Production of a recombinant phospholipase A2 in Escherichia coli using resonant acoustic mixing that improves oxygen transfer in shake flasks

Microbial Cell Factories

Research

Alejandro Olvera¹  Alejandro Alagón¹  Mauricio A. Trujillo-Roldán²  Sara Restrepo-Pineda²  Jesus González-Santana²  Greta I. Reynoso-Cereceda²  Norma A. Valdez-Cruz²  Saumel Pérez-Rodriguez²  Guadalupe Zavala³ 

[1] Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Mor, Mexico;Programa de Investigación de Producción de Biomoléculas, Unidad de Bioprocesos, Departamento de Biología Molecular y Biotecnología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, AP. 70228, CP 04510, Mexico City, Mexico;Unidad de Microscopía, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Mor, Mexico;

关键词: Resonant acoustic mixing;    Orbital mixing;    Shake flask;    Recombinant protein;    Inclusion bodies;    Escherichia coli;   

DOI  :  10.1186/s12934-017-0746-1

 received in 2017-03-06, accepted in 2017-07-20,  发布年份 2017

来源: Springer

【 摘 要 】

BackgroundShake flasks are widely used during the development of bioprocesses for recombinant proteins. Cultures of recombinant Escherichia coli with orbital mixing (OM) have an oxygen limitation negatively affecting biomass growth and recombinant-protein production. With the aim to improve mixing and aeration in shake flask cultures, we analyzed cultures subjected to OM and the novel resonant acoustic mixing (RAM) by applying acoustic energy to E. coli BL21-Gold (DE3): a producer of recombinant phospholipase A2 (rPLA2) from Micrurus laticollaris snake venom.ResultsComparing OM with RAM (200 rpm vs. 7.5g) at the same initial volumetric oxygen transfer coefficient (kLa ≈ 80 h−1) ~69% less biomass was obtained with OM compared with RAM. We analyzed two more conditions increasing agitation until maximal speed (12.5 and 20g), and ~1.6- and ~1.4-fold greater biomass was obtained as compared with cultures at 7.5g. Moreover, the specific growth rate was statistically similar in all cultures carried out in RAM, but ~1.5-fold higher than that in cultures carried out under OM. Almost half of the glucose was consumed in OM, whereas between 80 and 100% of the glucose was consumed in RAM cultures, doubling biomass per glucose yields. Differential organic acid production was observed, but acetate production was prevented at the maximal RAM (20g). The amount of rPLA2 in both, OM and RAM cultures, represented 38 ± 5% of the insoluble protein. A smaller proportion of α-helices and β-sheet of purified inclusion bodies (IBs) were appreciated by ATR-FTIR from cultures carried out under OM, than those from RAM. At maximal agitation by RAM, internal E. coli localization patterns of protein aggregation changed, as well as, IBs proteolytic degradation, in conjunction with the formation of small external vesicles, although these changes did not significantly affect the cell survival response.ConclusionsIn moderate-cell-density recombinant E. coli BL21-Gold (DE3) cultures, the agitation increases in RAM (up to the maximum) was not enough to avoid the classical oxygen limitation that happens in OM shake flasks. However, RAM presents a decrease of oxygen limitation, resulting in a favorable effect on biomass growth and volumetric rPLA2 production. While under OM a higher recombinant protein yield was obtained.

【 授权许可】

CC BY   
© The Author(s) 2017