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Christel Malden
This is an important follow-up study to a previous paper in which the authors reconstituted CO2 metabolism (autotrophy) in Escherichia coli. Here, the authors define a set of just three mutations that promote autotrophy, highlighting the malleability of E. coli metabolism. The authors make a convincing case that mutations in pgi are loss-of-function mutations that prevent metabolic efflux from the reductive pentose phosphate autocatalytic cycle, and their data suggest possible roles of mutations in two other genes - crp and rpoB. This research will be particularly interesting to synthetic biologists, systems biologists, and metabolic engineers aiming to develop synthetic autotrophic microorganisms.
Recently, with the increasing interest in platforms for sustainable production, synthetic biologists have been able to successfully manipulate different organisms to enable major metabolic transitions (Gassler et al. 2020; Gleizer et al. 2019; Kim et al. 2020; Chen et al. 2020; Keller et al. 2022; Bang et al. 2020; Satanowski et al. 2020; Yishai et al. 2018). These efforts resulted in the successful introduction of non-native C1 utilisation pathways, and even included successful transitions from heterotrophic into completely autotrophic metabolisms in bacteria (Gleizer et al. 2019) and yeast (Gassler et al. 2020). These previous studies introduced the reductive pentose phosphate (rPP) cycle, the most prominent carbon fixation pathway in nature (also known as the Calvin-Benson cycle), into Escherichia coli (E. coli) and Komagataella phaffii (previously Pichia pastoris). Indeed, we showed that E. coli was able to utilise the rPP cycle for the synthesis of all biomass carbon - thus converting it from a heterotroph to an autotroph (Gleizer et al. 2019). However, this transition required continuous culture in selective conditions for several months (adaptive lab evolution) and resulted in many uninterpretable mutations, exposing a large knowledge gap.
Adaptive lab evolution is an effective tool that enables integration of non-native metabolic pathways. In order to harness the power of adaptive lab evolution, a selection system must be implemented to direct the evolution towards the desired function. In our case, the selection for the activity of the non-native rPP cycle. To ensure that some level of carbon fixation by RuBisCO becomes essential for growth, we knocked out phosphofructokinase (pfkA and pfkB), and 6-phosphate-1-dehydrogenase (zwf) which creates a stoichiometric imbalance and growth arrest when growing on five carbon sugars such as xylose (Gleizer et al. 2019). This imbalance could be rescued by a metabolic bypass - the phosphorylation of ribulose-5-P by Prk and the subsequent carboxylation by RuBisCO, thereby coupling growth to the heterologously expressed rPP enzymes. Critically, this setup allowed us to evolve E. coli, a natural heterotroph, into an autotroph, using a non-native rPP cycle to utilise CO2 as a sole carbon-source. This system also requires formate dehydrogenase to oxidise formate for energy (Gleizer et al. 2019).
To isolate the essential genetic changes, we used a workflow that included three main stages as shown in Fig. 1. A) rational design-introduction of required heterologous genes and metabolic knockouts to enforce RuBisCO-dependent growth; B) adaptive lab evolution-revealing mutation candidates by growing the designed strain in autotrophic-selecting conditions over many generations until an autotrophic phenotype is obtained; C) evolution-inspired genetic engineering-introducing the most promising mutations revealed in stage B into the designed strain and testing it for autotrophic growth. Stages B and C were repeated until no further evolution was required, i.e. the designed strain in stage C had an autotrophic phenotype.
We introduced the H386Y pgi mutation into the rationally designed ancestor (Fig. 1, step C1), and the engineered strain was tested for growth in autotrophic conditions (methods). No growth was observed in those conditions, which meant that additional mutations were necessary in order to achieve autotrophy.
Genomic sequencing revealed that during the genetic manipulation for introducing the pgi mutation, another mutation in rpoB (A1245V) appeared in the genome. In parallel, reverting a variety of RNA polymerase mutations in other evolved strains back to their wild-type allele, showed that mutations in RNA polymerase are in fact essential to the phenotype. Therefore, despite the fact that it was unintentional, and different from the other rpoB mutations observed in the ALE experiments, we left the mutation in the genome and proceeded with this strain.
We verified the genotype using whole genome sequencing (methods). The results included the rationally designed knockouts (ΔpfkA, ΔpfkB, Δzwf), heterologous plasmids (energy and carbon-fixing modules) and the three introduced mutations (pgi*, crp* and rpoB*). During the genetic engineering process, two additional mutations occurred unintentionally in the genes uhpT and yejG. uhpT is a hexose transporter and is unlikely to have any effect on the autotrophic phenotype, especially because the mutation was an early stop codon (Q7*) and likely a loss of function. As the function of yejG is unknown, we wanted to ensure that it is not essential to the autotrophic phenotype. Therefore, we reverted the mutation back to its wild-type allele and found that indeed the cells were still autotrophic.
As described above, in order to select for autotrophic growth, the native E. coli was metabolically re-wired by 3 auxiliary genomic knockouts (ΔpfkA, ΔpfkB, Δzwf). These knockouts created a dependency on carboxylation by RuBisCO for growth even when consuming pentose sugars which were supplemented during the chemostat evolution (see methods, Antonovsky et al. 2016; Gleizer et al. 2019). This dependency was expected to direct the evolution towards increased usage of the synthetic rPP cycle and, eventually, to autotrophic growth.
We transformed a wild-type BW25113 E. coli strain (black bacteria) with the carboxylating and energy module plasmids (grey circles with coloured gene annotations), and inserted 3 auxiliary genomic knockouts (red octagon) to rewire metabolism toward carboxylating dependency. This strain was used for iterative evolution experiments in order to generate diverse autotrophic strains and reveal mutations candidates for rational design, i.e. consensus mutations. The identified autotrophic enabling mutations (thin grey circle) were then introduced in a wild type strain expressing the heterologous plasmids but without the auxiliary knockouts, the final strain was able to grow in autotrophic conditions. Dashed lines represent gene/mutation introduction.
We thus established a new engineered autotrophic E. coli strain containing a set of three essential and sufficient mutations, and showed that the bypass-prevention knockouts introduced before the ALE are themselves not necessary for the phenotype. Due to the small required number of genetic modifications, this strain provides an opportunity to derive guidelines for future engineering efforts that use the rPP cycle. Having this goal in mind, we focused our attention on identifying the relevant phenotypes of each of these mutations and explaining their adaptive advantage. Because the mutations were essential for the autotrophic growth, we could not use autotrophic conditions to compare them to a strain with wild-type alleles. Therefore, we chose the most suitable combinations of strains and conditions that could isolate the effect of the pertinent mutation.
Metabolic pathways that regenerate and synthesise more of their own metabolites are referred to as autocatalytic cycles. Within the autotrophic E. coli the rPP cycle is autocatalytic. Due to the inherent positive feedback mechanism, autocatalytic cycles tend to be unstable, and therefore the fluxes of entry and exit points (bifurcation/branch points) need to be balanced (Barenholz et al. 2017). Such tuning is needed since any disruption of the balance between cycling and branching fluxes could result in the depletion, or alternatively toxic accumulation of intermediate metabolites, and cycle arrest.
We previously predicted that mutations in branch points will be needed to stabilise the steady state flux within the rPP cycle (Barenholz et al. 2017). In line with this prediction we find that the mutation in phosphoglucoisomerase (Pgi), a key branch point in the rPP cycle, follows this design principle. Pgi consumes fructose-6-phosphate (F6P), one of the cycle intermediates, converting it into glucose-6-phosphate (G6P), a precursor for cell membrane biosynthesis. Thus, Pgi regulates flux out of the autocatalytic cycle (Fig. 4A).
In the different adaptive lab evolution experiments, we found three distinct mutations in the pgi gene. The first, H386Y, is a non-synonymous mutation occurring in one of the catalytic residues in the active site, and is part of the autotrophic enabling gene set. The second was a complete knockout, a 22KB chromosomal deletion, including also 16 other genes. The third was an early stop codon E72*. These observations led us to suspect the H386Y mutation in pgi decreases or even completely eliminates the activity of the enzyme.
Pgi is part of glycolysis/gluconeogenesis, where significant flux is required in wild type E. coli. Accordingly, wild type Pgi catalyses a reaction that is causing a strong efflux of F6P from the cycle, diminishing the regeneration of ribulose-1,5-bisphosphate (RuBP) which is usually not needed in the native metabolism but is essential for the rPP autocatalytic activity. In the mutated version of Pgi the efflux capacity is diminished, which can stabilise the cycle (Fig. 4C).
Following the same logic, we can expect an increase in the F6P substrate metabolite pool due to reduced consumption by Pgi. Therefore, we measured intracellular sugar-phosphates in the pgi mutant strain and a strain with a wild-type pgi allele by liquid chromatography-tandem mass spectrometry (LC-MS/MS). For this comparison, we used the RuBisCO-dependent ancestor strain as the genetic background. We found that the ratio of F6P to G6P was about 3 times higher in the pgi mutant strain relative to the wild-type (Fig. 4D). Furthermore, the pgi mutant had higher levels of metabolites within the rPP cycle (Fig. 4D), confirming the stabilising function of the mutation.
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