| dc.description.abstract | The production of recombinant proteins usingmicrobial cell factories such as Escherichia coli
(E. coli) is both a high value industry and a growing area of interest for the field of metabolomics.
While historical industrial methods for optimization are often based on empirical studies, the
field of metabolomics aims to investigate metabolic patterns within the cell in combination
with cellular phenotype observed from the outside. The combination of cultivation data and
metabolite concentrations could lead to an improved and better informed process of recombinant
protein production (RPP) optimization. The expression vectors which carry the DNA
for the desired protein, as well as the production demand itself, can cause stress to the E. coli
as they grow and produce the recombinant protein. The characterization and subsequent
understanding of these stresses will directly aid in the optimization of RPP.
In this study, selected expression vectors transformed into E. coli BL21 were investigated to
compare their growth patterns, yields and metabolism with the aim of gaining insight into
the metabolic consequences of inducible recombinant protein production of the fluorescent
protein mCherry. The variations in the expression vector included different number of copies
of the plasmid within the host, a strong vs. weak promoter for starting transcription, as well
as versions of the plasmid with and without the mCherry gene.
The growth characteristics and metabolic data revealed that a strong promoter has a larger
metabolic impact than a higher plasmid copy number. This effectwas seen to be compounded
for strains with both a strong promoter and a higher copy number. The strain with a low copy
number but a strong promoter showed the highest efficiency of substrate conversion to product,
but showed evidence of metabolic and phenotypic instability during production. The
variation observed for this strain is hypothesized to be related to regulatory consequences
that are likely associated with the improved RPP. Hypotheses discussed include glyoxylate
shunt activity, inclusion body formation and favorable valine stress. This Z1mCh strain is
suggested for future investigation, as the reasons for its success could be key to novel mechanisms
for RPP improvement. | |