Phospholipase D engineering for improving the biocatalytic synthesis of phosphatidylserine

Hou, Hai Juan; Gong, Jin Song; Dong, Yu; Qin, Jiufu; Li, Heng; Li, Hui; Lu, Zhen Ming; Zhang, Xiao Mei; Xu, Zheng; Shi, Jin Song

doi:10.1007/s00449-019-02116-7

Cited by 17 publications

(16 citation statements)

References 34 publications

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“…Owing to the low activity and complex culture conditions of PLD derived from Streptomyces, heterologous PLD expression has been widely explored. 4 The activity of PLD expressed in Bacillus subtilis, 28 Corynebacterium glutamate, 29 and E. coli has been reported as 1.5−1.9 U/mL. In E. coli, most of the PLD was expressed in an insoluble form.…”

Section: ■ Discussionmentioning

confidence: 99%

Rational Design of Phospholipase D to Improve the Transphosphatidylation Activity for Phosphatidylserine Synthesis

Liu²,

Song

et al. 2022

J. Agric. Food Chem.

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Phosphatidylserine (PS) has been widely used in the fields of food and medicine, among others, owing to its unique chemical structure and health benefits. However, the phospholipase D (PLD)-mediated enzymatic production of PS remains a challenge due to the low transphosphatidylation activity of PLD. Therefore, in the present study, we designed a maltose-binding protein (MBP) tag and a PLD co-expression method to achieve the expression of soluble PLD in Escherichia coli. A “reconstruct substrate pocket” strategy was then proposed based on the catalytic mechanism and molecular dynamics simulation, expanding the substrate pocket and manipulating the coordination of l-Ser within the active site. The best mutant (Sr MBPPLDMu6) exhibited a 2.04-fold higher transphosphatidylation/hydrolysis ratio than the wild-type Furthermore, under optimal conditions, Mu6 produced 58.6 g/L PS with 77.2% conversion, within 12 h on a 3 L scale, which demonstrates the potential of the proposed method for industrial application.

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Section: ■ Discussionmentioning

confidence: 99%

Rational Design of Phospholipase D to Improve the Transphosphatidylation Activity for Phosphatidylserine Synthesis

Liu²,

Song

et al. 2022

J. Agric. Food Chem.

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“…Promoter engineering has been applied to gene expression and regulation of various engineered hosts, such as E. coli, , Corynebacterium glutamicum, B. subtilis , − and P. pastoris , etc.…”

Section: Versatile Strategies To Improve the Integrated Expression Of...mentioning

confidence: 99%

Improving the Intensity of Integrated Expression for Microbial Production

et al. 2021

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Chromosomal integration of exogenous genes is preferred for industrially related fermentation, as plasmid-mediated fermentation leads to extra metabolic burden and genetic instability. Moreover, with the development and advancement of genome engineering and gene editing technologies, inserting genes into chromosomes has become more convenient; integration expression is extensively utilized in microorganisms for industrial bioproduction and expected to become the trend of recombinant protein expression. However, in actual research and application, it is important to enhance the expression of heterologous genes at the host genome level. Herein, we summarized the basic principles and characteristics of genomic integration; furthermore, we highlighted strategies to improve the expression of chromosomal integration of genes and pathways in host strains from three aspects, including chassis cell optimization, regulation of expression elements in gene expression cassettes, optimization of gene dose level and integration sites on chromosomes. Moreover, we reviewed and summarized the relevant studies on the application of integrated expression in the exploration of gene function and the various types of industrial microorganism production. Consequently, this review would serve as a reference for the better application of integrated expression.

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“…PLD belongs to the PLD superfamily. Members in the PLD superfamily contain one or two highly conserved HxKxxxxDxxxxxxGG/S (HKD) motifs, which are assumed to act as active centers [ 17 ]. Until now, there have been few reports on the crystal structure of PLDs.…”

Section: Introductionmentioning

confidence: 99%

A New Phospholipase D from Moritella sp. JT01: Biochemical Characterization, Crystallization and Application in the Synthesis of Phosphatidic Acid

Wang

Mao

Deng

et al. 2022

IJMS

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A new phospholipase D from marine Moritella sp. JT01 (MsPLD) was recombinantly expressed and biochemically characterized. The optimal reaction temperature and pH of MsPLD were determined to be 35 °C and 8.0. MsPLD was stable at a temperature lower than 35 °C, and the t1/2 at 4 °C was 41 days. The crystal structure of apo-MsPLD was resolved and the functions of a unique extra loop segment on the enzyme activity were characterized. The results indicated that a direct deletion or fastening of the extra loop segment by introducing disulfide bonds both resulted in a complete loss of its activity. The results of the maximum insertion pressure indicated that the deletion of the extra loop segment significantly decreased MsPLD’s interfacial binding properties to phospholipid monolayers. Finally, MsPLD was applied to the synthesis of phosphatidic acid by using a biphasic reaction system. Under optimal reaction conditions, the conversion rate of phosphatidic acid reached 86%. The present research provides a foundation for revealing the structural–functional relationship of this enzyme.

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Phospholipase D engineering for improving the biocatalytic synthesis of phosphatidylserine

Cited by 17 publications

References 34 publications

Rational Design of Phospholipase D to Improve the Transphosphatidylation Activity for Phosphatidylserine Synthesis

Rational Design of Phospholipase D to Improve the Transphosphatidylation Activity for Phosphatidylserine Synthesis

Improving the Intensity of Integrated Expression for Microbial Production

A New Phospholipase D from Moritella sp. JT01: Biochemical Characterization, Crystallization and Application in the Synthesis of Phosphatidic Acid

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