Engineered <i>Rhodosporidium toruloides</i> Strains Capable of Biosynthesizing a Non-natural Cofactor

doi:10.13523/j.cb.2201011

China Biotechnology

2022, Vol. 42

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

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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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

Received: 10 January 2022 Published: 17 June 2022

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Corresponding Authors: Zong-bao ZHAO E-mail: zhaozb@dicp.ac.cn

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	Shi-yu LIANG
	Li WAN
	Xiao-jia GUO
	Xue-ying WANG
	Li-ting LV
	Ying-han HU
	Zong-bao ZHAO

Cite this article:

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.

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

Fig.1 The reaction catalyzed by NCD synthetase CTP: Cytidine triphosphate; NMN: Nicotinamide mononucleotide; NCD: Nicotinamide cytosine dinucleotide; PPi: Pyrophosphoric acid

Table 1 Strains and plasmids used in this study

Table 2 Primers used in this study

Fig.2 PCR verification of R. toruloides transformants (a) Schematic of NCDS construct (b) Pretreatment result, GAPDH amplified by the primers GAPDH-F and GAPDH-R (c) PCR result of NCDS amplified by the primers NCDS-NF and NCDS-NR Lane M: DNA marker; Lane 1-5: Represent 5 individual transformants, NP11-NCDS1-5; Lane -: R. toruloides NP11; Lane +: The plasmid PZPK-P_PGK-NCDS-P2A-HYG-T_hsp; Lane H₂O: Distilled water

Fig.3 Detection of NcdS proteins in R. toruloides transformants by Western blot Lane M: Protein marker; Lane 1-5: Total protein sample from 5 individual transformants, NP11-NCDS1-5; Lane -: Total protein sample from R. toruloides NP11; Molecular weight: NcdS; 25.3 kDa

Fig.4 Principles and results of crude enzyme activity determination (a) Principle of the coupled colorimetric assay and activity determination (b) Crude enzyme activity of the transformants. Strain 1-5: Represent 5 individual transformants, NP11-NcdS1-5. Error bars represent means ± standard deviation. Significant differences of engineered strains and NP11 were calculated using the t test (^* P<0.05; ^** P<0.01; ^*** P<0.001)

Fig.5 Crude enzyme catalyzes the synthesis of NCD in vitro (a) HPLC profiles of NCD and crude enzyme catalytic system (b) The MS spectra of crude enzyme catalytic system. MS detection mode: Negative ion mode. NCD: Experimental monoisotopic mass, 638.091 0; Calculated molecular mass, 638.090 6

Fig.6 Determination of intracellular NCD in engineered strains (a) NCD concentrations in engineered strains. Strain 1-5: Represent 5 individual transformants, NP11-NcdS1-5. Error bars represent means ± standard deviation (b) The MS spectra of intracellular NCD(H) in NP11-NCDS1 and NP11. MS detection mode: Negative ion mode. Experimental monoisotopic mass: NCD, 638.088 6; NCDH, 640.103 8. Calculated molecular mass: NCD, 638.090 6; NCDH, 640.106 3

Table 3 Lipid production results by R. toruloides strains


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