Effect of flexible linker length on the activity of fusion protein 4-coumaroyl-CoA ligase::stilbene synthase

Guo, Huili; Yang, Yuliang; Xue, Feiqun; Zhang, Hong; Huang, Tiran; Liu, Wenbin; Liu, Huan; Zhang, Fenqiang; Yang, Mo; Liu, Chunmei; Lu, Hongyu; Zhang, Yansheng; Ma, Lanqing

doi:10.1039/c6mb00563b

Cited by 39 publications

(28 citation statements)

References 60 publications

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“…Meanwhile, the result showed that if the distance of protein is too far, it may cause negative effect on titer. Too long linkers might make the physical distance between two enzyme active sites so far, which may result in insufficient interaction . Therefore, a suitable linker might create the appropriate distance between DFR and LAR, allowing them to achieve maximal catalytic activity and reducing the loss of intermediates .…”

Section: Discussionmentioning

confidence: 99%

Enhancing Flavan-3-ol Biosynthesis in Saccharomyces cerevisiae

Sun

et al. 2021

J. Agric. Food Chem.

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Flavan-3-ols are a group of flavonoids that exert beneficial effects. This study aimed to enhance key metabolic processes related to flavan-3-ols biosynthesis. The engineered Saccharomyces cerevisiae strain E32 that produces naringenin from glucose was further engineered for de novo production of two basic flavan-3-ols, afzelechin (AFZ) and catechin (CAT). Through introduction of flavonoid 3-hydroxylase, flavonoid 3′-hydroxylase, dihydroflavonol 4-reductase (DFR), and leucoanthocyanidin reductase (LAR), de novo production of AFZ and CAT can be achieved. The combination of FaDFR from Fragaria × ananassa and VvLAR from Vitis vinifera was optimal. (GGGGS)2 and (EAAAK)2 linkers between DFR and LAR proved optimal for the production of AFZ and CAT, respectively. Optimization of promoters and the enhanced supply of NADPH further increased the production. By combining the best engineering strategies, the optimum strains produced 500.5 mg/L AFZ and 321.3 mg/L CAT, respectively, after fermentation for 90 h in a 5 L bioreactor. The strategies presented could be applied for a more efficient production of flavan-3-ols by various microorganisms.

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Section: Discussionmentioning

confidence: 99%

Enhancing Flavan-3-ol Biosynthesis in Saccharomyces cerevisiae

Sun

et al. 2021

J. Agric. Food Chem.

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“…The peptide linker, located in the functional domain, can increase structural folding, stability, expression, and biological activity of fusion proteins. [25] The (Gly-Gly-Gly-Gly-Ser) n ([G 4 S] n ) linker (n = 1, 2, and 3) has been widely used for fusion proteins because it consists of small and simple amino acids without side chains and does not participate in the protein secondary structure. [26] It was also reported that the peptide linker could functionally link two different enzymes, β-glucanase and xylanase.…”

Section: Resultsmentioning

confidence: 99%

Robust Biocatalysts Displayed on Crystalline Protein‐Layered Cells for Efficient and Sustainable Hydration of Carbon Dioxide

Koo

Choi

Song

et al. 2021

Adv Funct Materials

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Mineral carbonation is the most effective carbon capture technique, but carbon dioxide (CO 2 ) conversion is limited by the slow hydration rate of CO 2 (<10 −1 s −1 ). A biological solution exists: carbonic anhydrase (CA) efficiently hydrates CO 2 at a turnover rate of ≈10 6 s −1 under ambient conditions, making it an extremely attractive candidate for industrial post-combustion CO 2 capture. However, high cost and poor long-term stability impose a technical barrier to its practical uses. Here, a genetically engineered Corynebacterium glutamicum (C. glutamicum) is introduced as a robust cell display platform for the in situ stabilization and low-cost production of CA. The enzyme is displayed in the mycolic layer with porin B as an anchoring protein with (GGGGS) 2 as a spacer. The cell-displayed CA exhibits no significant inactivation of the CO 2 hydration activity for at least one month at 37 °C. Its denaturation rate constant at 50 °C (0.07) is an order of magnitude lower than that of free CA (0.52-0.54). This study demonstrates that a structurally robust cell template allows the effective stabilization of CA, suggesting the C. glutamicum-based cell display as a promising technique to achieve highly efficient, sustainable, and low-cost CO 2 capture for industrial applications.

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“…3). It was suggested that the length of the exible linkers might affect the activity of the fusion proteins [31]. Then, fusion genes were constructed and compared by linking the YsADH and TkNOX with GGGGS as well as other peptide linkers ((GGGGS) 2 , GSG and (GSG) 2) .…”

Section: Construction Of Ysadh/tknox/vshgb Fusion Proteins and Their mentioning

confidence: 99%

Efficient whole-cell oxidation of α,β-unsaturated alcohols to α,β-unsaturated aldehydes through the cascade biocatalysis of alcohol dehydrogenase, NADPH oxidase and hemoglobin

Qiao

Wang

Zeng

et al. 2021

Preprint

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Background: α,β-Unsaturated aldehydes are widely used in the organic synthesis of fine chemicals for application in products such as flavoring agents, fragrances and pharmaceuticals. In the selective oxidation of α,β-unsaturated alcohols to the corresponding α,β-unsaturated aldehydes, it remains challenging to overcome poor selectivity, overoxidation and a low atom efficiency in chemical routes. Results: An E. coli strain coexpressing the NADP+-specific alcohol dehydrogenase YsADH and the oxygen-dependent NADPH oxidase TkNOX was constructed; these components enabled the NADP+ regeneration and catalyzed the oxidation of 100 mM 3-methyl-2-buten-1-ol to 3-methyl-2-butenal with a yield of 21.3%. The oxygen supply was strengthened by introducing the hemoglobin protein VsHGB into recombinant E. coli cells and replacing the atmosphere of the reactor with pure oxygen, which increased the yield to 51.3%. To further improve catalytic performance, the E. coli cells expressing the multifunctional fusion enzyme YsADH-(GSG)-TkNOX-(GSG)-VsHGB were generated, which completely converted 250 mM 3-methyl-2-buten-1-ol to 3-methyl-2-butenal after 8 h of whole-cell oxidation. The reaction conditions for the cascade biocatalysis were optimized, in which supplementation with 0.2 mM FAD and 0.4 mM NADP+ was essential for maintaining high catalytic activity. Finally, the established whole-cell system could serve as a platform for the synthesis of valuable α,β-unsaturated aldehydes through the selective oxidation of various α,β-unsaturated alcohols. Conclusions: The construction of a strain expressing the fusion enzyme YsADH-(GSG)-TkNOX-(GSG)-VsHGB achieved efficient NADP+ regeneration and the selective oxidation of various α,β-unsaturated alcohols to the corresponding α,β-unsaturated aldehydes. Among the available redox enzymes, the fusion enzyme YsADH-(GSG)-TkNOX-(GSG)-VsHGB has become the most recent successful example to improve catalytic performance in comparison with its separate components.

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Effect of flexible linker length on the activity of fusion protein 4-coumaroyl-CoA ligase::stilbene synthase

Cited by 39 publications

References 60 publications

Enhancing Flavan-3-ol Biosynthesis in Saccharomyces cerevisiae

Enhancing Flavan-3-ol Biosynthesis in Saccharomyces cerevisiae

Robust Biocatalysts Displayed on Crystalline Protein‐Layered Cells for Efficient and Sustainable Hydration of Carbon Dioxide

Efficient whole-cell oxidation of α,β-unsaturated alcohols to α,β-unsaturated aldehydes through the cascade biocatalysis of alcohol dehydrogenase, NADPH oxidase and hemoglobin

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