Autotrophic growth of E. coli is achieved by a small number of genetic changes

Reviewer #1 (Public Review):

The main objective of this study is to achieve the development of a synthetic autotroph using adaptive laboratory evolution. To accomplish this, the authors conducted chemostat cultivation of engineered E. coli strains under xylose-limiting conditions and identified autotrophic growth and the causative mutations. Additionally, the mutational mechanisms underlying these causative mutations were also explored with drill down assays. Overall, the authors demonstrated that only a small number of genetic changes were sufficient (i.e., 3) to construct an autotrophic E. coli when additional heterologous genes were added. While natural autotrophic microorganisms typically exhibit low genetic tractability, numerous studies have focused on constructing synthetic autotrophs using platform microorganisms such as E. coli. Consequently, this research will be of interest to synthetic biologists and systems biologists working on the development of synthetic autotrophic microorganisms. The conclusions of this paper are mostly well supported by appropriate experimental methods and logical reasoning. However, further experimental validation of the mutational mechanisms involving rpoB and crp would enhance readers' understanding and provide clearer insights, despite acknowledgement that these genes impact a broad set of additional genes. Additionally, a similar study, 10.1371/journal.pgen.1001186, where pgi was deleted from the E. coli genome and evolved to reveal an rpoB mutation is relevant to this work and should be placed in the context of the presented findings.

The authors addressed rpoB and crp as one unit and performed validation. They cultivated the mutant strain and wild type in a minimal xylose medium with or without formate, comparing their growth and NADH levels. The authors argued that the increased NADH level in the mutant strain might facilitate autotrophic growth. Although these phenotypes appear to be closely related, their relationship cannot be definitively concluded based on the findings presented in this paper alone. Therefore, one recommendation is to explore investigating transcriptomic changes induced by the rpoB and crp mutations. Otherwise, conducting experimental verification to determine whether the NADH level directly causes autotrophic growth would provide further support for the authors' claim.

Reviewer #2 (Public Review):

Synthetic autotrophy of biotechnologically relevant microorganisms offers exciting chances for CO2 neutral or even CO2 negative production of goods. The authors' lab has recently published an engineered and evolved Escherichia coli strain that can grow on CO2 as its only carbon source. Lab evolution was necessary to achieve growth. Evolved strains displayed tens of mutations, of which likely not all are necessary for the desired phenotype.

In the present paper the authors identify the mutations that are necessary and sufficient to enable autotrophic growth of engineered E. coli. Three mutations were identified, and their phenotypic role in enhancing growth via the introduced Calvin-Benson-Bassham cycle were characterized. It was demonstrated that these mutations allow autotrophic growth of E. coli with the introduced CBB cycle without any further metabolic intervention. Autotrophic growth is demonstrated by 13C labelling with 13C CO2, measured in proteinogenic amino acids. In Figures 2B and S1, the labeling data are shown, with an interval of the "predicted range under 13CO2". Here, the authors should describe how this interval was derived.

The methodology is clearly described and appropriate.

The present results will allow other labs to engineer E. coli and other microorganisms further to assimilate CO2 efficiently into biomass and metabolic products. The importance is evident in the opportunity to employ such strain in CO2 based biotech processes for the production of food and feed protein or chemicals, to reduce atmospheric CO2 levels and the consumption of fossil resources.

Reviewer #3 (Public Review):

The authors previously showed that expressing formate dehydrogenase, rubisco, carbonic anhydrase, and phosphoribulokinase in Escherichia coli, followed by experimental evolution, led to the generation of strains that can metabolise CO2. Using two rounds of experimental evolution, the authors identify mutations in three genes - pgi, rpoB, and crp - that allow cells to metabolise CO2 in their engineered strain background. The authors make a strong case that mutations in pgi are loss-of-function mutations that prevent metabolic efflux from the reductive pentose phosphate autocatalytic cycle. The authors also argue that mutations in crp and rpoB lead to an increase in the NADH/NAD+ ratio, which would increase the concentration of the electron donor for carbon fixation. While this may explain the role of the crp and rpoB mutations, there is good reason to think that the two mutations have independent effects, and that the change in NADH/NAD+ ratio may not be the major reason for their importance in the CO2-metabolising strain.

Specific comments:

1. Deleting pgi rather than using a point mutation would allow the authors to more rigorously test whether loss-off-function mutants are being selected for in their experimental evolution pipeline. The same argument applies to crp.

2. Page 10, lines 10-11, the authors state "Since Crp and RpoB are known to physically interact in the cell (26-28), we address them as one unit, as it is hard to decouple the effect of one from the other". CRP and RpoB are connected, but the authors' description of them is misleading. CRP activates transcription by interacting with RNA polymerase holoenzyme, of which the Beta subunit (encoded by rpoB) is a part. The specific interaction of CRP is with a different RNA polymerase subunit. The functions of CRP and RpoB, while both related to transcription, are otherwise very different. The mutations in crp and rpoB are unlikely to be directly functionally connected. Hence, they should be considered separately.

3. A Beta-galactosidase assay would provide a very simple test of CRP H22N activity. There are also simple in vivo and in vitro assays for transcription activation (two different modes of activation) and DNA-binding. H22 is not near the DNA-binding domain, but may impact overall protein structure.

4. There are many high-resolution structures of both CRP and RpoB (in the context of RNA polymerase). The authors should compare the position of the sites of mutation of these proteins to known functional regions, assuming H22N is not a loss-of-function mutation in crp.

5. RNA-seq would provide a simple assay for the effects of the crp and rpoB mutations. While the precise effect of the rpoB mutation on RNA polymerase function may be hard to discern, the overall impact on gene expression would likely be informative.