Strong Reduction of Acetate Overflow in Escherichia coli By Systems Metabolic Engineering

The biotechnology industry has extensively exploited Escherichia coli for producing recombinant proteins, low molecular weight compounds etc. However, high growth/glucose uptake rate aerobic E. coli cultivations are accompanied by carbon wasting as acetate i.e. acetate overflow. Elimination of acetate overflow would improve many bioprocesses as acetate accumulation in the growth environment leads to numerous negative effects as it inhibits growth, diverts valuable carbon from biomass/product formation and is detrimental for target product synthesis. Despite decades of studies, mechanism and regulation of acetate overflow are still not completely understood. Hence, the aim of this work was to elucidate the mechanism and regulation behind acetate overflow using a comprehensive systems biology approach leading to the design of reduced overflow strains.
In this work, we continuously monitored specific growth rate (μ)-dependent acetate overflow dynamics of wild-type E. coli using advanced continuous cultivation methods (A-stat
and D-stat). Absolute quantitative exo-metabolome, transcriptome and proteome analyses coupled to metabolic flux analysis revealed that acetate overflow (at μ=0.27±0.02 h-1) in E. coli is triggered by carbon catabolite repression-mediated down-regulation of acetyl-CoA syntethase (Acs) resulting in decreased assimilation of acetate produced by phosphotransacetylase (Pta) and disruption of the PTA-ACS node. This was confirmed by two-substrate A-stat and D-stat experiments which showed that acetate consumption capability of E. coli decreased drastically, just as Acs expression, before the start of acetate overflow. These results suggested insufficient Acs activity for completely consuming the acetate produced by Pta, leading to disruption of acetate recycling in the PTA-ACS node where constant acetyl-phosphate or acetate regeneration is essential for E. coli chemotaxis, proteolysis, pathogenesis etc. regulation. The latter indicated that E. coli actually synthesizes acetate constantly at all μ under aerobic conditions and no acetate overflow occurs at low μ since acetate is fully recycled in the PTA- ACS node. This indeed turned out to be the case as both E. coli mutants (Î?acs and Î?cobB) unable to consume acetate in the PTA-ACS node excreted acetate at all μ studied, further
highlighting the relevance of Acs in acetate overflow regulation.
The following obvious effort to diminish acetate overflow based on the latter results was to increase the expression of Acs. However, a strain overexpressing acs did not lead to reduced
acetate overflow which could have arisen from a substantial part of the Acs protein pool being inactive as Acs activity in vivo is known to be actively repressed by post-translational acetylation.
Thus we next analyzed growth of E. coli lacking protein lysine acetyltransferase (Pka), known to inactivate Acs, which resulted in postponed start of acetate overflow. We surmised that acetate overflow could be further reduced by slightly increased levels of active Acs in Î?pka and
increased throughput of downstream pathways (e.g. TCA cycle) to recycle more acetate into acetyl-CoA and process the latter downstream to divert carbon away from acetate. Exactly this
was achieved by deleting the TCA cycle regulator arcA in Î?pka background leading to further postponed onset and strongly reduced acetate overflow by the coordinated activation of PTA- ACS and TCA cycles. The double deletion Î?pka Î?arcA strain showed 4-fold reduced acetate
overflow (2 vs 8% from total carbon) at fastest growth compared to wild-type, did not accumulate any other detrimental by-product besides acetate, showed identical μmax and only
~5% lower biomass yield compared to wild-type. Moreover, this strain would enable production of target compounds in the absence of acetate at considerably higher growth/glucose uptake rates most probably resulting in higher volumetric productivities (~22% higher gDCW L-1 h-1 compared to wild-type). We conclude that a fine-tuned coordination between increasing the recycling capabilities of acetate in the PTA-ACS node through higher pool of active acetate scavenging Acs protein and downstream metabolism throughput in the TCA cycle are necessary for diminishing acetate overflow in E. coli.
This work demonstrates that a simple genetic overexpression does not work in all cases for achieving the desired effects but the expression level of the relevant fraction of the protein pool (active Acs in this case) has to be fine-tuned together with downstream throughput (TCA).
To the best of our knowledge, this is the first successful application of modification of protein acetylation for metabolic engineering in E. coli. Furthermore, we see this work being a good example for proving the value of systems biology study of metabolism for successful metabolic engineering of strains with potential interest for industrial use.