Yeast Surface Display for <i>In Vitro</i> Biosynthetic Pathway Reconstruction

Luo, Biaobiao; Jin, Moonsoo M.; Li, Xiaohua; Makunga, Nokwanda P.; Hu, Xuebo

doi:10.1021/acssynbio.1c00175

Cited by 14 publications

(7 citation statements)

References 50 publications

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“…Many previous studies have demonstrated that the overexpression of the key enzymes involved in the MVA pathway could increase the yields of heterologous triterpenoids in yeast, and the down-regulation of 2,3-oxidosqualene metabolic flux towards ergosterol could redirect more metabolic flux towards the biosynthetic pathways of triterpenoids. 44,45 In order to further increase the production of PPD, we integrated IDI1 , ERG20 , ERG9, ERG1 and antisense ERG7 genes into the δ4 site of strain YP-3, resulting in strain YPU. After being cultivated for 6 d, the titer of PPD in YPU reached 93.1 mg L −1 , which was 2.2-fold that in YP-3 (42.4 mg L −1 ) (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Green Chemistry flux towards ergosterol could redirect more metabolic flux towards the biosynthetic pathways of triterpenoids. 44,45 In order to further increase the production of PPD, we integrated IDI1, ERG20, ERG9, ERG1 and antisense ERG7 genes into the δ4 site of strain YP-3, resulting in strain YPU. After being cultivated for 6 d, the titer of PPD in YPU reached 93.1 mg L −1 , which was 2.2-fold that in YP-3 (42.4 mg L −1 ) (Fig.…”

Section: Papermentioning

confidence: 99%

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Combining protein and metabolic engineering to achieve green biosynthesis of 12β-O-Glc-PPD inSaccharomyces cerevisiae

Zhou

Chen

et al. 2023

Green Chem.

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Section: Resultsmentioning

confidence: 99%

Section: Papermentioning

confidence: 99%

Combining protein and metabolic engineering to achieve green biosynthesis of 12β-O-Glc-PPD inSaccharomyces cerevisiae

Zhou

Chen

et al. 2023

Green Chem.

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“…However, preparation processes such as enzyme purification and matrix selection for immobilization are usually time-consuming and expensive [ 46 ]. The microbial surface-displayed strategy of fusing target enzymes with cell wall carrier proteins avoids laborious, time-consuming processes of immobilization [ 47 , 48 ]. Here, the CarCby whole cell biocatalyst presented a potential candidate for the biodegradation of carbaryl, a kind of carbamate pesticide.…”

Section: Resultsmentioning

confidence: 99%

Display of a novel carboxylesterase CarCby on Escherichia coli cell surface for carbaryl pesticide bioremediation

et al. 2022

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Background Carbamate pesticides have been widely used in agricultural and forestry pest control. The large-scale use of carbamates has caused severe toxicity in various systems because of their toxic environmental residues. Carbaryl is a representative carbamate pesticide and hydrolase/carboxylesterase is the initial and critical enzyme for its degradation. Whole-cell biocatalysts have become a powerful tool for environmental bioremediation. Here, a whole cell biocatalyst was constructed by displaying a novel carboxylesterase/hydrolase on the surface of Escherichia coli cells for carbaryl bioremediation. Results The carCby gene, encoding a protein with carbaryl hydrolysis activity was cloned and characterized. Subsequently, CarCby was displayed on the outer membrane of E. coli BL21(DE3) cells using the N-terminus of ice nucleation protein as an anchor. The surface localization of CarCby was confirmed by SDS–PAGE and fluorescence microscopy. The optimal temperature and pH of the engineered E. coli cells were 30 °C and 7.5, respectively, using pNPC4 as a substrate. The whole cell biocatalyst exhibited better stability and maintained approximately 8-fold higher specific enzymatic activity than purified CarCby when incubated at 30 °C for 120 h. In addition, ~ 100% and 50% of the original activity was retained when incubated with the whole cell biocatalyst at 4 ℃ and 30 °C for 35 days, respectively. However, the purified CarCby lost almost 100% of its activity when incubated at 30 °C for 134 h or 37 °C for 96 h, respectively. Finally, approximately 30 mg/L of carbaryl was hydrolyzed by 200 U of the engineered E. coli cells in 12 h. Conclusions Here, a carbaryl hydrolase-containing surface-displayed system was first constructed, and the whole cell biocatalyst displayed better stability and maintained its catalytic activity. This surface-displayed strategy provides a new solution for the cost-efficient bioremediation of carbaryl and could also have the potential to be used to treat other carbamates in environmental bioremediation.

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“…Currently, the host cells for surface display are mainly concentrated in phage, − bacteria, − yeast, ,− and Bacillus subtilis spores. − However, any traditional cell surface display system has shortcomings. The molecular weight of the polypeptide or protein displayed on the phage cannot be too large; otherwise, it will affect the assembly and infection of the phage.…”

Section: Introductionmentioning

confidence: 99%

Establishment of a Novel Cell Surface Display Platform Based on Natural “Chitosan Beads” of Yeast Spores

Wang

et al. 2022

J. Agric. Food Chem.

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Cell surface display technology, which expresses and anchors proteins on the surface of microbial cells, has broad application prospects in many fields, such as protein library screening, biocatalysis, and biosensor development. However, traditional cell surface display systems have disadvantages: the molecular weight of phage display proteins cannot be too large; bacterial display lacks the post-translational modification process for eukaryotic proteins; yeast display is prone to excessive protein glycosylation and misfolding of multisubunit proteins; and the compatibility of Bacillus subtilis spore display needs to be further improved. Therefore, it is extremely valuable to develop an efficient surface display platform with strong universality and stress resistance properties. Although yeast surface display systems have been extensively investigated, the establishment of a surface display platform using yeast spores has rarely been reported. In this study, a novel cell surface display platform based on natural "chitosan beads" of yeast spores was developed. The target protein in fusion with the chitosan affinity protein (CAP) exhibited strong binding capability with "chitosan beads" of yeast spores in vitro and in vivo. Moreover, this protein display system showed highly preferable enzymatic properties and stability. As an example, the displayed LXYL-P1-2-CAP demonstrated high thermostability and reusability (60% of the initial activity after seven cycles of reuse), high storage stability (75% of original activity after 8 weeks), and excellent tolerance to a concentration up to 75% (v/v) organic reagents. To prove the practicability of this surface display system, the semisynthesis of paclitaxel intermediate was demonstrated and its highest conversion rate was 92% using 0.25 mM substrate. This study provides a novel and useful platform for the surface display of proteins, especially for multimeric macromolecular proteins of eukaryotic origin.

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Yeast Surface Display for In Vitro Biosynthetic Pathway Reconstruction

Cited by 14 publications

References 50 publications

Combining protein and metabolic engineering to achieve green biosynthesis of 12β-O-Glc-PPD inSaccharomyces cerevisiae

Combining protein and metabolic engineering to achieve green biosynthesis of 12β-O-Glc-PPD inSaccharomyces cerevisiae

Display of a novel carboxylesterase CarCby on Escherichia coli cell surface for carbaryl pesticide bioremediation

Establishment of a Novel Cell Surface Display Platform Based on Natural “Chitosan Beads” of Yeast Spores

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