Functional reconstitution of a bacterial CO 2 concentrating mechanism in E. coli

  1. Avi I. Flamholz
  2. Eli Dugan
  3. Cecilia Blikstad
  4. Shmuel Gleizer
  5. Roee Ben-Nissan
  6. Shira Amram
  7. Niv Antonovsky
  8. Sumedha Ravishankar
  9. Elad Noor
  10. Arren Bar-Even
  11. Ron Milo
  12. David F. Savage

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Abstract

Many photosynthetic organisms employ a CO 2 concentrating mechanism (CCM) to increase the rate of CO 2 fixation via the Calvin cycle. CCMs catalyze ≈50% of global photosynthesis, yet it remains unclear which genes and proteins are required to produce this complex adaptation. We describe the construction of a functional CCM in a non-native host, achieved by expressing genes from an autotrophic bacterium in an engineered E. coli strain. Expression of 20 CCM genes enabled E. coli to grow by fixing CO 2 from ambient air into biomass, with growth depending on CCM components. Bacterial CCMs are therefore genetically compact and readily transplanted, rationalizing their presence in diverse bacteria. Reconstitution enabled genetic experiments refining our understanding of the CCM, thereby laying the groundwork for deeper study and engineering of the cell biology supporting CO 2 assimilation in diverse organisms.

One Sentence Summary

A bacterial CO 2 concentrating mechanism enables E. coli to fix CO 2 from ambient air.

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  1. eLife

    ###Reviewer #3:

    General assessment:

    The work presented is a major scientific achievement. This is the first functional reconstitution of any CO2 concentrating mechanism. The work has major implications for engineering of CCMs into crops for increasing yields: the authors have definitively identified a set of components that confer CCM activity in a heterologous host. As a bonus, the authors demonstrate a new way of generating a Rubisco-dependent E. coli.

    The writing is generally clear. The claims are well-supported by multiple lines of evidence. The engineered Rubisco-dependent E. coli showed clear improvements in growth phenotypes after introduction of H. neapolitanus CCM genes, which were then confirmed using thorough genetic and biochemical analyses.

    Major comment:

    The control EM images in Figure 5 should be present in the main figure, not a supplement. It is concerning that the positive control failed. It should be repeated, or, if possible, it would really help to show TEMs of WT H. neapolitanus. This would allow comparison of the putative carboxysomes to a native carboxysome and would greatly improve the quality and value of this figure.

  2. eLife

    ###Reviewer #2:

    The manuscript by Flamholz et al. is a significant and excellent piece of work that is very novel and would have wide appeal to a range of microbiologists and general biologists. The manuscript is well written and represents a very interesting and largely complete set of data.

    It was an ambitious goal to convert a model bacterium such as E. coli into a system that is able to grow with dependence on the CO2-fixing enzyme Rubisco, and a basic Calvin Cycle. The authors have achieved that, and as expected these engineered cells required a very high 10% CO2 for optimal growth. No LB media was required except for addition of some minimal salts and glycerol. Without added CO2 growth does not proceed with glycerol alone. Next, and Importantly, they then asked if they could add a basic CO2 concentrating mechanism (CCM) from a sulphur bacterium (Halothiobacillus) so that the E. coli cells could scavenge and accumulate enough inorganic carbon (CO2 /bicarbonate) to grow at air levels of CO2 (namely 0.04% CO2). Some 20 genes were required to make up this basic CCM work, namely a complete carboxysome operon, genes for a Ci pump (DabBA2), Rubisco genes, phosphoribulokinase, and engineered removal of both carbonic anhydrase genes from E.coli as well as riboseP-isomerase. The growth rate of cells at air was relatively slow, but shown to be at an expected rate based on modelling. Ultimately this work has implications towards the question of whether a basic CCM could function in a plant chloroplast and provides a boost to photosynthetic CO2 fixation. It seems to support this goal.

    Curiously, the complete 20-gene system did not initially allow growth at air CO2 levels, but did work after a series of directed evolution experiments in bioreactors that led to some minor mutations. It is noted that one of these changes was the transfer of a the high copy number origin from one plasmid to the other, while some were 'regulatory' elements within the pCCM and pCB plasmids, then designated as pCCM' and pCB' plasmids after mutations. The authors should provide more detail on the net result of these mutations, as to whether expression was altered upwards or downwards for the two key plasmids? QPCR would be adequate.

    One of the remarkable achievements in this manuscript is to mark out the necessary changes to convert an enteric bacterium into an organism that is dependent on Rubisco for CO2 fixation/carbon gain at limited CO2 levels (and glycerol as an initial carbon backbone). No more than 20 genes are required, possibly less, and clearly all the primary genes to assemble one example of a functional alpha-type carboxysome is now proven because of this experiment. Though there are likely to be some general chaperones required that the host provides.

  3. eLife

    ###Reviewer #1:

    The photosynthetic efficiency of C3 plants is largely limited by the catalytic inefficiency of rubisco, the CO2 fixing enzyme in the Calvin-Benson-Bassham cycle of photosynthesis. Since rubisco can also react with O2, bacteria, algae and C4 plants have evolved CO2 concentrating mechanisms (CCMs) to increase the concentration of CO2 around rubisco. The CCM promotes carboxylation and inhibits the competitive oxygenation reaction of rubisco. Transplanting CCMs into C3 crop plants is considered a promising strategy to improve rubisco's photosynthetic performance. Bacterial CCMs consist of two essential components: inorganic carbon transporters at the membrane and the proteinaceous shell organelle, carboxysomes. Reconstitution of carboxysomes in E. coli and tobacco have been previously reported, however, there is no report of a functioning reconstituted CCM.

    In this paper, the authors introduced 20 CCM-related genes from the proteobacterium H. neapolitanus into E. coli cells which have been engineered to be dependent on rubisco function for growth. Their results show that at most 20 genes are sufficient to generate a bacterial CCM which enables E. coli to grow at ambient CO2 concentration due to efficient fixation of CO2 by rubisco. This manuscript provides a useful platform for future investigations to establish the minimal number of genes required for transplanting the cyanobacterial CCM into non-native autotrophic hosts to improve their CO2 assimilation and growth.

    Major comments:

    1. For the benefit of a non-expert reader, the names of the 20 proteins and corresponding genes should listed in a Table, together with their function and the relevant references.

    2. In Figure 3-figure supplement 1A, the authors should discuss why the gene csos1D is present in both pCB and pCCM.

    3. In Figure 4B, the large variance in the OD600 after 4 days for CCMB1:pCB'+pCCM' cultures was explained as being due to genetic effects or non-genetic differences (line 1064). However, in Figure 3 - figure supplement 2B the measured growth kinetics did not show such big differences.

    4. The negative control in Figure 5-figure supplement 1 is too dark and difficult to compare with the other micrographs. Moreover, to observe recombinant carboxysomes in the positive control (WT:pHnCB10), the authors should have induced the cells using a lower concentration of IPTG as reported previously by Bonacci et. al. (PNAS 2012).

  4. eLife

    ##Preprint Review

    This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 3 of the manuscript. The major points agreed by the reviewers are included below, as well as the separate reviews.

    ###Summary:

    The work presented is a major scientific achievement. This is the first functional reconstitution of any CO2 concentrating mechanism (CCM). The work has major implications for engineering of CCMs into crops for increasing yields: the authors have definitively identified a set of components that confer CCM activity in a heterologous host. As a bonus, the authors demonstrate a new way of generating a Rubisco-dependent E. coli.

    Major points:

    1. The EM images shown in Figure 5-figure supplement 1 should be presented as a main figure, not a supplement. The negative control is too dark and difficult to compare with the other micrographs. Moreover, it is concerning that the positive control (WT:pHnCB10) failed. It should be repeated as it would allow comparison of the putative carboxysomes to a native carboxysome and would greatly improve the quality and value of this figure.

    2. For the benefit of a non-expert reader, the names of the 20 proteins and corresponding genes should listed in a Table, together with their function and the relevant references.

    3. In Figure 3-figure supplement 1A, the authors should discuss why the gene csos1D is present in both pCB and pCCM.

    4. In Figure 4B, the large variance in the OD600 after 4 days for CCMB1:pCB'+pCCM' cultures was explained as being due to genetic effects or non-genetic differences (line 1064). However, in Figure 3 - figure supplement 2B the measured growth kinetics did not show such big differences. Authors please explain.

    5. Would be nice if the authors can demonstrate that Rubisco localizes to the putative carboxysomes by performing an experiment such as immunogold labeling. It would improve the claim that the observed polyhedral bodies are in fact carboxysomes. We leave the decision of such an experiment to the authors.

  5. Version published to 10.1101/2020.05.27.119784 on bioRxiv