Galactose can be an inducer for production of therapeutic proteins by auto-induction using E. coli BL21 strains

Xu, Jinying; Banerjee, Akhilesh; Pan, Shih-Hsie; Li, Zheng Jian

doi:10.1016/j.pep.2012.02.014

Cited by 38 publications

(37 citation statements)

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“…Despite being frequently used for protein production using T7-based E. coli expression systems in shake flask cultures [27,34], only a few investigations have considered bioreactor cultures based on auto-induction complex medium formulations [29,30,35]. …”

Section: Resultsmentioning

confidence: 99%

High-throughput strategies for penicillin G acylase production in rE. colifed-batch cultivations

et al. 2014

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BackgroundPenicillin G acylase (PGA) is used industrially to catalyze the hydrolysis of penicillin G to obtain 6-aminopenicillanic acid. In Escherichia coli, the most-studied microorganism for PGA production, this enzyme accumulates in the periplasmic cell space, and temperature plays an important role in the correct synthesis of its subunits.ResultsThis work investigates the influence of medium composition, cultivation strategy, and temperature on PGA production by recombinant E. coli cells. Shake flask cultures carried out using induction temperatures ranging from 18 to 28°C revealed that the specific enzyme activity achieved at 20°C (3000 IU gDCW-1) was 6-fold higher than the value obtained at 28°C. Auto-induction and high cell density fed-batch bioreactor cultures were performed using the selected induction temperature, with both defined and complex media, and IPTG and lactose as inducers. Final biomass concentrations of 100 and 120 gDCW L-1, and maximum enzyme productivities of 7800 and 5556 IU L-1 h-1, were achieved for high cell density cultures using complex and defined media, respectively.ConclusionsTo the best of our knowledge, the volumetric enzyme activity and productivity values achieved using the complex medium are the highest ever reported for PGA production using E. coli. Overall PGA recovery yields of 64 and 72% after purification were achieved for crude extracts obtained from cells cultivated in defined and complex media, respectively. The complex medium was the most cost-effective for PGA production, and could be used in both high cell density and straightforward auto-induction protocols.

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Section: Resultsmentioning

confidence: 99%

High-throughput strategies for penicillin G acylase production in rE. colifed-batch cultivations

et al. 2014

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“…After the transformation of the plasmid mutant library, the E. coli TOP10 cells were spread on the 1.5% agar LB medium containing 50 μM kanamycin with an expected colony number of 500–800 per Petri dish. The dishes were incubated at 37 °C overnight and at room temperature for another day to ensure enough recombinant 6PGDH expression49. The colonies on plates were treated at 70 °C for 1 h to kill cells, deactivate E. coli mesophilic enzymes, and degrade metabolites and intracellular coenzymes.…”

Section: Methodsmentioning

confidence: 99%

High-Throughput Screening of Coenzyme Preference Change of Thermophilic 6-Phosphogluconate Dehydrogenase from NADP+ to NAD+

Huang

Chen

Zhong

et al. 2016

Sci Rep

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Coenzyme engineering that changes NAD(P) selectivity of redox enzymes is an important tool in metabolic engineering, synthetic biology, and biocatalysis. Here we developed a high throughput screening method to identify mutants of 6-phosphogluconate dehydrogenase (6PGDH) from a thermophilic bacterium Moorella thermoacetica with reversed coenzyme selectivity from NADP+ to NAD+. Colonies of a 6PGDH mutant library growing on the agar plates were treated by heat to minimize the background noise, that is, the deactivation of intracellular dehydrogenases, degradation of inherent NAD(P)H, and disruption of cell membrane. The melted agarose solution containing a redox dye tetranitroblue tetrazolium (TNBT), phenazine methosulfate (PMS), NAD+, and 6-phosphogluconate was carefully poured on colonies, forming a second semi-solid layer. More active 6PGDH mutants were examined via an enzyme-linked TNBT-PMS colorimetric assay. Positive mutants were recovered by direct extraction of plasmid from dead cell colonies followed by plasmid transformation into E. coli TOP10. By utilizing this double-layer screening method, six positive mutants were obtained from two-round saturation mutagenesis. The best mutant 6PGDH A30D/R31I/T32I exhibited a 4,278-fold reversal of coenzyme selectivity from NADP+ to NAD+. This screening method could be widely used to detect numerous redox enzymes, particularly for thermophilic ones, which can generate NAD(P)H reacted with the redox dye TNBT.

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“…Glycerol is included to (together with lactose) provide sustained growth during the induction phase (Studier 2005). In spite of the widespread use of auto-induction in shaker flask experiments, only few bioreactor cultures carried out with auto-induction media in batch (Giomarelli et al 2006; Blommel and Fox 2007a, b) or fed-batch mode (Jia et al 2011; Xu et al 2012) have been reported so far. In these studies, bioreactor media contain basically the same carbon and nitrogen sources, but the total carbon source concentration was increased by factors of approximately two (Blommel et al 2007a, b), three (Giomarelli et al 2006) or four (Xu et al 2012) in comparison to the sum of glucose, glycerol and lactose concentrations present in the original auto-induction ZYM-5052 complex formulation (Studier 2005).…”

Section: Introductionmentioning

confidence: 99%

“…In spite of the widespread use of auto-induction in shaker flask experiments, only few bioreactor cultures carried out with auto-induction media in batch (Giomarelli et al 2006; Blommel and Fox 2007a, b) or fed-batch mode (Jia et al 2011; Xu et al 2012) have been reported so far. In these studies, bioreactor media contain basically the same carbon and nitrogen sources, but the total carbon source concentration was increased by factors of approximately two (Blommel et al 2007a, b), three (Giomarelli et al 2006) or four (Xu et al 2012) in comparison to the sum of glucose, glycerol and lactose concentrations present in the original auto-induction ZYM-5052 complex formulation (Studier 2005). Consequently, maximum optical densities (600 nm) in the range of ~ 20 to 40 (Giomarelli et al 2006; Blommel et al 2007a, b; Jia et al 2011) were reported for batch experiments and of 140 for fed-batch cultures (Xu et al 2012).…”

Section: Introductionmentioning

confidence: 99%

“…In these studies, bioreactor media contain basically the same carbon and nitrogen sources, but the total carbon source concentration was increased by factors of approximately two (Blommel et al 2007a, b), three (Giomarelli et al 2006) or four (Xu et al 2012) in comparison to the sum of glucose, glycerol and lactose concentrations present in the original auto-induction ZYM-5052 complex formulation (Studier 2005). Consequently, maximum optical densities (600 nm) in the range of ~ 20 to 40 (Giomarelli et al 2006; Blommel et al 2007a, b; Jia et al 2011) were reported for batch experiments and of 140 for fed-batch cultures (Xu et al 2012). Nevertheless, none of these studies aimed to compare induction strategies.…”

Section: Introductionmentioning

confidence: 99%

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Non-conventional induction strategies for production of subunit swine erysipelas vaccine antigen in rE. coli fed-batch cultures

et al. 2013

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In spite of the large number of reports on fed-batch cultivation of E. coli, alternative cultivation/induction strategies remain to be more deeply exploited. Among these strategies, it could be mentioned the use of complex media with combination of different carbon sources, novel induction procedures and feed flow rate control matching the actual cell growth rate. Here, four different carbon source combinations (glucose, glycerol, glucose + glycerol and auto-induction) in batch media formulation were compared. A balanced combination of glucose and glycerol in a complex medium formulation led to: fast growth in the batch-phase; reduced plasmid instability by preventing early expression leakage; and protein volumetric productivity of 0.40 g.L-1.h-1. Alternative induction strategies were also investigated. A mixture of lactose and glycerol as supplementary medium fully induced a high biomass population, reaching a good balance between specific protein production (0.148 gprot.gDCW-1) and volumetric productivity (0.32 g.L-1.h-1). The auto-induction protocol showed excellent results on specific protein production (0.158 gprot.gDCW-1) in simple batch cultivations. An automated feed control based on the on-line estimated growth rate was implemented, which allowed cells to grow at higher rates than those generally used to avoid metabolic overflow, without leading to acetate accumulation. Some of the protocols described here may provide a useful alternative to standard cultivation and recombinant protein production processes, depending on the performance index that is expected to be optimized. The protocols using glycerol as carbon source and induction by lactose feeding, or glycerol plus glucose in batch medium and induction by lactose pulse led to rSpaA production in the range of 6 g.L-1, in short fed-batch processes (16 to 20 h) with low accumulation of undesired side metabolites.

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Galactose can be an inducer for production of therapeutic proteins by auto-induction using E. coli BL21 strains

Cited by 38 publications

References 31 publications

High-throughput strategies for penicillin G acylase production in rE. colifed-batch cultivations

High-throughput strategies for penicillin G acylase production in rE. colifed-batch cultivations

High-Throughput Screening of Coenzyme Preference Change of Thermophilic 6-Phosphogluconate Dehydrogenase from NADP+ to NAD+

Non-conventional induction strategies for production of subunit swine erysipelas vaccine antigen in rE. coli fed-batch cultures

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