Protein engineering of cellobiose dehydrogenase from Phanerochaete chrysosporium in yeast Saccharomyces cerevisiae InvSc1 for increased activity and stability

Blažić, Marija; Balaž, Ana Marija; Tadić, Vojin; Draganić, Bojana; Ostafe, Raluca; Fischer, Rainer; Prodanović, Radivoje

doi:10.1016/j.bej.2019.03.025

Cited by 15 publications

(6 citation statements)

References 37 publications

(50 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In general, our experiments confirmed that the replacement of methionine residues can improve stability against oxidative damage as shown previously, 42 , 58 but it also appears to be coupled to a loss in structural stability not immediately evident at short time scale, especially when these substitutions are within the hydrophobic core of the protein. It has been shown previously that mutations, especially in the core of a protein, frequently result in a decrease of the thermodynamic stability.…”

Section: Results and Discussionsupporting

confidence: 90%

“… 40 Two recent studies described the negative effects of H 2 O 2 on the stability of CDH, and the authors showed that the replacement of methionine residues can improve the chemical stability of CDH. 41 , 42 However, both studies focused on the effects of externally supplied H 2 O 2 , and only limited data are available on the effects of turnover-related ROS production. In general, engineering turnover stability of enzymes is much less explored compared to directed evolution efforts toward higher activities, increased solvent/temperature stabilities, or novel activities.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Engineering the Turnover Stability of Cellobiose Dehydrogenase toward Long-Term Bioelectronic Applications

Geiss

Reichhart

Pejker

et al. 2021

ACS Sustainable Chem. Eng.

View full text Add to dashboard Cite

Cellobiose dehydrogenase (CDH) is an attractive oxidoreductase for bioelectrochemical applications. Its two-domain structure allows the flavoheme enzyme to establish direct electron transfer to biosensor and biofuel cell electrodes. Yet, the application of CDH in these devices is impeded by its limited stability under turnover conditions. In this work, we aimed to improve the turnover stability of CDH by semirational, high-throughput enzyme engineering. We screened 13 736 colonies in a 96-well plate setup for improved turnover stability and selected 11 improved variants. Measures were taken to increase the reproducibility and robustness of the screening setup, and the statistical evaluation demonstrates the validity of the procedure. The selected CDH variants were expressed in shaking flasks and characterized in detail by biochemical and electrochemical methods. Two mechanisms contributing to turnover stability were found: (i) replacement of methionine side chains prone to oxidative damage and (ii) the reduction of oxygen reactivity achieved by an improved balance of the individual reaction rates in the two CDH domains. The engineered CDH variants hold promise for the application in continuous biosensors or biofuel cells, while the deduced mechanistic insights serve as a basis for future enzyme engineering approaches addressing the turnover stability of oxidoreductases in general.

show abstract

Section: Results and Discussionsupporting

confidence: 90%

Section: Introductionmentioning

confidence: 99%

Engineering the Turnover Stability of Cellobiose Dehydrogenase toward Long-Term Bioelectronic Applications

Geiss

Reichhart

Pejker

et al. 2021

ACS Sustainable Chem. Eng.

View full text Add to dashboard Cite

show abstract

“…Hence, the native secretory leader sequence gave three times higher expression than a signal sequence of α-factor [39]. Also, in research conducted earlier in our laboratory, the ability of S. cerevisiae to produce cellobiose dehydrogenase was confirmed, but the yields of the expressed enzymes were not high [40]. The results of work [39,40], both using the native secretion signal of CDH, are very different due to the use of different yeast strains.…”

Section: Cdhmentioning

confidence: 87%

“…Also, in research conducted earlier in our laboratory, the ability of S. cerevisiae to produce cellobiose dehydrogenase was confirmed, but the yields of the expressed enzymes were not high [40]. The results of work [39,40], both using the native secretion signal of CDH, are very different due to the use of different yeast strains. In work [39], the protease-free S. cerevisiae BJ5465 strain was used that can give high amounts of protein after long fermentation times.…”

Section: Cdhmentioning

confidence: 89%

State of the Art Technologies for High Yield Heterologous Expression and Production of Oxidoreductase Enzymes: Glucose Oxidase, Cellobiose Dehydrogenase, Horseradish Peroxidase, and Laccases in Yeasts P. pastoris and S. cerevisiae

Crnoglavac Popović,

Stanišić,

Prodanović

2024

Fermentation

Self Cite

View full text Add to dashboard Cite

Oxidoreductase (OXR) enzymes are in high demand for biocatalytic applications in the food industry and cosmetics (glucose oxidase (GOx) and cellobiose dehydrogenase (CDH)), bioremediations (horseradish peroxidase (HRP) and laccase (LAC)), and medicine for biosensors and miniature biofuel cells (GOx, CDH, LAC, and HRP). They can be used in a soluble form and/or within the yeast cell walls expressed as chimeras on the surface of yeast cells (YSD), such as P. pastoris and S. cerevisiae. However, most of the current studies suffer from either low yield for soluble enzyme expression or low enzyme activity when expressed as chimeric proteins using YSD. This is always the case in studies dealing with the heterologous expression of oxidoreductase enzymes, since there is a requirement not only for multiple OXR gene integrations into the yeast genome (super transformations), and codon optimization, but also very careful design of fermentation media composition and fermentation conditions during expression due to the need for transition metals (copper and iron) and metabolic precursors of FAD and heme. Therefore, scientists are still trying to find the optimal formula using the above-mentioned approaches; most recently, researcher started using protein engineering and directed evolution to increase in the yield of recombinant enzyme production. In this review article, we will cover all the current state-of-the-art technologies and most recent advances in the field that yielded a high expression level for some of these enzymes in specially designed expression/fermentation systems. We will also tackle and discuss new possibilities for further increases in fermentation yield using cutting-edge technologies such as directed evolution, protein and strain engineering, high-throughput screening methods based on in vitro compartmentalization, flow cytometry, and microfluidics.

show abstract

“…The disadvantages of wild-type chitinases can be overcome by combining heterologous expression with the redesign of chitinases using molecular evolution, which involves consecutive rounds of mutation and selection to isolate clones with improved properties. Directed evolution has been applied to a variety of enzymes with industrial applications, such as glucose oxidase [ 9 , 10 ], P450 monooxygenases [ 11 ], cellulases [ 12 ], peroxidases [ 13 ], cellobiose dehydrogenase [ 14 ], transaminases [ 15 ], esterases [ 16 ], lipases [ 17 ], proteases [ 18 ], and many more. However, there are only a few examples of the directed evolution of chitinases [ 19 , 20 , 21 ].…”

Section: Introductionmentioning

confidence: 99%

A High-Throughput Screening System Based on Fluorescence-Activated Cell Sorting for the Directed Evolution of Chitinase A

Menghiu

Ostafe

Prodanović

et al. 2021

IJMS

Self Cite

View full text Add to dashboard Cite

Chitinases catalyze the degradation of chitin, a polymer of N-acetylglucosamine found in crustacean shells, insect cuticles, and fungal cell walls. There is great interest in the development of improved chitinases to address the environmental burden of chitin waste from the food processing industry as well as the potential medical, agricultural, and industrial uses of partially deacetylated chitin (chitosan) and its products (chito-oligosaccharides). The depolymerization of chitin can be achieved using chemical and physical treatments, but an enzymatic process would be more environmentally friendly and more sustainable. However, chitinases are slow-acting enzymes, limiting their biotechnological exploitation, although this can be overcome by molecular evolution approaches to enhance the features required for specific applications. The two main goals of this study were the development of a high-throughput screening system for chitinase activity (which could be extrapolated to other hydrolytic enzymes), and the deployment of this new method to select improved chitinase variants. We therefore cloned and expressed the Bacillus licheniformis DSM8785 chitinase A (chiA) gene in Escherichia coli BL21 (DE3) cells and generated a mutant library by error-prone PCR. We then developed a screening method based on fluorescence-activated cell sorting (FACS) using the model substrate 4-methylumbelliferyl β-d-N,N′,N″-triacetyl chitotrioside to identify improved enzymes. We prevented cross-talk between emulsion compartments caused by the hydrophobicity of 4-methylumbelliferone, the fluorescent product of the enzymatic reaction, by incorporating cyclodextrins into the aqueous phases. We also addressed the toxicity of long-term chiA expression in E. coli by limiting the reaction time. We identified 12 mutants containing 2–8 mutations per gene resulting in up to twofold higher activity than wild-type ChiA.

show abstract

Protein engineering of cellobiose dehydrogenase from Phanerochaete chrysosporium in yeast Saccharomyces cerevisiae InvSc1 for increased activity and stability

Cited by 15 publications

References 37 publications

Engineering the Turnover Stability of Cellobiose Dehydrogenase toward Long-Term Bioelectronic Applications

Engineering the Turnover Stability of Cellobiose Dehydrogenase toward Long-Term Bioelectronic Applications

State of the Art Technologies for High Yield Heterologous Expression and Production of Oxidoreductase Enzymes: Glucose Oxidase, Cellobiose Dehydrogenase, Horseradish Peroxidase, and Laccases in Yeasts P. pastoris and S. cerevisiae

A High-Throughput Screening System Based on Fluorescence-Activated Cell Sorting for the Directed Evolution of Chitinase A

Contact Info

Product

Resources

About