Graphical Abstract Highlights d Conversion of obligate heterotroph to full autotrophy over laboratory timescales d Non-native Calvin cycle operation generates biomass carbon from CO 2 in E. coli d Formate is oxidized by heterologous formate dehydrogenase to provide reducing power d Chemostat-based directed evolution led to complete trophic mode change in z200 days In BriefMetabolic rewiring and directed evolution led to the emergence of E. coli clones that use CO 2 as their sole carbon source, while formate is oxidized to provide all the reducing power and energy demands. SUMMARYThe living world is largely divided into autotrophs that convert CO 2 into biomass and heterotrophs that consume organic compounds. In spite of widespread interest in renewable energy storage and more sustainable food production, the engineering of industrially relevant heterotrophic model organisms to use CO 2 as their sole carbon source has so far remained an outstanding challenge. Here, we report the achievement of this transformation on laboratory timescales. We constructed and evolved Escherichia coli to produce all its biomass carbon from CO 2 . Reducing power and energy, but not carbon, are supplied via the one-carbon molecule formate, which can be produced electrochemically. Rubisco and phosphoribulokinase were co-expressed with formate dehydrogenase to enable CO 2 fixation and reduction via the Calvin-Benson-Bassham cycle. Autotrophic growth was achieved following several months of continuous laboratory evolution in a chemostat under intensifying organic carbon limitation and confirmed via isotopic labeling.

Many photosynthetic organisms employ a CO2 concentrating mechanism (CCM) to increase the rate of CO2 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 E. coli strain engineered to depend on rubisco carboxylation for growth. Expression of 20 CCM genes enabled E. coli to grow by fixing CO2 from ambient air into biomass, with growth in ambient air depending on the components of the CCM. 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 CO2 assimilation in diverse organisms.

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22One Sentence Summary 33 A bacterial CO2 concentrating mechanism enables E. coli to fix CO2 from ambient air. 34 (Desmarais et al., 2019) expressing rubisco and its associated chaperones (green), carboxysome structural proteins 96 (purple), and an inorganic carbon transporter (orange). 98Using a genome-wide screen in the CO2-fixing proteobacterium H. neapolitanus, we recently 99 demonstrated that a 20-gene cluster encodes all activities required for the CCM, at least in 100principle (Desmarais et al., 2019). These genes include rubisco large and small subunits, the 101 carboxysomal carbonic anhydrase, seven structural proteins of the ɑ-carboxysome (Bonacci et 102 al., 2012), an energy-coupled inorganic carbon transporter (Desmarais et al., 2019; Scott et al., 103 2019), three rubisco chaperones (Aigner et al., 2017; Mueller-Cajar, 2017; Wheatley et al., 2014), 104and four genes of unknown function ( Figure 1C). We aimed to test whether these genes are 105 sufficient to establish a functioning CCM in E. coli. 106 107 Figure 2. CCMB1 depends on rubisco 108 carboxylation for growth on glycerol. (A) Ribose-109 5-phosphate (Ri5P) is required for nucleotide 110 biosynthesis. Deletion of ribose-phosphate 111 isomerase (Δrpi) in CCMB1 blocks ribulose-5-112 phosphate (Ru5P) metabolism in the pentose 113 phosphate (PP) pathway. Expression of rubisco (H. 114 neapolitanus cbbLS) and phosphoribulokinase (S. 115 elongatus PCC7942 prk) on the p1A plasmid (B) 116 permits Ru5P metabolism, thus enabling growth on 117 M9 glycerol media in 10% CO2 (C). Mutating the 118 rubisco active site (p1A cbbL -) abrogates growth, as 119 does mutating ATP-binding residues of prk (p1A 120 prk -). (D) CCMB1:p1A grows well under 10% CO2, 121 but fails to grow in ambient air. Cells grown on M9 122 glycerol media throughout. The algorithmic design 123 of CCMB1 is described in figure supplement 1 and 124 the mechanism of rubisco-dependence is 125 diagrammed in figure supplement 2. Figure 126 supplement 3 shows CCMB1:p1A growth 127 phenotypes on various media and figure 128 supplement 4 demonstrates that rubisco 129 oxygenation is not required for growth by 130 demonstrating growth in the absence of O2. 131 Acronyms: ribulose 1,5-bisphosphate (RuBP), 3-132 phosphoglycerate (3PG).133

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