scholarly journals Media optimization for SHuffle T7 Escherichia coli expressing SUMO-Lispro proinsulin by response surface methodology

2022 ◽  
Vol 22 (1) ◽  
Author(s):  
Aida Bakhshi Khalilvand ◽  
Saeed Aminzadeh ◽  
Mohammad Hossein Sanati ◽  
Fereidoun Mahboudi
Keyword(s):  
Escherichia Coli ◽  
Cell Density ◽  
Shake Flask ◽  
High Cell Density ◽  
Fold Increase ◽  
Soluble Expression ◽  
High Yield ◽  
High Cell ◽  
E Coli ◽  
High Cell Density Fermentation

Abstract Background SHuffle is a suitable Escherichia coli (E. coli) strain for high yield cytoplasmic soluble expression of disulfide-bonded proteins such as Insulin due to its oxidative cytoplasmic condition and the ability to correct the arrangement of disulfide bonds. Lispro is an Insulin analog that is conventionally produced in E. coli as inclusion bodies (IBs) with prolonged production time and low recovery. Here in this study, we aimed to optimize cultivation media composition for high cell density fermentation of SHuffle T7 E. coli expressing soluble Lispro proinsulin fused to SUMO tag (SU-INS construct) to obtain high cell density fermentation. Results Factors including carbon and nitrogen sources, salts, metal ions, and pH were screened via Plackett–Burman design for their effectiveness on cell dry weight (CDW) as a measure of cell growth. The most significant variables of the screening experiment were Yeast extract and MgCl2 concentration, as well as pH. Succeedingly, The Central Composite Design was utilized to further evaluate and optimize the level of significant variables. The Optimized media (OM-I) enhanced biomass by 2.3 fold in the shake flask (2.5 g/L CDW) that reached 6.45 g/L (2.6 fold increase) when applied in batch culture fermentation. The efficacy of OM-I media for soluble expression was confirmed in both shake flask and fermentor. Conclusion The proposed media was suitable for high cell density fermentation of E. coli SHuffle T7 and was applicable for high yield soluble expression of Lispro proinsulin.

scholarly journals Process and Metabolic Strategies for Improved Production of Escherichia coli-Derived 6-Deoxyerythronolide B

2002 ◽  
Vol 68 (7) ◽  
pp. 3287-3292 ◽  
Author(s):  
Blaine Pfeifer ◽  
Zhihao Hu ◽  
Peter Licari ◽  
Chaitan Khosla
Keyword(s):  
Escherichia Coli ◽  
Cell Density ◽  
Shake Flask ◽  
High Cell Density ◽  
Saccharopolyspora Erythraea ◽  
Efficient Production ◽  
High Cell ◽  
E Coli ◽  
Heterologous Biosynthesis ◽  
Fermentation Strategy

ABSTRACT Recently, the feasibility of using Escherichia coli for the heterologous biosynthesis of complex polyketides has been demonstrated. In this report, the development of a robust high-cell-density fed-batch procedure for the efficient production of complex polyketides is described. The effects of various physiological conditions on the productivity and titers of 6-deoxyerythronolide B (6dEB; the macrocyclic core of the antibiotic erythromycin) in recombinant cultures of E. coli were studied in shake flask cultures. The resulting data were used as a foundation to develop a high-cell-density fermentation procedure by building upon procedures reported earlier for recombinant protein production in E. coli. The fermentation strategy employed consistently produced ∼100 mg of 6dEB per liter, whereas shake flask conditions generated between 1 and 10 mg per liter. The utility of an accessory thioesterase (TEII from Saccharopolyspora erythraea) for enhancing the productivity of 6dEB in E. coli was also demonstrated (increasing the final titer of 6dEB to 180 mg per liter). In addition to reinforcing the potential for using E. coli as a heterologous host for wild-type- and engineered-polyketide biosynthesis, the procedures described in this study may be useful for the production of secondary metabolites that are difficult to access by other routes.

scholarly journals Development of a semi-defined medium for high cell density cultivation of Escherichia coli in shake flasks: Part 1

2016 ◽  
Author(s):  
Wenfa Ng ◽  
Yen-Peng Ting
Keyword(s):  
Escherichia Coli ◽  
Cell Density ◽  
Yeast Extract ◽  
High Cell Density ◽  
Defined Medium ◽  
High Cell ◽  
High Cell Density Cultivation ◽  
E Coli ◽  
Carbon And Nitrogen ◽  
Shake Flasks

Sufficient quantities of cells of consistent characteristics are needed for studying biological processes (at the population level) in many areas of applied microbiology. However, generating the requisite biomass by cell culture is usually the rate-limiting step of a project given the relatively low biomass yield of many commercial culture media in shake flasks. This work reports the formulation of a semi-defined medium that enabled aerobic high cell density cultivation of Escherichia coli DH5α (ATCC 53868) in shake flasks. The formulated medium (FM) comprises: a buffer system (K2HPO4: 12.54 g/L and KH2PO4: 2.31 g/L); vitamins and trace elements (yeast extract: 12.0 g/L); salts (NaCl: 5.0 g/L and MgSO4: 0.24 g/L); and carbon and nitrogen sources (D-Glucose: 6.0 g/L and NH4Cl: 1.5 g/L). Notable characteristics of this medium are: high buffer capacity (89 mM phosphate), 1:1 molar ratio between D-Glucose and NH4Cl, and yeast extract providing trace elements and a secondary source of carbon and nitrogen. Preliminary data revealed that an OD600nm of 9 was attained after 24 hours of cultivation at 37 oC, with glucose and NH4Cl as the main nutrients. At 48 hours, the OD600nm reached a maximum value of 11 with yeast extract providing the necessary nutrients for cell growth and biomass formation. The broth’s pH varied between 5.5 and 7.8 during cultivation. For comparison, the maximum OD600nm of E. coli grown in three commonly used complex media: Nutrient Broth, LB Lennox, and Tryptic Soy Broth (TSB) were 1.4, 3.2 and 9.2, respectively, under identical culture conditions. Finally, FM maintained the viability of a larger population of cells for three days - compared to a population collapse observed in TSB after one day. Collectively, the present findings suggested that the formulated medium might find use as a high cell density aerobic growth medium for E. coli in shake flasks. Part 2 of this work describes improvements in medium performance - specifically, higher cell yield as well as a shorter diauxic lag phase and total culture period – achieved through a small reduction in D-Glucose and NH4Cl concentrations in the medium composition. An abstract preprint of Part 2 is available at https://peerj.com/preprints/117/

scholarly journals Development of a semi-defined medium for high cell density cultivation of Escherichia coli in shake flasks: Part 1

2017 ◽  
Author(s):  
Wenfa Ng
Keyword(s):  
Escherichia Coli ◽  
Cell Density ◽  
Yeast Extract ◽  
High Cell Density ◽  
Defined Medium ◽  
High Cell ◽  
High Cell Density Cultivation ◽  
E Coli ◽  
Carbon And Nitrogen ◽  
Shake Flasks

Sufficient quantities of cells of consistent characteristics are needed for studying biologicalprocesses (at the population level ) in many areas of applied microbiology. However, generating the requisite biomass by cell culture is usually the rate-limiting step of a project given the relatively low biomass yield of many commercial culture media in shake flask culture systems. This work reports the formulation of a semi-defined medium that enabled aerobic high cell density cultivation of Escherichia coli DH5α (ATCC 53868) in shake flasks. The formulated medium (FM) comprises: a buffer system (K2HPO4 : 12.54 g/L and KH2 PO4 : 2.31 g/L); vitamins and trace elements (yeast extract: 12.0 g/L); salts (NaCl: 5.0 g/L and MgSO4 : 0.24 g/L); and carbon and nitrogen sources (D-Glucose: 6.0 g/L and NH4Cl: 1.5 g/L). Notable characteristics of this medium are: high buffer capacity (89 mM phosphate), 1:1 molar ratio between D-Glucose and NH4Cl, and yeast extract providing trace elements and a secondary source of carbon and nitrogen. Preliminary data revealed an OD 600nm of 9 after 24 hours of cultivation at 37 oC, presumably with glucose and NH4Cl as the main nutrients. At 48 hours, an OD 600nm of 11 was attained with yeast extract providing the necessary nutrients for cell growth and biomass formation. The broth’s pH varied between 5.5 and 7.8 during cultivation. On the other hand, the maximum OD 600nm of E. coli grown in three commonly used complex media: Nutrient Broth, LB Lennox, and Tryptic Soy Broth (TSB) were 1.4, 3.2 and 9.2, respectively, under identical culture conditions. Finally, FM maintained the viability of a larger population of cells for three days, compared to a population collapse in TSB broth after one day. Collectively, the results suggested that the formulated medium might find use as a high cell density aerobic growth medium for E. coli in shake flasks. Part 2 of this work describes improvements in medium performance ; specifically, higher cell yield as well as a shorter diauxic lag phase and total culture period achieved through a small reduction in D-Glucose and NH4Cl concentrations in the medium composition. An abstract preprint of Part 2 is available at https://peerj.com/preprints/117/

scholarly journals Development of a semi-defined medium for high cell density cultivation of Escherichia coli in shake flask culture system

2015 ◽  
Author(s):  
Wenfa Ng ◽  
Yen-Peng Ting
Keyword(s):  
Escherichia Coli ◽  
Cell Density ◽  
Yeast Extract ◽  
High Cell Density ◽  
Growth Media ◽  
Buffer System ◽  
High Cell ◽  
High Cell Density Cultivation ◽  
E Coli ◽  
Carbon And Nitrogen

Microbes in environmental studies should be cultured in growth media with characteristics as close to their original habitat as possible, and which also allows a high cell density to be attained for providing enough cells in subsequent experiments. This in-progress report describes the formulation of a medium with an environmentally-relevant composition, and which also affords aerobic high cell density cultivation of Escherichia coli DH5α in shake flasks. The formulated medium comprises four components: a buffer system (K2HPO4: 12.54 g/L and KH2PO4: 2.31 g/L), vitamins (yeast extract: 12.0 g/L), salts (NaCl: 5.0 g/L and MgSO4: 0.24 g/L), and carbon and nitrogen sources (D-Glucose: 6.0 g/L and NH4Cl: 1.5 g/L). Notable characteristics of this medium were: high capacity phosphate buffer system (89 mM phosphate); 1:1 molar ratio between D-Glucose and NH4Cl; and yeast extract providing trace elements and a secondary carbon and nitrogen source. Growth experiments revealed that an OD600nm of 9 was attained after 24 hours of cultivation at 37 oC. This phase of growth was largely fuelled by glucose and NH4Cl. After 48 hours, the OD600nm reached 11, which was fuelled by the mixture of carbohydrates, lipids and proteins in yeast extract. Broth’s pH varied between 5.5 and 7.8 during cultivation, which was in the range conducive for growth of E. coli. In comparison, the OD600nm of E. coli reached 1.4, 3.2, and 9.2 for three commonly used complex media; Nutrient Broth, LB Lennox, and Tryptic Soy Broth, respectively, over 48 hours under identical culture conditions. In addition, the formulated medium was able to maintain a large viable cell population for a longer period of time (three days) relative to Tryptic Soy Broth. Thus, preliminary data suggested that the formulated medium holds potential for use as a high cell density aerobic growth medium for Gram-negative bacteria.

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