Efficient production of levan using a recombinant yeast <i>Saccharomyces cerevisiae</i> hypersecreting a bacterial levansucrase

Ko, Hyunjun; Bae, Jung Hoon; Sung, Bong Hyun; Kim, Mi Jin; Kim, Chul Ho; Oh, Baek Rock; Sohn, Jae Hak

doi:10.1007/s10295-019-02206-1

Cited by 25 publications

(16 citation statements)

References 39 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The resin was washed with water, then with 0.9% NaCl, and again with water. The prepared resin was stored in 20% ethanol solution at 4 °C, and the binding yield of levan onto the agarose was measured via high performance liquid chromatography (HPLC) [ 46 ].…”

Section: Methodsmentioning

confidence: 99%

A novel protein fusion partner, carbohydrate-binding module family 66, to enhance heterologous protein expression in Escherichia coli

Kang

Kim

et al. 2021

Microb Cell Fact

Self Cite

View full text Add to dashboard Cite

Background Proteins with novel functions or advanced activities developed by various protein engineering techniques must have sufficient solubility to retain their bioactivity. However, inactive protein aggregates are frequently produced during heterologous protein expression in Escherichia coli. To prevent the formation of inclusion bodies, fusion tag technology has been commonly employed, owing to its good performance in soluble expression of target proteins, ease of application, and purification feasibility. Thus, researchers have continuously developed novel fusion tags to expand the expression capacity of high-value proteins in E. coli. Results A novel fusion tag comprising carbohydrate-binding module 66 (CBM66) was developed for the soluble expression of heterologous proteins in E. coli. The target protein solubilization capacity of the CBM66 tag was verified using seven proteins that are poorly expressed or form inclusion bodies in E. coli: four human-derived signaling polypeptides and three microbial enzymes. Compared to native proteins, CBM66-fused proteins exhibited improved solubility and high production titer. The protein-solubilizing effect of the CBM66 tag was compared with that of two commercial tags, maltose-binding protein and glutathione-S-transferase, using poly(ethylene terephthalate) hydrolase (PETase) as a model protein; CBM66 fusion resulted in a 3.7-fold higher expression amount of soluble PETase (approximately 370 mg/L) compared to fusion with the other commercial tags. The intact PETase was purified from the fusion protein upon serial treatment with enterokinase and affinity chromatography using levan-agarose resin. The bioactivity of the three proteins assessed was maintained even when the CBM66 tag was fused. Conclusions The use of the CBM66 tag to improve soluble protein expression facilitates the easy and economic production of high-value proteins in E. coli.

show abstract

Section: Methodsmentioning

confidence: 99%

A novel protein fusion partner, carbohydrate-binding module family 66, to enhance heterologous protein expression in Escherichia coli

Kang

Kim

et al. 2021

Microb Cell Fact

Self Cite

View full text Add to dashboard Cite

show abstract

“…S. cerevisiae EBY100 used in our systems was not an invertase defective strain in sucrose utilization, and only part of the substrate is used for transformation, while the other part is used by the host. This has been reported in the production of levan using a recombinant invertase defective S. cerevisiae [31]. It indicated the levan productivity of the yeast-developing system can be further increased by choosing different strains of invertase.…”

Section: Levan Biosynthesismentioning

confidence: 80%

Development and Characterization of Two Types of Surface Displayed Levansucrases for Levan Biosynthesis

et al. 2021

View full text Add to dashboard Cite

Levan has wide applications in chemical, cosmetic, pharmaceutical and food industries. The free levansucrase is usually used in the biosynthesis of levan, but the poor reusability and low stability of free levansucrase have limited its large-scale use. To address this problem, the surface-displayed levansucrase in Saccharomyces cerevisiae were generated and evaluated in this study. The levansucrase from Zymomonas mobilis was displayed on the cell surface of Saccharomyces cerevisiae EBY100 using a various yeast surface display platform. The N-terminal fusion partner is based on a-agglutinin, and the C-terminal one is Flo1p. The yield of levan produced by these two whole-cell biocatalysts reaches 26 g/L and 34 g/L in 24 h, respectively. Meanwhile, the stability of the surface-displayed levansucrases is significantly enhanced. After six reuses, these two biocatalysts retained over 50% and 60% of their initial activities, respectively. Furthermore, the molecular weight and polydispersity test of the products suggested that the whole-cell biocatalyst of levansucrase displayed by Flo1p has more potentials in the production of levan with low molecular weight which is critical in certain applications. In conclusion, our method not only enable the possibility to reuse the enzyme, but also improves the stability of the enzyme.

show abstract

“…The levan yield varies greatly among levansucrases from different types of microorganisms. Halomonas levansucrase could produce 18 g/L levan after optimization [44], levansucrase from recombinant Saccharomyces could produce 76 g/L levan [45], B. licheniformis 8-37-0-1 levansucrase produced a maximal yield of 7.1 mg/mL levan [32], and Brenneria goodwinii levansucrase reached a yield of 185 g/L levan [16]. In this investigation, the levan yield of BM-2 levansucrases was higher than those reported in most of the studies; accordingly, the enzyme might be more suitable for industrial production.…”

Section: Optimization Of Enzyme Dose Sucrose Concentration and Reacmentioning

confidence: 99%

Cloning and Expression of Levansucrase Gene of Bacillus velezensis BM-2 and Enzymatic Synthesis of Levan

Zhang

Zhao

et al. 2021

Processes

View full text Add to dashboard Cite

Levan is a versatile and valuable fructose homopolymer, and a few bacterial strains have been found to produce levan. Although levan products have numerous specific functions, their application and promotion were limited by the production capacity and production cost. Bacillus velezensis BM-2 is a levan-synthesizing strain, but its levan production is too low to apply. In this study, the levansucrase gene of B. velezensis BM-2 was cloned to plasmid pET-32a-Acma-zz, and the recombinant plasmids were transferred to Escherichia coli BL21. A transformed clone was selected to express and secrete the fusion enzymes with an Acma-tag efficiently. The expressed products were further purified by a self-developed separating material called bacterial enhancer matrix (BEM) particles. The purification efficiency was 93.4%, with a specific activity of 16.589 U/mL protein. The enzymatic reaction results indicated that the optimal reaction temperature is 50 °C, the optimal pH of the acetate buffer is 5.6, and the buffer system greatly influenced the enzyme activity. The enzyme activity was enhanced to 130% in the presence of 5 mM Ca2+, K+, Zn2+, and Mn2+, whereas it was almost abolished in the case of Cu2+ and Fe3+. The values of Km, kcat, and kcat/Km were 17.41 mM, 376.83 s−1, and 21.64 mM−1s−1, respectively. The enzyme amount of 20 U/g sucrose was added to the system containing 400 g/L sucrose, and the levan products with a concentration of 120 g/L reached after an incubation of 18 h, which was 8 times that of the yield before optimization. The results of molecular docking analysis indicated that the Asp86 might act as a nucleophilic catalytic residue for sucrose, Arg246 and Asp247 act as transition state stabilizer of transfructosylation, and Glu340 and Arg306 were recognized as general acid donors. They formed the catalytic-groups triad. The unique properties and catalytic activity of the levansucrase suggest that it deserves further research and might have good industrial application prospects.

show abstract

Efficient production of levan using a recombinant yeast Saccharomyces cerevisiae hypersecreting a bacterial levansucrase

Cited by 25 publications

References 39 publications

A novel protein fusion partner, carbohydrate-binding module family 66, to enhance heterologous protein expression in Escherichia coli

A novel protein fusion partner, carbohydrate-binding module family 66, to enhance heterologous protein expression in Escherichia coli

Development and Characterization of Two Types of Surface Displayed Levansucrases for Levan Biosynthesis

Cloning and Expression of Levansucrase Gene of Bacillus velezensis BM-2 and Enzymatic Synthesis of Levan

Contact Info

Product

Resources

About