A recursive pathway for isoleucine biosynthesis arises from enzyme promiscuity

Reviewer #1 (Public review):

As presented in this short report, the focus is to only establish that acetohydroxyacid synthase II can have underground activity to generate 2-ketobutyrate (from glyoxylate and pyruvate). Additionally, the gene that encodes this protein has an inactivating point mutation in the lab strain of E. coli. In strains lacking the conventional Ile biosynthesis pathway, this enzyme gets reactivated (after short-term laboratory evolution) and putatively can contribute to producing sufficient 2-ketobutyrate, which can feed into Ile production. This is clearly a very interesting observation and finding, and the paper focuses on this single point.

However, the manuscript as it currently stands is 'minimal', and just barely shows that this reaction/pathway is feasible. There is no characterization of the restored enzyme's activity, rate, or specificity. Additionally, there is no data presented on how much isoleucine can be produced, even at saturating concentrations of glyoxylate or pyruvate. This would greatly benefit from more rigorous characterization of this enzyme's activity and function, as well as better demonstration of how effective this pathway is in generating 2-ketobutyrate (and then its subsequent condensation with pyruvate).

Reviewer #2 (Public review):

Summary:

The manuscript by Rainaldi et al. reports a new sub-pathway for isoleucine biosynthesis by demonstrating the promiscuous activity of the native enzyme acetohydroxyacid synthase II (AHAS II). AHAS-II is primarily known to catalyze the condensation of 2-ketobutyrate (2KB) with pyruvate to form a further downstream intermediate, AHB, in the isoleucine biosynthesis pathway. However, the catalysis of pyruvate and glyoxylate condensation to produce 2KB via the ilvG encoded AHAS II is reported in this manuscript for the first time.

Using an isoleucine/2KB auxotrophic E. coli strain, the authors report (i) repair of the inactivating frameshift mutation in the ilvG gene, which encodes AHAS-II, supports growth in glyoxylate-supplemented media, (ii) the promiscuity of AHAS-II in glyoxylate and pyruvate condensation, resulting in the formation of isoleucin precursors (2-KB), aiding the biosynthesis of isoleucine, and (iii) comparable efficiency of the recursive AHAS-II route to the canonical routes of isoleucin biosynthesis via computational Flux-based analysis.

Strengths:

The authors have used laboratory evolution to uncover a non-canonical metabolic route. The metabolomics and FBA have been used to strengthen the claim.

Weaknesses:

While the manuscript proposes an interesting metabolic route for the isoleucine biosynthesis, the data lack key controls, biological replicates, and consistency. The figures and methods are presented inadequately. In the current state, the data fails to support the claims made in the manuscript.

Author response:

We gratefully acknowledge the comments on our manuscript and the time you took to read and understand our work. Nevertheless, it is the opinion of these authors that the evidence provided in the submitted paper is strong and we performed multiple replicates of the experiments. In particular, gene deletion and complementation is the accepted gold standard for studies in physiology. In the isoleucine auxotroph (IMaux) strain carrying an ilvG deletion, growth is only possible if ilvG is reintroduced on a plasmid and induced. Additionally, isotopic labeling clearly demonstrates the activity of the proposed pathway. Regardless, we agree with the reviewers that the paper and the scientific community would benefit from an in vitro characterization of the promiscuity of IlvG, so we will perform this experiment and resubmit the paper for further revision, and in this revision also provide more detail on the replicates performed.