Enhanced Production of a Thermostable Carbonic Anhydrase in Escherichia coli by Using a Modified NEXT Tag

Hwang, In Seong; Kim, Joo Hyeon; Jo, Byung Hoon

doi:10.3390/molecules26195830

Cited by 6 publications

(4 citation statements)

References 32 publications

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“…coli and S. cerevisiae, such as NEXT, MBP, and Ser-Lys-Ile-Lys (SKIK) . To improve the expression of GfCPS/KS without losing its activity, a series of short peptide tags (SK, SKI, and SKIK) were inserted into the N-termination of GfCPS/KS, resulting in strains JR15–17.…”

Section: Resultsmentioning

confidence: 99%

“…Production by Enzyme Engineering. It has been reported that some peptide tags could markedly improve the expression level of proteins in E. coli and S. cerevisiae, such as NEXT, 36 MBP, 37 and Ser-Lys-Ile-Lys (SKIK). 38 To improve the expression of GfCPS/KS without losing its activity, a series of short peptide tags (SK, SKI, and SKIK) were inserted into the N-termination of GfCPS/KS, resulting in strains JR15−17.…”

Section: Improving the Ent-kaurenementioning

confidence: 99%

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Heterologous Biosynthesis of Kauralexin A1 in Saccharomyces cerevisiae through Metabolic and Enzyme Engineering

Chen,

Wang,

et al. 2024

J. Agric. Food Chem.

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Kauralexin A1 (KA1) is a key intermediate of the kauralexin A series metabolites of maize phytoalexins. However, their application is severely limited by their low abundance in maize. In this study, an efficient biosynthetic pathway was constructed to produce KA1 in Saccharomyces cerevisiae. Also, metabolic and enzyme engineering strategies were applied to construct the hightiter strains, such as chassis modification, screening synthases, the colocalization of enzymes, and multiple genomic integrations. First, the KA1 precursor ent-kaurene was synthesized using the efficient diterpene synthase GfCPS/KS from Fusarium fujikuroi, and optimized to reach 244.36 mg/L in shake flasks, which displayed a 200-fold increase compared to the initial strain. Then, the KA1 was produced under the catalysis of ZmCYP71Z18 from Zea mays and SmCPR1 from Salvia miltiorrhiza, and the titer was further improved by integrating the fusion protein into the genome. Finally, an ent-kaurene titer of 763.23 mg/L and a KA1 titer of 42.22 mg/L were achieved through a single-stage fed-batch fermentation in a 5 L bioreactor. This is the first report of the heterologous biosynthesis of maize diterpene phytoalexins in S. cerevisiae, which lays a foundation for further pathway reconstruction and biosynthesis of the kauralexin A series maize phytoalexins.

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Section: Resultsmentioning

confidence: 99%

Section: Improving the Ent-kaurenementioning

confidence: 99%

Heterologous Biosynthesis of Kauralexin A1 in Saccharomyces cerevisiae through Metabolic and Enzyme Engineering

Chen,

Wang,

et al. 2024

J. Agric. Food Chem.

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“…coli with minimal or no impact on enzyme activity [ 32 ]. Tag systems such as MBP (maltose binding protein) [ 33 ] and NEXT (originating from N-terminus of Hydrogenovibrio marinus CA) [ 34 ] are widely used. NEXT tag was shown to increase the soluble expression level in E. coli BL21 (DE3) by up to 5.6–8.3 fold [ 32 ].…”

Section: Resultsmentioning

confidence: 99%

Streamlining heterologous expression of top carbonic anhydrases in Escherichia coli: bioinformatic and experimental approaches

Wei,

Lunin,

Alahuhta

et al. 2024

Microb Cell Fact

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Background Carbonic anhydrase (CA) enzymes facilitate the reversible hydration of CO2 to bicarbonate ions and protons. Identifying efficient and robust CAs and expressing them in model host cells, such as Escherichia coli, enables more efficient engineering of these enzymes for industrial CO2 capture. However, expression of CAs in E. coli is challenging due to the possible formation of insoluble protein aggregates, or inclusion bodies. This makes the production of soluble and active CA protein a prerequisite for downstream applications. Results In this study, we streamlined the process of CA expression by selecting seven top CA candidates and used two bioinformatic tools to predict their solubility for expression in E. coli. The prediction results place these enzymes in two categories: low and high solubility. Our expression of high solubility score CAs (namely CA5-SspCA, CA6-SazCAtrunc, CA7-PabCA and CA8-PhoCA) led to significantly higher protein yields (5 to 75 mg purified protein per liter) in flask cultures, indicating a strong correlation between the solubility prediction score and protein expression yields. Furthermore, phylogenetic tree analysis demonstrated CA class-specific clustering patterns for protein solubility and production yields. Unexpectedly, we also found that the unique N-terminal, 11-amino acid segment found after the signal sequence (not present in its homologs), was essential for CA6-SazCA activity. Conclusions Overall, this work demonstrated that protein solubility prediction, phylogenetic tree analysis, and experimental validation are potent tools for identifying top CA candidates and then producing soluble, active forms of these enzymes in E. coli. The comprehensive approaches we report here should be extendable to the expression of other heterogeneous proteins in E. coli.

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“…The NEXT tag has been employed as a fusion tag to address the challenges associated with both the low-level expression and poor solubility of taCA [18,19]. The NEXT tag, a 53 amino acid-length peptide with a molecular mass of 5.5 kDa, originated from the N-terminal extension sequence of α-type CA (hmCA) in the marine bacterium Hydrogenovibrio marinus [18,20].…”

Section: Introductionmentioning

confidence: 99%

Improved Solubility and Stability of a Thermostable Carbonic Anhydrase via Fusion with Marine-Derived Intrinsically Disordered Solubility Enhancers

2024

IJMS

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Carbonic anhydrase (CA), an enzyme catalyzing the reversible hydration reaction of carbon dioxide (CO2), is considered a promising biocatalyst for CO2 reduction. The α-CA of Thermovibrio ammonificans (taCA) has emerged as a compelling candidate due to its high thermostability, a critical factor for industrial applications. However, the low-level expression and poor in vitro solubility have hampered further utilization of taCA. Recently, these limitations have been addressed through the fusion of the NEXT tag, a marine-derived, intrinsically disordered small peptide that enhances protein expression and solubility. In this study, the solubility and stability of NEXT-taCA were further investigated. When the linker length between the NEXT tag and the taCA was shortened, the expression level decreased without compromising solubility-enhancing performance. A comparison between the NEXT tag and the NT11 tag demonstrated the NEXT tag’s superiority in improving both the expression and solubility of taCA. While the thermostability of taCA was lower than that of the extensively engineered DvCA10, the NEXT-tagged taCA exhibited a 30% improvement in long-term thermostability compared to the untagged taCA, suggesting that enhanced solubility can contribute to enzyme thermostability. Furthermore, the bioprospecting of two intrinsically disordered peptides (Hcr and Hku tags) as novel solubility-enhancing fusion tags was explored, demonstrating their performance in improving the expression and solubility of taCA. These efforts will advance the practical application of taCA and provide tools and insights for enzyme biochemistry and bioengineering.

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Enhanced Production of a Thermostable Carbonic Anhydrase in Escherichia coli by Using a Modified NEXT Tag

Cited by 6 publications

References 32 publications

Heterologous Biosynthesis of Kauralexin A1 in Saccharomyces cerevisiae through Metabolic and Enzyme Engineering

Heterologous Biosynthesis of Kauralexin A1 in Saccharomyces cerevisiae through Metabolic and Enzyme Engineering

Streamlining heterologous expression of top carbonic anhydrases in Escherichia coli: bioinformatic and experimental approaches

Improved Solubility and Stability of a Thermostable Carbonic Anhydrase via Fusion with Marine-Derived Intrinsically Disordered Solubility Enhancers

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