Strains Capable of Biosynthesizing a Non-natural Cofactor. China Biotechnology, 2022, 42(5): 58-68.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2201011 或 https://manu60.magtech.com.cn/biotech/CN/Y2022/V42/I5/58

图1 NCD合成酶催化的反应

表1 本文所用菌株和质粒

表2 本文所用引物

图2 转化子菌落PCR鉴定

图3 工程菌NcdS蛋白表达验证

图4 粗酶活测定原理与结果

图5 粗酶液体外催化合成NCD

图6 工程菌胞内NCD测定

表3 菌株油脂生产数据

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