RETRACTED: Genetic and metabolic engineering approaches for the production and delivery of L-asparaginases: An overview

Vidya, Jalaja; Sajitha, Syed; Ushasree, Mrudula Vasudevan; Sindhu, Raveendran; Madhavan, Aravind; Pandey, Ashok

doi:10.1016/j.biortech.2017.05.057

Cited by 26 publications

(13 citation statements)

References 50 publications

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“…Furthermore, L-asparaginase genes have been widely expressed in many microbial host systems, and a variety of strategies have been used to improve L-asparaginase yields in these strains 20 . Meena et al .…”

Section: Introductionmentioning

confidence: 99%

Simultaneous cell disruption and semi-quantitative activity assays for high-throughput screening of thermostable L-asparaginases

Zhang

et al. 2018

Sci Rep

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L-asparaginase, which catalyses the hydrolysis of L-asparagine to L-aspartate, has attracted the attention of researchers due to its expanded applications in medicine and the food industry. In this study, a novel thermostable L-asparaginase from Pyrococcus yayanosii CH1 was cloned and over-expressed in Bacillus subtilis 168. To obtain thermostable L-asparaginase mutants with higher activity, a robust high-throughput screening process was developed specifically for thermophilic enzymes. In this process, cell disruption and enzyme activity assays are simultaneously performed in 96-deep well plates. By combining error-prone PCR and screening, six brilliant positive variants and four key amino acid residue mutations were identified. Combined mutation of the four residues showed relatively high specific activity (3108 U/mg) that was 2.1 times greater than that of the wild-type enzyme. Fermentation with the mutant strain in a 5-L fermenter yielded L-asparaginase activity of 2168 U/mL.

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Section: Introductionmentioning

confidence: 99%

Simultaneous cell disruption and semi-quantitative activity assays for high-throughput screening of thermostable L-asparaginases

Zhang

et al. 2018

Sci Rep

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“…Strain engineering, mostly by means of genetic modification, provides an effective approach to improve production of target products ( Idiris et al, 2010 ; Gottardi et al, 2017 ; Hossain et al, 2017 ). Recently, metabolic engineering strategies – for example, promoter exchange – have been widely used to rebalance metabolic flux related to synthesis of target products in rational designs of expression host strains ( Tai and Stephanopoulos, 2013 ; Qiu et al, 2014 ; Willenbacher et al, 2016 ; Vidya et al, 2017 ; Wang et al, 2017 ; Wen et al, 2017 ). Strain engineering using a metabolic engineering strategy aims to improve production of secondary metabolites of interest, and heterologous recombinant proteins as terminal products were seldom reported or analyzed.…”

Section: Introductionmentioning

confidence: 99%

Decreased Rhamnose Metabolic Flux Improved Production of Target Proteins and Cell Flocculation in Pichia pastoris

Yan

Zhao

et al. 2018

Front. Microbiol.

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Previously, several genes, including LRA1–LRA4 and LRAR, involved in rhamnose utilization pathway, were discovered in Pichia pastoris GS115; among them, LRA3 and LRA4 were considered as key rate-determining step enzymes. A P. pastoris expression platform based on the strong rhamnose-inducible promoter PLRA3 did not meet the demands of industrial application due to poor production of recombinant proteins. To enhance recombinant protein production of this expression platform, a genetically engineered strain, P. pastoris GS115m, with decreased rhamnose metabolic flux was developed from P. pastoris GS115 by replacement of the rhamnose-inducible promoter PLRA4 with another much weaker rhamnose-inducible promoter, PLRA2. Grown in MRH and YPR media using rhamnose as the main carbon source, the engineered strain showed decreased growth rate and maximal biomass compared with the parental strain. More importantly, grown in rhamnose-containing MRH and YPR media, the recombinant engineered strain harboring a β-galactosidase gene lacB, whose expression was regulated by rhamnose-inducible PLRA3, yielded substantial increases, of 2.5- and 1.5-fold, respectively, in target protein production over the parental strain. Additionally, grown in MRH and YPR media, the engineered strain had remarkable cell flocculation and rapid sedimentation with the increasing of cell density, providing an effective and convenient separation of the fermentation supernatant from strain cells. The engineered strain is a promising expression host for industrial production of target proteins due to its advantages over the parental strain as follows: (i) improved production of recombinant proteins, and (ii) remarkable cell flocculation and rapid sedimentation.

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“…However, the low yield of l ‐asparaginase has restricted the application of this enzyme. Some researchers have used gene manipulation, such as signal peptide screening, promoter mutation, N‐terminal deletion, and some fermentation strategy, such as DO‐stat fermentation strategy, Box–Behnken based optimization, process parameter optimization to improve l ‐asparaginase yields . But there were few reports with designed systems from gene expression level to fermentation for the l ‐asparaginase production, and the present study focused on this research.…”

Section: Discussionmentioning

confidence: 99%

“…Based on this medium, dissolved oxygen was optimized in this study. Dissolved oxygen (DO), a key factor that influences fermentation due to its effects on the tricarboxylic acid cycle of cell, restricting the carbon flux of cell, and thereby affecting biomass and product formation . Further, different phases of the fermentation of B. subtilis have different requirements of dissolved oxygen .…”

Section: Discussionmentioning

confidence: 99%

Design of a high‐efficiency synthetic system for l‐asparaginase production in Bacillus subtilis

Zhang

et al. 2019

Engineering in Life Sciences

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l‐asparaginase has high application value in medicine and food industry, but the low yield limits its application. In this study, we designed a synthetic system in Bacillus subtilis to produce l‐asparaginase by improving gene expression and optimizing the fermentation agitation speed. Gene expression was improved by respectively increasing transcription levels and translation speeds through screening promoters and RBS sequences. With the optimal promoter, P43, and the synthetic RBS sequence, the yield obtained in a shake flask was 371.87 U/mL, which was 2.09 times that with the original strain. To further enhance production in a 5‐L fermenter, a multistage agitation speed control strategy was adopted, involving agitation at 600 rpm for the first 12 h, followed by a gradual increase in speed to 900 rpm, which resulted in the highest yield of l‐asparaginase, 5321 U/mL, after 42 h of fermentation.

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RETRACTED: Genetic and metabolic engineering approaches for the production and delivery of L-asparaginases: An overview

Cited by 26 publications

References 50 publications

Simultaneous cell disruption and semi-quantitative activity assays for high-throughput screening of thermostable L-asparaginases

Simultaneous cell disruption and semi-quantitative activity assays for high-throughput screening of thermostable L-asparaginases

Decreased Rhamnose Metabolic Flux Improved Production of Target Proteins and Cell Flocculation in Pichia pastoris

Design of a high‐efficiency synthetic system for l‐asparaginase production in Bacillus subtilis

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