Application of extremophile cell factories in industrial biotechnology

Wang, Yuzhou; Qian, Jinyi; Shi, Tianqiong; Wang, Yuetong; Ding, Qiang; Ye, Chao

doi:10.1016/j.enzmictec.2024.110407

Cited by 4 publications

(4 citation statements)

References 106 publications

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“…Realizing the industrial potential of these microbes will require rapid development of amenable genetic tools. As it currently stands, there is a lack of broadly applicable genetic tools, including selectable markers, promoters, plasmid origins, and transformation protocols, as broadly utilized mesophilic tools are not functional in these thermophilic microbes (Y. Wang et al., 2024 ; Ye et al., 2023 ). While there are a growing number of tools being generated for use in thermophilic microbes (Adalsteinsson et al., 2021 ; Le & Sun, 2022 ; Riley et al., 2019 ; Walker et al., 2020 ; Wang et al., 2022 ; Wu et al., 2023 ; Yang et al., 2023 ), developing new tools and implementing CRISPR mediated genome engineering and/or thermostable serine recombinase-assisted genome engineering (SAGE) mediated integration would greatly simplify testing production of various industrial chemicals in these currently underutilized microbes (Fenster & Eckert, 2021 ; Wu et al., 2023 ).…”

Section: Discussionmentioning

confidence: 99%

Application of functional genomics for domestication of novel non-model microbes

Bales,

Vergara,

Eckert

2024

Journal of Industrial Microbiology and Biotechnology

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With the expansion of domesticated microbes producing biomaterials and chemicals to support a growing circular bioeconomy, the variety of waste and sustainable substrates that can support microbial growth and production will also continue to expand. The diversity of these microbes also requires a range of compatible genetic tools to engineer improved robustness and economic viability. As we still do not fully understand the function of many genes in even highly studied model microbes, engineering improved microbial performance requires introducing genome-scale genetic modifications followed by screening or selecting mutants that enhance growth under prohibitive conditions encountered during production. These approaches include adaptive laboratory evolution, random or directed mutagenesis, transposon-mediated gene disruption, or CRISPR interference (CRISPRi). Although any of these approaches may be applicable for identifying engineering targets, here we focus on using CRISPRi to reduce the time required to engineer more robust microbes for industrial applications. One-Sentence Summary The development of genome scale CRISPR-based libraries in new microbes enables discovery of genetic factors linked to desired traits for engineering more robust microbial systems.

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Section: Discussionmentioning

confidence: 99%

Application of functional genomics for domestication of novel non-model microbes

Bales,

Vergara,

Eckert

2024

Journal of Industrial Microbiology and Biotechnology

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“…These environments encompass extreme temperatures, significant variations in salinity, high pressures, the presence of heavy metals, and drastic pH levels ( Sysoev et al, 2021 ). Extremophilic proteins hold a unique position in biotechnology due to their exceptional stability under harsh environmental conditions ( Wang et al, 2024 ). They contribute to sustainable biotechnology on Earth and offer valuable insights into the potential existence of life on other planets ( Allers, 2010 ; Schultz et al, 2023 ; Wang et al, 2024 ).…”

Section: Introductionmentioning

confidence: 99%

“…Extremophilic proteins hold a unique position in biotechnology due to their exceptional stability under harsh environmental conditions ( Wang et al, 2024 ). They contribute to sustainable biotechnology on Earth and offer valuable insights into the potential existence of life on other planets ( Allers, 2010 ; Schultz et al, 2023 ; Wang et al, 2024 ). The efficient expression and cost-effective purification of recombinant proteins are paramount in today’s pharmaceutical and biotechnology industries ( Puetz and Wurm, 2019 ).…”

Section: Introductionmentioning

confidence: 99%

A systematic analysis of affinity tags in the haloarchaeal expression system, Haloferax volcanii for protein purification

Karan,

Renn,

Allers

et al. 2024

Front. Microbiol.

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Extremophilic proteins are valuable in various fields, but their expression can be challenging in traditional hosts like Escherichia coli due to misfolding and aggregation. Haloferax volcanii (H. volcanii), a halophilic expression system, offers a solution. This study examined cleavable and non-cleavable purification tags at both the N- and C-termini when fused with the superfolder green fluorescent protein (sfGFP) in H. volcanii. Our findings reveal that an N-terminal 8xHis-tag or Strep-tag®II significantly enhances protein production, purity, and yield in H. volcanii. Further experiments with mCherry and halophilic alcohol dehydrogenase (ADH) showed improved expression and purification yields when the 8xHis-tag or Strep-tag®II was positioned at the C-terminus for mCherry and at the N-terminus for ADH. Co-positioning 8xHis-tag and Twin-Strep-tag® at the N-terminus of sfGFP, mCherry, and ADH yielded significantly enhanced results. These findings highlight the importance of thoughtful purification tag design and selection in H. volcanii, providing valuable insights for improving protein production and purification with the potential to advance biotechnological applications.

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“…They grow more e ciently and are not sensitive to sterilization, making them more productive in the industry. Especially its high synthesis capacity carries excellent potential in the eld of enzyme production (Wang 2024). Oligotrophic bacteria can be found in numbers up to 10 3 -10 5 in environments containing dissolved carbon at µg L − 1 concentrations (Kundu et al 2020).…”

Section: Introductionmentioning

confidence: 99%

Expression of Recombinant Taq DNA Polymerase in Oligotrophic Klebsiella oxytoca: A Novel Approach to Industrial Enzyme Production

Tankus,

Balci,

Dedeakayogullari

et al. 2024

Preprint

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The demand for high-efficiency DNA polymerases in molecular biology and diagnostic applications has led to the exploration of novel microbial hosts for enzyme production. This study investigates the expression of recombinant Taq DNA polymerase in Klebsiella oxytoca, an oligotrophic bacterium known for its minimal nutrient requirements and robust growth in diverse environments. By leveraging the metabolic versatility and adaptive capabilities of K. oxytoca, we aimed to establish a cost-effective and sustainable method for producing Taq polymerase at an industrial scale. The recombinant K. oxytoca was engineered using a plasmid vector containing the Taq polymerase gene under the control of a strong promoter. Optimal expression conditions were identified, including the appropriate induction time and temperature, leading to high yields of active enzyme. The Taq DNA polymerase was successfully expressed in a standard LB medium and at a concentration of 0.1% (v/v). Expressed Taq DNA polymerases were characterized by SDS-PAGE and PCR activity analyses were performed. The same processes were also carried out in scale-up studies, and it was investigated whether TaqDNA polymerase production in Klebsiella oxytoca was suitable for industry. This approach not only reduces production costs but also aligns with green chemistry principles by utilizing a host organism that thrives on minimal resources. Our findings suggest that oligotrophic K. oxytoca is a promising candidate for the biotechnological production of recombinant enzymes, offering an innovative pathway for enhancing industrial enzyme manufacturing processes.

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Application of extremophile cell factories in industrial biotechnology

Cited by 4 publications

References 106 publications

Application of functional genomics for domestication of novel non-model microbes

Application of functional genomics for domestication of novel non-model microbes

A systematic analysis of affinity tags in the haloarchaeal expression system, Haloferax volcanii for protein purification

Expression of Recombinant Taq DNA Polymerase in Oligotrophic Klebsiella oxytoca: A Novel Approach to Industrial Enzyme Production

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