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Engineering Escherichia coli to improve culture performance and reduce formation of by‐products during recombinant protein production under transient intermittent anaerobic conditions
Author(s) -
Lara Alvaro R.,
VazquezLimón Consuelo,
Gosset Guillermo,
Bolívar Francisco,
LópezMunguía Agustín,
Ramírez Octavio T.
Publication year - 2006
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.20954
Subject(s) - fermentation , escherichia coli , formate , zymomonas mobilis , mutant , bioreactor , biochemistry , anaerobic exercise , lactate dehydrogenase , recombinant dna , green fluorescent protein , strain (injury) , formate dehydrogenase , chemistry , biology , enzyme , gene , ethanol fuel , catalysis , physiology , organic chemistry , anatomy
Abstract Three E. coli strains, named VAL22, VAL23, and VAL24, were engineered at the level of mixed‐acid fermentation pathways to improve culture performance under transient anaerobic conditions. VAL22 is a single mutant with an inactivated poxB gene that codes for pyruvate oxidase which converts pyruvate to acetate. VAL23 is a double mutant unable to produce lactate and formate due to deletions of the ldhA and pflB genes that code for lactate dehydrogenase and pyruvate‐formate lyase, respectively. VAL24 is a triple mutant with ldhA and pflB deleted and poxB inactivated. Engineered strains were cultured under oscillating dissolved oxygen tension (DOT) in a scale‐down system, to simulate gradients occurring in large‐scale bioreactors. Kinetic and stoichiometric parameters of constant (10%) and oscillating DOT cultures of the engineered strains were compared with those of the parental strain, W3110. All strains expressed recombinant green fluorescent protein (GFP) as a protein model. Mutant strains showed improved specific growth rate, reduced by‐product formation, and reduced specific glucose uptake rate compared to the parental strain, when cultured under oscillating DOT. In particular, lactate and formate production was abolished and acetate accumulation was reduced by 9–12%s. VAL24 showed the best performance, as specific growth and GFP production rates, and maximum GFP concentration were not affected by DOT gradients and were at least twofold higher than those of W3110 under constant DOT. Under oscillating DOT, VAL24 wasted about 40% less carbon into fermentation by‐products than W3110. It was demonstrated that, although E. coli responds rapidly to DOT fluctuations by deviating to fermentative metabolism, such pathways can be eliminated as they are not necessary for bacterial survival during the short circulation times typical of large‐scale cultures. The approach shown here opens new possibilities for designing metabolically engineered strains, with reduced sensitivity to DOT gradients and improved performance under typical conditions of large‐scale cultures. © 2006 Wiley Periodicals, Inc.