Effect of NH4+ and glycerol on cytidine 5′-diphosphocholine synthesis in Saccharomyces cerevisiae

Tang, Jiapeng; Yao, Yuelan; Ying, Hanjie; Xiong, Jing; Zhang, Lei; Li, Zhenjiang; Bai, Jianxin; Zhang, Yeyong; Ouyang, Pingkai

doi:10.1016/j.biortech.2009.04.045

Cited by 16 publications

(7 citation statements)

References 24 publications

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“…The highest molar yield of CDP‐choline to CMP ever reported by the permeabilized S. cerevisiae was 85.6% (Tang et al, 2010, 2009) with a CDP‐choline production per unit wet biomass of 0.054 g/g WW (Tang et al, 2010). For P. pastoris , the wet weight of biomass is about 3.0 times the DW (data not shown); therefore, the CDP‐choline production per unit wet biomass can be calculated as 0.19 g/g WW.…”

Section: Resultsmentioning

confidence: 99%

“…Currently, CDP‐choline is produced using CMP (or its precursor) and choline phosphate (or its precursor) as substrates. As these substrates penetrate into intact cells in an inefficient manner (Kimura, Morita, & Tochikura, 1971), the permeabilized cells of Escherichia coli (Fujio & Maruyama, 1997; Fujio, Teshiba, & Maruyama, 1997; Lee et al, 2009; Liu et al, 2017) or Saccharomyces cerevisiae (Kariya, Kimuta & Tochikura, 1976; Tang et al, 2010, 2009) are usually used as the source of catalytic enzymes.…”

Section: Introductionmentioning

confidence: 99%

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Engineering substrate and energy metabolism for living cell production of cytidine‐5′‐diphosphocholine

Ren

Liu

et al. 2020

Biotech & Bioengineering

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Cytidine‐5′‐diphosphocholine (CDP‐choline) is a widely used neuroprotective drug for multiple indications. In industry, CDP‐choline is synthesized by a two‐step cell culture/permeabilized cell biotransformation method because substrates often do not enter cells in an efficient manner. This study develops a novel one‐step living cell fermentation method for CDP‐choline production. For this purpose, the feasibility of Pichia pastoris as a chassis was demonstrated by substrate feeding and CDP‐choline production. Overexpression of choline phosphate cytidylyltransferase and choline kinase enhanced the choline transformation pathway and improved the biosynthesis of CDP‐choline. Furthermore, co‐overexpression of ScHnm1, which is a heterologous choline transporter, highly improved the utilization of choline substrates, despite its easy degradation in cells. This strategy increased CDP‐choline titer by 55‐folds comparing with the wild‐type (WT). Overexpression of cytidine‐5′‐monophosphate (CMP) kinase and CDP kinase in the CMP transformation pathway showed no positive effects. An increase in the ATP production by citrate stimulation or metabolic pathway modification further improved CDP‐choline biosynthesis by 120%. Finally, the orthogonal optimization of key substrates and pH was carried out, and the resulting CDP‐choline titer (6.0 g/L) at optimum conditions increased 88 times the original titer in the WT. This study provides a new paradigm for CDP‐choline bioproduction by living cells.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Engineering substrate and energy metabolism for living cell production of cytidine‐5′‐diphosphocholine

Ren

Liu

et al. 2020

Biotech & Bioengineering

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“…The molar yield to CMP and choline chloride were 83.8 and 63.7%, respectively, whereas the highest CDP-choline titer ever reported by the CGS process was 26.9 mM, with a molar yield of 82.3%, at a rate of 1.1 mM L −1 h −1 . 12 Although improvement was achieved on the final yield of CDP-choline, the starting material, CMP, is still expen-sive for mass production. For this reason, we tried to apply more economically efficient material to produce CDP-choline.…”

Section: Introductionmentioning

confidence: 99%

Design and optimizing a new CDP‐choline in vitro multienzyme producing process starts from d‐ribose

Zheng

Liu

et al. 2021

Biotech and App Biochem

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This work designs an in vitro multienzyme system to produce CDP-choline from d-ribose and develop an optimization procedure for one-pot multienzyme catalytic system. The entire process integrated 10 enzymes, and an efficient acetate kinase/acetyl phosphate-based ATP regeneration module was applied. Then, some optimizations to this system were made including selecting optimum enzyme building blocks and improving expression parameters. The process improved the final yield of CDP-choline from 0.2 to 6 g/L (CDP-choline titer 12.2 mM). This new one-pot CDP-choline producing system has a potential for industrial use, and the optimization procedure shed light on improving other one-pot multienzyme system for industrial production of energy rich compounds.

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“…Industrial CDP‐choline production was once through chemosynthesis, yet it was not suitable for large‐batch production because of toxic reagent and low conversion rate of substrates . Since that, biocatalysis processes such as the orotic acid‐glucose‐ C. ammoniagenes ‐ EscherichiaEscherichia coli (OGCE) process and the CMP‐glucose‐ Saccharomyces cerevisiae (CGS) became an alternative approach . However, these whole‐cell‐based methods are not efficient and are characterized by low CDP‐choline titers and productivity.…”

Section: Introductionmentioning

confidence: 99%

Rational Design of an Efficient Halotolerant Enzymatic System for In Vitro One‐Pot Synthesis of Cytidine Diphosphate Choline

Wang

Zheng

Cao

et al. 2018

Biotechnology Journal

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Salt accumulation often impedes cytidine diphosphate choline (CDP-choline) in vitro biosynthetic process. In this work a halotolerant in vitro enzymatic system is developed to solve this problem. It applies a halotolerant choline-phosphate cytidylyltransferase (CCT) obtained from rational design instructed by a unique strategy, which refers to one of the features of naturally occurring halophilic enzymes. By increasing acidic residues on protein surface where is most variable with respect to amino acid in the sequence alignment with other CCT, the mutants are obtained. The mutants represent higher catalytic activities and IC50 values (inhibit activity by 50%) at high-salt concentrations. Furthermore, when the halotolerant CCT is applied to in vitro one-pot biosynthesis of CDP-choline, the maximum titer and productivity are 161 ± 3.5 mM and 6.2 ± 0.1 mM L h , respectively. When acetate concentration increases, it still keeps relatively high reaction rate and is 2.2-fold higher than process using wild-type CCT (3.87 mM L h comparing with 1.74 mM L h ). This halotolerant system has great potential for industrial use, and the rational design concept can be applied to modify other enzymes, addressing the salt accumulation problem in in vitro systems, and gives insight into resolving by-product inhibition during reaction.

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Effect of NH4+ and glycerol on cytidine 5′-diphosphocholine synthesis in Saccharomyces cerevisiae

Cited by 16 publications

References 24 publications

Engineering substrate and energy metabolism for living cell production of cytidine‐5′‐diphosphocholine

Engineering substrate and energy metabolism for living cell production of cytidine‐5′‐diphosphocholine

Design and optimizing a new CDP‐choline in vitro multienzyme producing process starts from d‐ribose

Rational Design of an Efficient Halotolerant Enzymatic System for In Vitro One‐Pot Synthesis of Cytidine Diphosphate Choline

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