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Microbial cell factories
Oct 10, 2019;18 (1): 169.

Construction of a novel MK-4 biosynthetic pathway in Pichia pastoris through heterologous expression of HsUBIAD1.

Xiaowen Sun, Hui Liu, Peng Wang, Li Wang, Wenfeng Ni, Qiang Yang, Han Wang, Hengfang Tang, Genhai Zhao, Zhiming Zheng
Author Information
  1. Xiaowen Sun: Key Laboratory of High Magnetic Field and Ion Beam Physical Biology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, 230031, Anhui, People's Republic of China.
  2. Hui Liu: Key Laboratory of High Magnetic Field and Ion Beam Physical Biology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, 230031, Anhui, People's Republic of China.
  3. Peng Wang: Key Laboratory of High Magnetic Field and Ion Beam Physical Biology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, 230031, Anhui, People's Republic of China.
  4. Li Wang: Key Laboratory of High Magnetic Field and Ion Beam Physical Biology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, 230031, Anhui, People's Republic of China.
  5. Wenfeng Ni: Key Laboratory of High Magnetic Field and Ion Beam Physical Biology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, 230031, Anhui, People's Republic of China.
  6. Qiang Yang: Key Laboratory of High Magnetic Field and Ion Beam Physical Biology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, 230031, Anhui, People's Republic of China.
  7. Han Wang: Key Laboratory of High Magnetic Field and Ion Beam Physical Biology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, 230031, Anhui, People's Republic of China.
  8. Hengfang Tang: Key Laboratory of High Magnetic Field and Ion Beam Physical Biology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, 230031, Anhui, People's Republic of China.
  9. Genhai Zhao: Key Laboratory of High Magnetic Field and Ion Beam Physical Biology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, 230031, Anhui, People's Republic of China. zhgh327@ipp.ac.cn.
  10. Zhiming Zheng: Key Laboratory of High Magnetic Field and Ion Beam Physical Biology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, 230031, Anhui, People's Republic of China. zhengzhiming2014@163.com. ORCID
PMID: 31601211 DOI: 10.1186/s12934-019-1215-9

Abstract

BACKGROUND: With a variety of physiological and pharmacological functions, menaquinone is an essential prenylated product that can be endogenously converted from phylloquinone (VK) or menadione (VK) via the expression of Homo sapiens UBIAD1 (HsUBIAD1). The methylotrophic yeast, Pichia pastoris, is an attractive expression system that has been successfully applied to the efficient expression of heterologous proteins. However, the menaquinone biosynthetic pathway has not been discovered in P. pastoris.
RESULTS: Firstly, we constructed a novel synthetic pathway in P. pastoris for the production of menaquinone-4 (MK-4) via heterologous expression of HsUBIAD1. Then, the glyceraldehyde-3-phosphate dehydrogenase constitutive promoter (P) appeared to be mostsuitable for the expression of HsUBIAD1 for various reasons. By optimizing the expression conditions of HsUBIAD1, its yield increased by 4.37 times after incubation at pH 7.0 and 24 °C for 36 h, when compared with that under the initial conditions. We found HsUBIAD1 expressed in recombinant GGU-23 has the ability to catalyze the biosynthesis of MK-4 when using VK and VK as the isopentenyl acceptor. In addition, we constructed a ribosomal DNA (rDNA)-mediated multi-copy expression vector for the fusion expression of SaGGPPS and PpIDI, and the recombinant GGU-GrIG afforded higher MK-4 production, so that it was selected as the high-yield strain. Finally, the yield of MK-4 was maximized at 0.24 mg/g DCW by improving the GGPP supply when VK was the isopentenyl acceptor.
CONCLUSIONS: In this study, we constructed a novel synthetic pathway in P. pastoris for the biosynthesis of the high value-added prenylated product MK-4 through heterologous expression of HsUBIAD1 and strengthened accumulation of GGPP. This approach could be further developed and accomplished for the biosynthesis of other prenylated products, which has great significance for theoretical research and industrial application.

Keywords

Aromatic prenyltransferase Geranylgeranyl diphosphate synthase HsUBIAD1 Menaquinone Pichia pastoris

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Grants

  1. 1608085QC46/Natural Science Foundation of Anhui Province
  2. 1908085MB43/Natural Science Foundation of Anhui Province
  3. 1908085MB48/Natural Science Foundation of Anhui Province
  4. 17030801036/Major Projects of Science and Technology in Anhui Province
  5. 1804b06020342/Key research and development plan of Anhui Province

MeSH Term

Biosynthetic Pathways
Dimethylallyltranstransferase
Gene Expression Regulation, Fungal
Pichia
Recombinant Proteins
Vitamin K 2

Chemicals

Recombinant Proteins
Vitamin K 2
menatetrenone
Dimethylallyltranstransferase
UBIAD1 protein, human
Journal Article

Word Cloud

Created with Highcharts 10.0.0expressionHsUBIAD1pastorisMK-4VKheterologouspathwayPprenylatedPichiaconstructednovelbiosynthesismenaquinoneproductviabiosyntheticsyntheticproductionconditionsyield0recombinantisopentenylacceptorGGPPBACKGROUND:varietyphysiologicalpharmacologicalfunctionsessentialcanendogenouslyconvertedphylloquinonemenadioneHomosapiensUBIAD1methylotrophicyeastattractivesystemsuccessfullyappliedefficientproteinsHoweverdiscoveredRESULTS:Firstlymenaquinone-4glyceraldehyde-3-phosphatedehydrogenaseconstitutivepromoterappearedmostsuitablevariousreasonsoptimizingincreased437timesincubationpH724 °C36 hcomparedinitialfoundexpressedGGU-23abilitycatalyzeusingadditionribosomalDNArDNA-mediatedmulti-copyvectorfusionSaGGPPSPpIDIGGU-GrIGaffordedhigherselectedhigh-yieldstrainFinallymaximized24 mg/gDCWimprovingsupplyCONCLUSIONS:studyhighvalue-addedstrengthenedaccumulationapproachdevelopedaccomplishedproductsgreatsignificancetheoreticalresearchindustrialapplicationConstructionAromaticprenyltransferaseGeranylgeranyldiphosphatesynthaseMenaquinone

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