Co-expressing Leucine Responsive Regulatory protein (Lrp) enhances recombinant L-Asparaginase-II production in Escherichia coli

Mahalik, Shubhashree; Sharma, Ashish K.; Das, Debdulal; Mittra, Debashrita; Mukherjee, Kakali

doi:10.1016/j.jbiotec.2022.04.012

Cited by 5 publications

(4 citation statements)

References 39 publications

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“…To counteract the negative effects of CSR, plasmid-based co-expression increased lrp activity. After 32 h of batch culture, a test culture with an additional plasmid expressing lrp under an arabinose promoter yielded 50% more recombinant L-ASNase than a control culture with only one plasmid expressing the recombinant protein [148].…”

Section: Optimization Of L-asparaginase Production In Escherichia Colimentioning

confidence: 99%

Engineering and Expression Strategies for Optimization of L-Asparaginase Development and Production

Shishparenok,

Gladilina,

Zhdanov

2023

IJMS

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Genetic engineering for heterologous expression has advanced in recent years. Model systems such as Escherichia coli, Bacillus subtilis and Pichia pastoris are often used as host microorganisms for the enzymatic production of L-asparaginase, an enzyme widely used in the clinic for the treatment of leukemia and in bakeries for the reduction of acrylamide. Newly developed recombinant L-asparaginase (L-ASNase) may have a low affinity for asparagine, reduced catalytic activity, low stability, and increased glutaminase activity or immunogenicity. Some successful commercial preparations of L-ASNase are now available. Therefore, obtaining novel L-ASNases with improved properties suitable for food or clinical applications remains a challenge. The combination of rational design and/or directed evolution and heterologous expression has been used to create enzymes with desired characteristics. Computer design, combined with other methods, could make it possible to generate mutant libraries of novel L-ASNases without costly and time-consuming efforts. In this review, we summarize the strategies and approaches for obtaining and developing L-ASNase with improved properties.

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Section: Optimization Of L-asparaginase Production In Escherichia Colimentioning

confidence: 99%

Engineering and Expression Strategies for Optimization of L-Asparaginase Development and Production

Shishparenok,

Gladilina,

Zhdanov

2023

IJMS

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“…The role of the lrp gene was also evaluated, concluding that its co-expression is a suitable target to enhance expression. Achieving a maximum volumetric efficiency of 458.43 mg/L, this result in a 1.5-fold improvement compared to the native expression level (Mahalik et al, 2022). In another study conducted by Sharma et al (Sharma et al, 2020), studied post-induction upregulated genes as potential candidates for the generation of Cellular Stress Response (CSR) using E. coli as strain model.…”

Section: Metabolic Engineering In Hosts To Improve the Heterologous E...mentioning

confidence: 99%

Current state of molecular and metabolic strategies for the improvement of L-asparaginase expression in heterologous systems

et al. 2023

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Heterologous expression of L-asparaginase (L-ASNase) has become an important area of research due to its clinical and food industry applications. This review provides a comprehensive overview of the molecular and metabolic strategies that can be used to optimize the expression of L-ASNase in heterologous systems. This article describes various approaches that have been employed to increase enzyme production, including the use of molecular tools, strain engineering, and in silico optimization. The review article highlights the critical role that rational design plays in achieving successful heterologous expression and underscores the challenges of large-scale production of L-ASNase, such as inadequate protein folding and the metabolic burden on host cells. Improved gene expression is shown to be achievable through the optimization of codon usage, synthetic promoters, transcription and translation regulation, and host strain improvement, among others. Additionally, this review provides a deep understanding of the enzymatic properties of L-ASNase and how this knowledge has been employed to enhance its properties and production. Finally, future trends in L-ASNase production, including the integration of CRISPR and machine learning tools are discussed. This work serves as a valuable resource for researchers looking to design effective heterologous expression systems for L-ASNase production as well as for enzymes production in general.

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“…Metabolic engineering in hosts to improve the heterologous expression of L-ASNase Mahalik et al (2022) explains that the first studies on the optimization of recombinant protein production focused on the individual steps of the protein biosynthesis route, particularly on the transcription, which was considered the step limiting the speed in the protein expression. This bottleneck was solved with the arrival of strong and strictly controlled promoters, which have been mentioned in this review.…”

Section: Genetic Engineering Of Host Strains To Improve the Expressio...mentioning

confidence: 99%

“…When fis was co-expressed together with L-ASNase at 6 hours of induction, the volumetric efficiency of L-ASNase increased 3-fold, compared to the yield with the native form of the host. In another iteration of this methodology, Mahalik et al (2022) evaluated the role of the lrp gene in the expression of L-ASNase, concluding that its co-expression is a suitable target to improve expression. This work achieved a maximum volumetric efficiency of 458.43 mg/L of equivalent to a 1.5-fold increase compared to the production of L-ASNase without lrp .…”

Section: Genetic Engineering Of Host Strains To Improve the Expressio...mentioning

confidence: 99%

Current state of molecular and metabolic strategies for the improvement of L-asparaginase expression in heterologous systems

Lefin

Miranda

Beltrán

et al. 2022

Preprint

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L-asparaginase is one of the world’s most in-demand industrial enzymes, mainly due to its therapeutic properties and its use in the food industry. Over the years studies have been conducted to improve its specific activity and reduce side effects, driving new discoveries that promise safer, more efficient, and cheaper drugs for consumers. However, heterologous protein modifications or expression continue to be a problem during production. Therefore, addressing bottlenecks when attempting to express modified and/or heterologous proteins using the rational design of heterologous expression systems with modified hosts could be a promising strategy to improve L-asparaginase production. Problems such as inadequate protein folding, metabolic load on host cells, codon usage bias, repetitive sequences affecting translation, heavy molecular weight and/or multi-membrane domains formation; need great attention during the development of “bio-better” proteins. In this article, we address the current molecular and metabolic strategies that have been developed to improve the heterologous expression of L-asparaginase.

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Co-expressing Leucine Responsive Regulatory protein (Lrp) enhances recombinant L-Asparaginase-II production in Escherichia coli

Cited by 5 publications

References 39 publications

Engineering and Expression Strategies for Optimization of L-Asparaginase Development and Production

Engineering and Expression Strategies for Optimization of L-Asparaginase Development and Production

Current state of molecular and metabolic strategies for the improvement of L-asparaginase expression in heterologous systems

Current state of molecular and metabolic strategies for the improvement of L-asparaginase expression in heterologous systems

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