Recombinant Protein Production and Plasmid Stability in Escherichia coli Biofilms

• Tipo: Outras Publicações
• Data: 2020

Autores

Luciana Gomes (Autor) FEUP; Monteiro, GA (Autor) Outra; Filipe Mergulhão (Autor) FEUP

Outras Informações

ID Authenticus: P-00S-GHE

DOI: 10.5194/biofilms9-65

Abstract (EN): <jats:p>&lt;p&gt;&lt;em&gt;Escherichia coli&lt;/em&gt; biofilms have a great biotechnological potential since this organism has been one of the preferred hosts for recombinant protein production for the past decades and it has been successfully used in metabolic engineering for the production of high-value compounds.&lt;/p&gt; &lt;p&gt;In a previous study, we have demonstrated that the non-induced enhanced green fluorescent protein (eGFP) expression from &lt;em&gt;E. coli&lt;/em&gt; biofilm cells was 30-fold higher than in the planktonic state without any optimization of cultivation parameters [1]. The aim of the present work was to evaluate the effect of chemical induction with isopropyl &amp;#946;-D-1-thiogalactopyranoside (IPTG) on the expression of eGFP by planktonic and biofilm cells of &lt;em&gt;E. coli&lt;/em&gt; JM109(DE3) transformed with a plasmid containing a T7 promoter.&lt;/p&gt; &lt;p&gt;It was shown that induction negatively affected the growth and viability of planktonic cultures, and eGFP production did not increase. Recombinant protein production was not limited by gene dosage or by transcriptional activity. Results suggest that plasmid maintenance at high copy number imposes a metabolic burden that precludes high level expression of the recombinant protein. In biofilm cells, the inducer avoided the overall decrease in the amount of expressed eGFP, although this was not correlated with the gene dosage. Higher specific production levels were always attained with biofilm cells and it seems that while induction of biofilm cells shifts their metabolism towards the maintenance of recombinant protein concentration, in planktonic cells the cellular resources are directed towards plasmid replication and growth [2].&lt;/p&gt; &lt;p&gt;It is expected that this work will be of great value to elucidate the mechanisms of induction on recombinant protein production, especially in biofilm cells which have shown potential to be used as protein factories.&lt;/p&gt; &lt;p&gt;&amp;#160;&lt;/p&gt; &lt;p&gt;&amp;#160;&lt;/p&gt; &lt;p&gt;References:&lt;/p&gt; &lt;p&gt;[1] Gomes, L.C., &amp; Mergulh&amp;#227;o, F.J. (2017) Heterologous protein production in &lt;em&gt;Escherichia coli&lt;/em&gt; biofilms: A non-conventional form of high cell density cultivation. &lt;em&gt;Process Biochemistry, 57, 1-8&lt;/em&gt;. https://doi.org/10.1016/j.procbio.2017.03.018&lt;/p&gt; &lt;p&gt;[2] Gomes, L., Monteiro, G., &amp; Mergulh&amp;#227;o, F. (2020). The Impact of IPTG Induction on Plasmid Stability and Heterologous Protein Expression by &lt;em&gt;Escherichia coli&lt;/em&gt; Biofilms. &lt;em&gt;International Journal of Molecular Sciences, 21(2), 576&lt;/em&gt;. https://doi.org/10.3390/ijms21020576&lt;/p&gt;</jats:p>

Idioma: Inglês

Tipo (Avaliação Docente): Científica