Improving the Biosynthesis of β-Carotene in <i>Yarrowia lipolytica</i> by Introducing an Artificial Isopentenol Utilization Pathway

doi:10.13523/j.cb.2012054

China Biotechnology

2021, Vol. 41

Issue (4): 37-46 DOI: 10.13523/j.cb.2012054

Improving the Biosynthesis of β-Carotene in Yarrowia lipolytica by Introducing an Artificial Isopentenol Utilization Pathway

ZHU Hang-zhi¹,JIANG Shan¹,CHEN Dan²,LIU Peng-yang¹,WAN Xia^1,^3,^4,^**(

)

1 Oil Crops Research Institute of Chinese Academy of Agricultural Sciences, Wuhan 430062, China
2 School of Chemical Engineering and Pharmacy, Wuhan Institute of Technology, Wuhan 430205, China
3 Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture, Wuhan 430062, China
4 National and Local Joint Engineering Laboratory of Oil Lipid Processing Technology, Wuhan 430062, China

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Abstract

Objective: The natural synthesis pathway of isopentenyl pyrophosphate, the precursor of isoprene compounds in microorganisms, is subject to strict metabolic regulation, which limits the efficient biosynthesis of isoprene compounds, and the use of the artificial isopentenol utilization pathway (IUP) is independent of the biological endogenous metabolic pathways. By introducing IUP into microorganisms, a large amount of isopentenol pyrophosphate can be synthesized, thereby promoting the large amount of synthesis of isoprene compounds. Methods: Introducing the artificial IUP into the oleaginous yeast Yarrowia lipolytica to strengthen the isopentenyl pyrophosphate biosynthesis and promote the efficient accumulation of β-carotene. Results: The tertiary structures of two key proteins involved in IUP, ScCK (Choline kinase from Saccharomyces cerevisiae) and AtIPK (isopentenyl phosphate kinase from Arabidopsis thaliana), are predicted to be acidic hydrophilic proteins without transmembrane domain and signal peptide. Both proteins have loose and unstable structural features and contain a variety of post-translational features. Modification sites and multiple interacting proteins are involved in phosphorylation. The β-carotene synthesis pathway was constructed by introducing carRP and carB, and strengthening the expression of thmgR and ggs1 in Yarrowia lipolytica by using homologous recombination technique, and 2.68 mg/L β-carotene was accumulated in the engineered strain. The ura on the genome were removed by the Cre-loxP system, and then IUP was integrated into the genome of this engineered strain. The yield of β-carotene was achieved at the levels of 410.2 mg/L in the engineered strain, which was nearly 200 folds greater than that from the original engineered strain. The fermentation conditions were as follows: cells were incubated for 96 h in the medium containing 20 mmol/L isoprenol and the ratio of carbon to nitrogen was set as 4/3 in the medium. Conclusion: IUP can promote the efficient accumulation of β-carotene in Y. lipolytica. This study provides a new strategy for the efficient biosynthesis of β-carotene and other isoprene compounds by using IUP.

Key words： Isopentenol utilization pathway Isopentenyl pyrophosphate β-Carotene Yarrowia lipolytica

Received: 25 December 2020 Published: 30 April 2021

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Corresponding Authors: Xia WAN E-mail: wanxia@oilcrops.cn

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	Hang-zhi ZHU
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	Peng-yang LIU
	Xia WAN

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ZHU Hang-zhi,JIANG Shan,CHEN Dan,LIU Peng-yang,WAN Xia. Improving the Biosynthesis of β-Carotene in Yarrowia lipolytica by Introducing an Artificial Isopentenol Utilization Pathway. China Biotechnology, 2021, 41(4): 37-46.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2012054 OR https://manu60.magtech.com.cn/biotech/Y2021/V41/I4/37

Table 1 Plasmids and strains used in this study

Fig.1 The tertiary of homologous modeling and protein interaction network of ScCK (a) and AtIPK (b)

Fig.2 IUP enhances the synthesis of BC in Y. lipolytica (a)Isopentenol Utilization Pathway, IDI, isopentenyl diphosphate isomerase, carRP, phytoene synthase/lycopene cyclase, carB, phytoene dehydrogenase (b)Effects of idi, ScCK, AtIPK overexpression and coexpression in YlBC_p on BC production (c)Effect of IUP in YlBC on BC production (d)qRT-PCR reveals the expression levels of transcription of target differential expressed proteins of IUP

Fig.3 The influences of culture condition on BC production of YlBC-IUP Effects of isoprenol concentration (a), carbon-nitrogen ratio (b), and culture time (c) on BC production, respectively


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