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

ABSTRACT: 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.

SUBMITTER: Shishparenok AN

PROVIDER: S-EPMC10607044 | biostudies-literature | 2023 Oct

REPOSITORIES: biostudies-literature

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

Shishparenok Anastasiya N AN Gladilina Yulia A YA Zhdanov Dmitry D DD

International journal of molecular sciences 20231016 20

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, ...[more]

PMID: 37894901

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Project description:BackgroundMannosylglycerate (MG) is one of the most widespread compatible solutes among marine microorganisms adapted to hot environments. This ionic solute holds excellent ability to protect proteins against thermal denaturation, hence a large number of biotechnological and clinical applications have been put forward. However, the current prohibitive production costs impose severe constraints towards large-scale applications. All known microbial producers synthesize MG from GDP-mannose and 3-phosphoglycerate via a two-step pathway in which mannosyl-3-phosphoglycerate is the intermediate metabolite. In an early work, this pathway was expressed in Saccharomyces cerevisiae with the goal to confirm gene function (Empadinhas et al. in J Bacteriol 186:4075-4084, 2004), but the level of MG accumulation was low. Therefore, in view of the potential biotechnological value of this compound, we decided to invest further effort to convert S. cerevisiae into an efficient cell factory for MG production.ResultsTo drive MG production, the pathway for the synthesis of GDP-mannose, one of the MG biosynthetic precursors, was overexpressed in S. cerevisiae along with the MG biosynthetic pathway. MG production was evaluated under different cultivation modes, i.e., flask bottle, batch, and continuous mode with different dilution rates. The genes encoding mannose-6-phosphate isomerase (PMI40) and GDP-mannose pyrophosphorylase (PSA1) were introduced into strain MG01, hosting a plasmid encoding the MG biosynthetic machinery. The resulting engineered strain (MG02) showed around a twofold increase in the activity of PMI40 and PSA1 in comparison to the wild-type. In batch mode, strain MG02 accumulated 15.86 mgMG g DCW -1 , representing a 2.2-fold increase relative to the reference strain (MG01). In continuous culture, at a dilution rate of 0.15 h-1, there was a 1.5-fold improvement in productivity.ConclusionIn the present study, the yield and productivity of MG were increased by overexpression of the GDP-mannose pathway and optimization of the mode of cultivation. A maximum of 15.86 mgMG g DCW -1 was achieved in batch cultivation and maximal productivity of 1.79 mgMG g DCW -1 h-1 in continuous mode. Additionally, a positive correlation between MG productivity and growth rate/dilution rate was established, although this correlation is not observed for MG yield.

Dataset Information

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

Publications

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

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