Application of adaptive laboratory evolution to overcome a flux limitation in an Escherichia coli production strain

Abstract

Gene deletion strategies using flux balance analysis (FBA) have improved the growth-coupled production of various compounds. However, the productivities were often below the expectation because the cells failed to adapt to these genetic perturbations. Here, we demonstrate the productivity of the succinate of the designed gene deletion strain was improved by adaptive laboratory evolution (ALE). Although FBA predicted deletions of adhE-pykAF-gldA-pflB lead to produce succinate from glycerol with a yield of 0.45 C-mol/C-mol, the knockout mutant did not produce only 0.08 C-mol/Cmol, experimentally. After the ALE experiments, the highest succinate yield of an evolved strain reached to the expected value. Genome sequencing analysis revealed all evolved strains possessed novel mutations in ppc of I829S or R849S. In vitro enzymatic assay and metabolic profiling analysis revealed that these mutations desensitizing an allosteric inhibition by L-aspartate and improved the flux through Ppc, while the activity of Ppc in the unevolved strain was tightly regulated by L-aspartate. These result demonstrated that the evolved strains achieved the improvement of succinate production by expanding the flux space of Ppc, realizing the predicted metabolic state by FBA.

Prediction of Rate‐Limiting Reactions for Growth‐Associated Production Using a Constraint‐Based Approach

“…Comparing the results of the laboratory evolution experiment, the sakeME successfully predicted PPC as the rate‐limiting step for succinate production. In addition, GLYK, in which a mutation was observed in an evolved strain, was also included in the predicted candidate by the sakeME.…”

“…To evaluate the prediction accuracy of sakeME, we applied the method to predict the possible rate‐limiting reactions in the growth‐coupled production of 1,4‐BDO or succinate in E. coli . The rate‐limiting reactions for the production of 1,4‐BDO or succinate in E. coli have been previously identified experimentally …”

“…Succinate can be produced by the endogenous pathways in E. coli . The FBA predicted that the growth was associated with succinate production by the combinatorial deletion of adhE , pykAF , gldA , and pflB . The predicted flux distribution at the optimal growth state suggested that the cell must produce succinate to maintain the nicotinamide adenine dinucleotide (NADH) production/consumption balance (Figure A).…”

“…Klamt and Mahadevan revealed the topological requirements that must be fulfilled for coupling of growth with product synthesis by the gene knockout strategy. Although several studies have successfully reported the various targets, such as 3‐hydroxypropionic acid, 2,3‐butanediol, 1,4‐butanediol (1,4‐BDO), and succinate using FBA, many of the strains do not achieve the predicted ideal performance . These inconsistencies arise because the FBA simplifies the complicated metabolic system by considering only the mass balance based on the steady‐state assumption.…”

Chasing bacterial chassis for metabolic engineering: a perspective review from classical to non‐traditional microorganisms

“…Recently, ALE has been applied together with flux balance analysis to generate an E . coli strain with increased succinate production from glycerol (Tokuyama et al ., ), and l ‐serine, an amino acid usually considered to be toxic (Mundhada et al ., ). The potential of ALE for evolving industrially relevant production phenotypes has been mostly limited to E .…”