Abstract
Recombinant protein production for medical, academic, or industrial applications is essential for our current life. Recombinant proteins are obtained mainly through microbial fermentation, with Escherichia coli being the host most used. In spite of that, some problems are associated with the production of recombinant proteins in E. coli, such as the formation of inclusion bodies, the metabolic burden, or the inefficient translocation/transport system of expressed proteins. Optimizing transcription of heterologous genes is essential to avoid these drawbacks and develop competitive biotechnological processes. Here, expression of YFP reporter protein is evaluated under the control of four promoters of different strength (PT7lac, Ptrc, Ptac, and PBAD) and two different replication origins (high copy number pMB1′ and low copy number p15A). In addition, the study has been carried out with the E. coli BL21 wt and the ackA mutant strain growing in a rich medium with glucose or glycerol as carbon sources. Results showed that metabolic burden associated with transcription and translation of foreign genes involves a decrease in recombinant protein expression. It is necessary to find a balance between plasmid copy number and promoter strength to maximize soluble recombinant protein expression. The results obtained represent an important advance on the most suitable expression system to improve both the quantity and quality of recombinant proteins in bioproduction engineering.
Highlights
Expression of heterologous and autologous genes is a routine method employed in several biotechnological fields such as metabolic engineering, in vivo biocatalysis, or in recombinant proteins or other high-value metabolite production (Sanchez-Garcia et al, 2016; Badenhorst and Bornscheuer, 2018; McCarty and Ledesma-Amaro, 2019)
To evaluate the influence of replication origin on expression of recombinant proteins in E. coli BL21, p15A (∼10 copies/cell) and a high copy number derived from pMB1 ori (500–700 copies/cell), denoted as pMB1, were selected (Selzer et al, 1983; Lin-Chao et al, 1992)
One of the advantages of E. coli as a host is the wide variety of expression vectors available
Summary
Expression of heterologous and autologous genes is a routine method employed in several biotechnological fields such as metabolic engineering, in vivo biocatalysis, or in recombinant proteins or other high-value metabolite production (Sanchez-Garcia et al, 2016; Badenhorst and Bornscheuer, 2018; McCarty and Ledesma-Amaro, 2019). The metabolic burden is associated with energetic and precursor constraints due to the transcription and translation of non-essential proteins for the host cell. This limitation is reflected in the alteration of physiological parameters, such as growth rate, and in the downregulation of several essential metabolic pathways for the cell (Mairhofer et al, 2013; Tan et al, 2020; Li and Rinas, 2021). In order to minimize metabolic imbalance, several commercially available plasmids have been engineered to design each biotechnological process, choosing between different promoters and origins of replication, both responsible for the expression level of the gene/s of interest (Zaslaver et al, 2006; De Mey et al, 2007; Wang et al, 2009; Rosano and Ceccarelli, 2014; Yang et al, 2016; Jervis et al, 2019; Rosano et al, 2019)
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