Design and analysis of synthetic carbon fixation pathways based on novel enzymatic reactions

Design and analysis of synthetic carbon fixation pathways based on novel enzymatic reactions | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Design and analysis of synthetic carbon fixation pathways based on novel enzymatic reactions View ORCID Profile Vittorio Rainaldi , View ORCID Profile Sarah D’Adamo , View ORCID Profile Nico J. Claassens doi: https://doi.org/10.1101/2025.11.21.689712 Vittorio Rainaldi 1 Laboratory of Microbiology, Wageningen University , Stippeneng 4, 6708 WE, Wageningen, the Netherlands 2 Bioprocess Engineering, Wageningen University , Droevendaalsesteeg 1, 6708 PB, Wageningen, the Netherlands Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Vittorio Rainaldi For correspondence: nico.claassens{at}wur.nl vittorio.rainaldi{at}wur.nl Sarah D’Adamo 2 Bioprocess Engineering, Wageningen University , Droevendaalsesteeg 1, 6708 PB, Wageningen, the Netherlands Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Sarah D’Adamo Nico J. Claassens 1 Laboratory of Microbiology, Wageningen University , Stippeneng 4, 6708 WE, Wageningen, the Netherlands Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Nico J. Claassens For correspondence: nico.claassens{at}wur.nl vittorio.rainaldi{at}wur.nl Abstract Full Text Info/History Metrics Supplementary material Data/Code Preview PDF Abstract Biological carbon fixation is currently limited to seven naturally occurring pathways, each with its own limitations and constraints. In recent years, computational analyses of known biochemical reaction networks have identified dozens of theoretical carbon fixation pathways, some of which may have the potential to outperform their natural counterparts. This mix-and-match approach, however, cannot account for those reactions that have not been reported to occur in nature, which heavily limits the possible solution space. Here, we use a bioretrosynthetic approach coupled with expert biochemical knowledge to identify several novel pathways that leverage enzyme promiscuity and the latent biochemical reaction space. We analyze the thermodynamic, stoichiometric, and kinetic parameters of these pathways and compare them to the ubiquitous Calvin-Benson-Bassham cycle and previously proposed synthetic CO 2 fixation cycles, highlighting advantages and disadvantages. We identify several promising pathways that could potentially outcompete the Calvin cycle and other previously proposed synthetic CO 2 fixation pathways in predicted biomass yield and/or overall pathway activity. In addition, unlike most of the previously proposed efficient mix-and-match pathways, the pathways proposed in this work do not require vitamin B 12 , which is an advantage for future implementation in plants or microalgae that typically lack B 12 biosynthesis. This work highlights the need for enzyme engineering and design in the quest for efficient biological carbon fixation. Introduction The Calvin-Benson-Bassham (CBB) cycle is responsible for over 99% of net primary productivity on earth 1 . Despite its dominance, it is a relatively inefficient pathway, requiring a high energy investment in terms of ATP to fix carbon. Furthermore, its key enzyme, rubisco, is rather slow when compared to other naturally occurring carboxylases and it presents a detrimental side activity with oxygen 2 , 3 , which results in additional energy costs and partial carbon loss during the cellular process known as photorespiration 4 . Many synthetic carbon fixation pathways have been designed so far by using a mix-and-match approach that combines enzymes from disparate sources with fast and efficient carboxylases to generate theoretical cycles that have not been described in nature 5 – 7 . Nevertheless, a recent quantitative analysis of natural and synthetic carbon fixation pathways by Lӧwe and Kremling showed that most pathways are in fact inferior to the CBB cycle or only marginally more efficient in terms of biomass yield and pathway activity 7 , likely making the massive engineering efforts required to implement them unjustified. Almost all of the current synthetic pathway designs, however, are limited by the fact that they only use known enzymatic activities from databases such as KEGG, which vastly limits the possible solution space. Erb, Jones, and Bar-Even have previously put forward a proposal for categorizing pathways based on five levels, with naturally occurring pathways in level one, and synthetic pathways based on new-to-nature enzyme mechanisms in level five 8 . Despite recent progress, designing enzymes with novel mechanisms remains highly challenging 9 , 10 . For this reason, we focused on level four pathways, namely those that are based on unreported promiscuous enzyme activities and reactions from the latent biochemical space. In this study, we use a combination of bioretrosynthesis and expert biochemical knowledge to generate novel pathway designs and analyze their thermodynamic 11 , stoichiometric 12 , and kinetic 13 parameters, comparing them to the CBB cycle and highlighting advantages and disadvantages. All these pathways require engineering of at least one enzyme to change its substrate or cofactor specificity or other relevant parameters such as oxygen tolerance. Oxygen tolerance is particularly important in the context of engineering CO 2 fixation in plants and algae, since these organisms produce oxygen as part of photosynthesis, which would lead to the rapid inactivation of oxygen-sensitive enzymes. However, oxygen tolerance is also important in hydrogen-based microbial bioproduction from CO 2 , as CO 2 fixation and bioproduction pathways require ATP, which is usually generated from O 2 -dependent respiration. Overall, this paper provides a novel approach to pathway design and highlights the need for enzyme engineering to achieve efficient carbon fixation, while offering suggestions regarding possible starting points to engineer the proposed pathways. Results The β-glutamate cycle We recently proposed and showed the partial in vivo implementation of a new family of carbon fixation pathways called the reductive methylaspartate (rMASP) cycles using the standard mix-and-match approach 14 . While these cycles are promising, they suffer from two main issues. First, the enzyme crotonyl-CoA carboxylase/reductase (CCR) is relatively inefficient because the NADPH invested for its reductive carboxylation activity is later only partially recovered in the form of a reduced quinone, leading to a loss of energy (3 protons or roughly 1 ATP). Second, these cycles require vitamin B 12 as a cofactor for two enzymatic reactions. Vitamin B 12 is a highly complex cofactor, which cannot be synthesized by most organisms, importantly including all higher plants and many algae 15 , thus limiting the applicability of pathways containing B 12 -dependent enzymes mostly to bacteria. To overcome both issues, we designed a variant of the rMASP cycles that we name the β-glutamate cycle based on its characteristic metabolite ( Fig. 1 ). According to our computational analysis, the β-glutamate cycle shows favorable parameters when compared to the CBB cycle, including higher max-min (thermodynamic) driving force (MDF), higher theoretical biomass yield, and comparable pathway activity. These advantages increase in ambient CO 2 environments, with a ∼40% increase in yield (assuming 20% Rubisco oxygenation activity for the CBB cycle at ambient CO 2 ) (Table S1). Download figure Open in new tab Figure 1: The β-glutamate cycle. In this figure and following ones, colored spheres represent different carbon groups and colored shapes represent cofactors or other molecules as represented in the legend. Both input and output molecules are framed by boxes. 1, succinyl-CoA synthetase; 2, succinyl-CoA reductase; 3, succinate semialdehyde dehydrogenase; 4, 4-hydroxybutyrate CoA transferase; 5, 4-hydroxybutyryl-CoA dehydratase; 6, crotonyl-CoA carboxylase; 7, glutaconate CoA transferase; 8, glutaconate ammonia lyase; 9, glutamate 2,3-aminomutase; 10, glutamate dehydrogenase; 11, 2-oxoglutarate carboxylase; 12, isocitrate dehydrogenase; 13, isocitrate lyase. In this figure and following ones the enzymes marked in red have never been reported in the literature. Bioprospecting or engineering efforts will likely be required to establish their activities. The β-glutamate cycle does not use CCR but instead depends on the promiscuous activity of methylcrotonyl-CoA gamma carboxylase (MCC) 16 , 17 , which we will refer to as crotonyl-CoA gamma carboxylase (CCC). This biotin-dependent carboxylase is involved in leucine degradation across diverse phyla including bacteria 18 , 19 and humans 20 . Its promiscuous side activity on the slightly smaller crotonyl-CoA has been reported in the literature with about 90% of the reaction rate when compared to the native substrate. Since the carboxylation happens in gamma, as opposed to alpha, it produces glutaconyl-CoA instead of ethylmalonyl-CoA, thus obviating the need for a B 12 dependent mutase to “straighten” the carbon skeleton. The cycle proceeds with the removal of the CoA from glutaconyl-CoA via a CoA transferase or CoA ligase to yield glutaconate, which is further aminated to 3-aminoglutarate, also known as β-glutamate, which gives its name to the cycle. This amination reaction could be promiscuously performed by aspartate/methylaspartate ammonia lyase 21 , though most likely it would require engineering to expand the substrate binding pocket to accommodate one extra carbon. An alternative to this route could be the direct amination of glutaconyl-CoA, for example by a promiscuous β-alanyl-CoA ammonia lyase, an enzyme found in anaerobic bacteria and used for β-alanine fermentation 22 . The resulting β-glutamyl-CoA would once again require a CoA transferase or CoA ligase to remove the CoA and yield β-glutamate. At this point, β-glutamate must undergo isomerization to produce glutamate. This reaction is catalyzed by the naturally occurring enzyme glutamate 2,3-aminomutase 23 , which is present in bacteria and archaea, respectively for glutamate fermentation and for the production of β-glutamate as an osmolyte 24 . This reaction, however, is highly challenging, requiring a radical mechanism based on S-adenosyl-methionine (SAM), making the enzyme oxygen-sensitive. Nevertheless, there are oxygen-tolerant variants reported to catalyze the same reaction aerobically, though using lysine as a substrate as opposed to glutamate 25 – 27 . Not much is known about this enzyme class, so it is difficult to know whether it would be easier to engineer oxygen-tolerance in a glutamate aminomutase or change substrate specificity in a lysine aminomutase. It must be noted, however, that while there are many examples of oxygen-tolerant radical SAM enzymes 28 , 29 , e.g. bioB and lipA from E. coli , there seems to be a tradeoff between activity and oxygen tolerance, with oxygen-sensitive variants showing higher activity. For this reason, it may not be possible to obtain an oxygen-tolerant aminomutase with comparable kinetic parameters to oxygen-sensitive isoforms. The 3-hydroxyglutaryl-CoA lyase-methylcitrate (3HGL-MC) cycle We reasoned that the product of CCC, glutaconyl-CoA, could also undergo hydration rather than amination, yielding 3-hydroxyglutaryl-CoA. This promiscuous reaction is indeed reported to occur efficiently 30 . This metabolite could then be split by 3HMG-CoA lyase to acetyl-CoA and malonate semialdehyde, with the latter being reduced to propionyl-CoA and subsequently going through the methylcitrate cycle for propionyl-CoA assimilation 31 , 32 . The resulting succinate can then be activated to succinyl-CoA and reduced to crotonyl-CoA to restart the cycle ( Fig. 2 ). More pathways based on CCC and glutaconyl-CoA hydration have previously been reported 5 , 33 , however the authors describe a hydration in C2 rather than C3, which produces 2-hydroxyglutaryl-CoA. Unfortunately, the enzyme that catalyzes this reaction is based on a ketyl radical mechanism and hence extremely oxygen-sensitive 34 , 35 . To the best of our knowledge there has never been a report of ketyl radical enzymes operating in aerobic conditions, making these proposed routes infeasible for aerobic implementation, while their anaerobic implementation would require an external electron acceptor other than oxygen for respiration, making the pathways biotechnologically not relevant. The 3HGL-MC cycle, on the other hand, does not use any oxygen-sensitive enzymes, since hydration in C3 occurs easily due to the differences in the pKa between the C2 protons and the C3 protons 36 . Download figure Open in new tab Figure 2: The 3-hydroxyglutaryl-CoA lyase-methylcitrate cycle. 1, methylmalonyl-CoA mutase; 2-6 are as in figure 1 ; 7, glutaconyl-CoA hydratase; 8, 3-hydroxyglutaryl-CoA lyase; 9, malonate semialdehyde reductase; 10, propionyl-CoA synthase; 11, methylcitrate synthase; 12, methylcitrate dehydratase; 13, aconitase; 14, methylisocitrate lyase; 15, pyruvate carboxylase. This pathway is predicted to have about 25% higher biomass yield at ambient CO 2 , with roughly double the activity (Table S1, Fig. 6 ). Moreover, the addition of acetyl-CoA carboxylase and malonyl-CoA reductase would make the cycle autocatalytic, much like the CBB cycle and reverse tricarboxylic acid (rTCA) cycle. Autocatalytic cycles are expected to be more stable and resilient to metabolic perturbations 37 , potentially simplifying engineering efforts. Two other variants of this pathway, the 3HGL-PCC cycle (Fig. S1) and the 3HGM cycle (Fig. S2) can be envisioned, however their strict B 12 requirement limits their potential application to some bacteria and algae 15 . The α-ketobutyrate carboxylase (ABC) cycles While the pathways based on crotonyl-CoA carboxylation discussed above use either the condensation of two acetyl-CoA molecules or the reduction of succinyl-CoA to form a C4 metabolite for carboxylation, a different strategy can be envisioned based on the threonine biosynthesis pathway. The in vivo implementation of the Serine Threonine Cycle (STC) for full formatotrophic growth was recently reported in E. coli 38 . The STC relies on the condensation of glycine derived from threonine cleavage with externally supplied formate and an additional carboxylation reaction based on pyruvate carboxylase to generate the C4 molecule aspartate, which is the precursor for threonine. To convert this formate assimilation pathway into a CO 2 fixation pathway design, we reasoned that threonine could instead undergo deamination to produce α-ketobutyrate. The latter C4 metabolite is an α-keto acid highly similar to pyruvate and thus amenable to carboxylation via an engineered pyruvate carboxylase. While we could find no reports of a biotin-dependent α-ketobutyrate carboxylase in the literature, there is at least one study showing partial promiscuous activity of pyruvate carboxylase on α-ketobutyrate 39 . This limited activity could be used as the basis for mutagenesis or rational engineering in an approach similar to the one used to change the substrate specificity of propionyl-CoA carboxylase to glycolyl-CoA 40 . An α-ketobutyrate carboxylase (ABC) would form the basis of the ABC cycles we discuss in this work. While pyruvate carboxylation produces oxaloacetate, α-ketobutyrate carboxylation would produce 3-methyloxaloacetate (also known as 2-methyl-3-oxosuccinate or oxalopropionate). This compound can undergo reductive amination or transamination to methylaspartate 41 , reduction to 3-methylmalate (erythro or threo), or condensation with acetyl-CoA to methylcitrate, though we did not investigate the latter option further, since the resulting cycle is rather complex and inefficient (Fig. S3). In the first case, methylaspartate can be isomerized to glutamate via a B 12 -dependent glutamate mutase (GM) and then proceed via the same steps as the previously described rMASP cycles 14 . The ABC-GM cycle (Fig. S4) compares favorably to the CBB at ambient CO 2 in terms of biomass yield, though its strict requirement for B 12 limits its applicability to microorganisms. To bypass the B 12 requirement, we propose a cycle where methylaspartate is deaminated to mesaconate and hydrated to citramalate, which is then activated to citramalyl-CoA and split to pyruvate and acetyl-CoA via a citramalyl-CoA lyase (CCL). The resulting ABC-CCL cycle ( Fig. 3 ) compares favorably to the CBB cycle in terms of theoretical biomass yield and predicted pathway activity at ambient CO 2 , is insensitive to oxygen and does not require B 12 as a cofactor (Table S1, Fig. 6 ). Conversion of methyloxaloacetate to mesaconate could proceed via different routes with slightly different energetic demands (Fig. S5). Download figure Open in new tab Figure 3: The α -ketobutyrate carboxylase-citramalyl-CoA lyase cycle. 1-9 are as in figure 4 . 10, methylaspartate ammonia lyase; 11, mesaconase; 12, citramalate CoA transferase; 13, citramalyl-CoA lyase; 14, pyruvate carboxylase. A third option could use the promiscuous activity of malate dehydrogenase (MDH) for the reduction of methyloxaloacetate to erythro-methylmalate, which can further be activated to a CoA ester and split to propionyl-CoA and glyoxylate. This variant, known as the ABC-PCC cycle (Fig. S6) also requires B 12 for the conversion of methylmalonyl-CoA to succinyl-CoA following the carboxylation of propionyl-CoA. Notably, all three proposed ABC cycles only require one enzyme to be engineered, and their overlap with the STC shows that they could be engineered relatively easily by applying some of the lessons learned during the STC engineering and evolution in E. coli 38 . Nevertheless, our theoretical analysis shows that threonine biosynthesis may limit the overall activity of these pathways, especially through the bottleneck enzyme homoserine dehydrogenase (Fig. S7). It is unclear whether faster variants of this enzyme can be found or engineered, and if the thermodynamic constraints of this biosynthetic route can be overcome, e.g. by optimizing enzymes and/or metabolite concentration. More research will be needed to ascertain whether this theoretical prediction will be verified experimentally. The Ribulose-5-phosphate carboxylase-Entner-Doudoroff (RED) cycle While all the cycles presented so far have some advantages over the CBB, it should be noted that that they are all based on completely different architectures, thus requiring extensive engineering efforts to establish in organisms that already use the CBB cycle, including algae and plants. For this reason, we set out to establish a carbon fixation cycle that is similar in architecture to the CBB cycle but presents more favorable characteristics in terms of biomass yield and pathway kinetics. The GND-Entner-Doudoroff (GED) cycle has been previously proposed as a promising alternative to the CBB based on natural enzymes 42 , however it is severely limited by the low activity and affinity for CO 2 of its signature enzyme, 6-phosphogluconate dehydrogenase (GND). This enzyme normally catalyzes the oxidative decarboxylation of 6-phosphogluconate, coupling the thermodynamically highly favorable decarboxylation reaction to the unfavorable reduction of NADP. This same mechanism can be found in at least two other central metabolic enzymes, namely the malic enzyme, which produces pyruvate and NADPH from malate and NADP, and isocitrate dehydrogenase, which produces α-ketoglutarate and NADPH from isocitrate and NADP. Notably, in both of these cases there is a biotin-dependent carboxylase to overcome the unfavorable thermodynamics and kinetics of these reactions in the direction of carboxylation, which is then coupled to a dehydrogenase/reductase. Pyruvate is carboxylated to oxaloacetate, which is then reduced to malate, while α-ketoglutarate is carboxylated to oxalosuccinate and then reduced to isocitrate 43 – 45 . A similar idea could be applied to the product of GND, ribulose-5-phosphate. Here, we propose the RED cycle ( Fig. 4 ), which is based on a hypothetical biotin-dependent ribulose-5-phosphate carboxylase. This enzyme would generate 3-keto-6-phosphogluconate, which could then be reduced to 6-phosphogluconate. For this latter reaction GND itself could be used, provided the residues involved in the decarboxylation half-reaction are inactivated. Download figure Open in new tab Figure 4: The Ribulose-5-phosphate carboxylase-Entner-Doudoroff (RED) cycle. 1, ribulose-5-phosphate carboxylase; 2, 6-phosphogluconate dehydrogenase; 3, Entner-Doudoroff dehydratase; 4, Entner-Doudoroff aldolase; 5, PEP synthetase; 6, enolase; 7, phosphoglycerate mutase, 8, phosphoglycerate kinase; 9, glyceraldehyde-3-phosphate dehydrogenase; 10, triose phosphate isomerase; 11, fructose bisphosphate aldolase; 12, fructose bisphosphatase; 13, transketolase; 14, sedoheptulose bisphosphate aldolase; 15, sedoheptulose bisphosphatase; 16, ribulose-5-phosphate epimerase; 17, ribulose-5-phosphate isomerase. 2PG, 2-phosphoglycerate; 3K6PG, 3-keto-6-phosphogluconate; 3PG, 3-phosphoglycerate; 6PG, 6-phosphogluconate; BPG, 1,3-bisphosphoglycerate; DHAP, dihydroxyacetone phosphate; E4P, erythrose-4-phosphate; F6P, fructose-6-phosphate; FBP, fructose-1,6-bisphosphate; GAP, glyceraldehyde-3-phosphate; KDPG, 2-keto-3-deoxy-phosphogluconate, PEP, phosphoenolpyruvate; R5P, ribose-5-phosphate; Ru5P, ribulose-5-phosphate; S7P, sedoheptulose-7-phosphate; SBP, sedoheptulose-1,7-bisphosphate; Xu5P, xylulose-5-phosphate. To the best of our knowledge, there is no reported enzyme taking a similar substrate for the carboxylation reaction, meaning that an extensive engineering campaign will be necessary to change the substrate specificity. This is probably beyond what is achievable with current strategies based on mutagenesis and selection, however it may become possible in the future to apply novel computational enzyme design approaches to redesign the substrate pocket. The RED cycle is insensitive to the presence of oxygen, does not require CoA or exotic cofactors, it is favorable in terms of efficiency and kinetics at ambient CO 2 (Table S1, Fig. 6 ), and it has a high degree of overlap with the CBB cycle, making it a good candidate for implementation in algae and plants. However, it should be noted that these organisms do not express lower glycolysis enzymes in their chloroplasts, which could increase the engineering complexity. The Dehydratase-less Ribulose-5-phosphate carboxylase-Entner-Doudoroff (DRED) Cycle While the RED cycle is a promising alternative to the CBB cycle in terms of pathway activity, its ATP consumption is higher, and consequently its overall predicted biomass yield is lower under high CO 2 conditions (Table S1, Fig. 6 ). This is due to the costly pyruvate kinase reaction, which consumes two ATP equivalents per pyruvate. To solve this issue, we propose a variant of the RED cycle that we name the DRED cycle ( Fig. 5 ). This variant does not rely on the Entner-Doudoroff dehydratase (Edd) but instead uses an isomerase to convert 3-keto-6-phosphogluconate to 2-keto-6-phosphogluconate. This metabolite could subsequently serve as the substrate for the Entner-Doudoroff aldolase (Eda), producing glyceraldehyde-3-phosphate (GAP) and hydroxypyruvate. The latter can be reduced to glycerate, which is then activated to 2-phosphoglycerate and enters gluconeogenesis to generate another GAP. The advantage of this sequence of reactions is that glycerate kinase only consumes one ATP equivalent. As a result, this cycle is predicted to be slightly more efficient than the CBB cycle even at high CO 2 concentrations. Additionally, our ECM analysis predicts a slightly higher pathway activity (Table S1, Fig. 6 ), all while eschewing the oxygenation side reaction of Rubisco. Download figure Open in new tab Figure 5: The Dehydratase-less-Ribulose-5-phosphate carboxylase-Entner-Doudoroff (DRED) cycle. 2, 3-keto-6-phosphogluconate isomerase; 3, 2-keto-6-phosphogluconate aldolase; 4, hydroxypyruvate reductase; 5, glycerate kinase; 6, phosphoglycerate mutase, 7, phosphoglycerate kinase; 8, glyceraldehyde-3-phosphate dehydrogenase; 9, triose phosphate isomerase; 10, fructose bisphosphate aldolase; 11, fructose bisphosphatase; 12, transketolase; 13, sedoheptulose bisphosphate aldolase; 14, sedoheptulose bisphosphatase; 15, ribulose-5-phosphate epimerase; 16, ribulose-5-phosphate isomerase. 2K6PG, 2-keto-6-phosphogluconate; All other abbreviations are as in figure 5 . Download figure Open in new tab Figure 6: Plot of theoretical biomass yield vs predicted pathway activity for each pathway presented in this paper and previously proposed pathways. All pathways except the CBB cycle were modeled using ambient CO 2 concentrations, see methods. This figure includes only pathways that require enzyme engineering for their implementation (other than the CBB cycle). We also include comparisons to the previously proposed malyl-CoA-tartronyl-CoA cycle, also known as LATCH 49 (Fig. S10) and the beta alanine variant of the malonyl-CoA-oxaloacetate-glyoxylate (MOG) cycle 50 (Fig. S11). For additional comparisons to B12-dependent pathways proposed in this paper and previously proposed pathways, including the lactyl-CoA mutase variant of the MOG cycle 50 , 51 (Fig. S12) and several mix-and-match CO 2 fixation pathways 6 , 42 , 52 – 54 , see figure S13. Despite these advantages, the cycle requires the engineering of three enzymes, as opposed to only one for the RED cycle. Additionally, hydroxypyruvate is a moderately toxic metabolite which can inhibit bacterial growth at mM concentrations. While its effect on plants is not known, careful balancing of pathway enzymes is likely sufficient to keep the levels of hydroxypyruvate low and avoid its toxicity. A similar way to bypass pyruvate kinase was proposed in the context of the GED cycle by Arren Bar-Even 46 , albeit using a 6-phosphogluconate-2-dehydrogenase (EC 1.1.1.43) instead of an isomerase, which is also a possibility here (Fig. S8). Coupling the CO 2 reduction (CORE) cycle to formatotrophic pathways An interesting alternative to CO 2 fixation cycles was recently proposed by Satanowski, Marchal et al., who theorized a number of oxygen tolerant CO 2 reduction (CORE) cycles and implemented one of them in E. coli 47 . These CORE cycles are proposed to be able to reduce CO 2 (also at ambient concentrations) to formate, which would allow for further coupling for formate assimilation routes, effectively generating CO 2 fixation routes via formate as intermediate. The experimentally validated CORE variant relies on the promiscuous activity of a β-keto-acid cleavage enzyme (BKACE) and yields one molecule of formate from one molecule of bicarbonate with the input of one NADPH and 1-3 ATP. While the CORE cycle was originally proposed as a photorespiration bypass, the resulting formate could be fully coupled to growth via a formatotrophic pathway such as the serine cycle, which can also operate at ambient CO 2 , unlike the reductive glycine pathway 48 . We have modeled the resulting pathway to compare it with our proposed CO 2 fixation cycles ( Fig. 6 ). Based on our results, the CORE+Serine cycle (Fig. S9) shows a comparable yield to the CBB in high CO 2 and a roughly 20% higher yield in ambient CO 2 . Assuming the limiting BKACE activity can be improved, the CORE+Serine cycle could be a highly relevant alternative to conventional CO 2 fixation cycles, since it is oxygen-tolerant, does not require B 12 , and mostly relies on reactions that are natively present in plants, or that have been shown to be functional after heterologous expression. Discussion In this paper, we proposed several novel carbon fixation pathways based on non-natural enzymatic reactions. We analyzed the pathways based on thermodynamic driving force, predicted biomass yield, and predicted pathway activity. This last parameter is computed based on the measured or estimated k cat and K M values for the enzymes. Unfortunately, the available enzymatic data in databases such as BRENDA is both limited and noisy, since different authors perform enzymatic assays in wildly different conditions in terms of pH, buffers, temperatures, etc. We carefully curated the chosen values, also based on the previous work by Lowe and Kremling 55 , however a relatively large fraction of the enzymes was only measured in single studies, reducing their reliability. Where multiple data points are available, it is not unusual to see a variation of one order of magnitude or more in reported k cat and K M . Furthermore, in vitro kinetics measurements, even when performed properly, are based on a purified enzyme in a dilute buffer, which is a completely different scenario compared to the densely packed cytosol of living organisms, where thousands of enzymes and metabolites are present. Recent work has shown k cat measurements in vivo based on different growth conditions and proteomics data 56 . These results show that while many in vitro measurements are also accurate in vivo, there are several outliers even for well characterized enzymes in central metabolism, with a relatively weak correlation R 2 of 0.62. All these problems are compounded when it comes to relatively understudied enzymes, and especially their promiscuous activities. Finally, the kinetic parameters of not yet existing enzymes are obviously impossible to obtain, and we have simply taken representative values for similar enzymes on related substrates. In this work, we used the ECM (enzyme cost minimization) method to predict pathway kinetics. This method assumes an optimal distribution of metabolites and enzymes, and does not take into account enzyme degradation, allosteric inhibition, competitive inhibition, and regulation. They also do not consider all the enzymes that are necessary for cell maintenance and growth, assuming all flux is going via the pathway of interest to generate the desired product. In practice, all these factors mean that the predicted pathway activity is at best an upper bound for the real pathway activity in a real-life scenario. Taking the CBB into account, measured values for CO 2 fixation rates in exponentially growing bacteria are in the order of 3-9 nmol/min/mg (total) protein 57 . This is a two order of magnitude difference below our computed value of around 0.5 µmol/min/mg (pathway) protein. This discrepancy is partially explained when considering the fact that CBB enzymes account for about 10% of total protein 58 . The predicted pathway activity for the CBB, then, is still one order of magnitude above measured values, despite the fact that all of its enzymes are well-characterized in dozens of studies. For the other pathways, greater uncertainty is expected due to the limited reliability of the available data. It is unclear whether this uncertainty leads to an overestimate or an underestimate of the total pathway activity, however, it is likely that for most of the candidate enzymes, kinetically superior isoforms could be found through a screening process. This points to the fact that pathways other than the CBB may be underestimated in our analysis. Further research will be needed to determine real life (i.e. in vivo) kinetic parameters for several enzymes, leading to better estimates. Considering all of this, our proposed pathways are especially promising for potential future applications in terms of yield, as they can outperform the CBB cycle. All the 6 proposed pathways can potentially outperform the yield of the CBB cycle at ambient CO 2 , whereas only the DRED cycle is predicted to outperform the CBB in terms of pathway activity at high CO 2 . Also, the beta-glutamate, 3HGL-MC and DRED cycles have relatively high yield gains versus the CBB cycle at ambient CO 2 . Overall, this makes the β-glutamate, DRED and 3HGL-MC cycles promising to explore, given a potentially attractive yield, and reasonable predicted pathway activity. However, given the required three new-to-nature activities, the β-glutamate cycle and especially the DRED cycle would be a tremendous engineering effort already in bacteria, and even more so in microalgae or plants. The ABC-CCL and 3HGL-MC cycles strike a balance between yield, activity, and relative ease of implementation, requiring only one engineered enzyme. The RED cycle has a more moderate yield advantage at ambient CO 2 (15% vs. CBB cycle) but could also be relevant to explore based on its overlap with the CBB cycle and high predicted pathway activity, however it requires the highly challenging ribulose-5-phosphate carboxylase activity, which is shared with the DRED cycle. The best pathways in this work are predicted to have comparable performance to the best previously proposed mix-and-match pathways, notably the CETCH and THETA cycles 6 , 52 . However, both these cycles require B 12 -dependent mutases, unlike the cycles proposed in this work. Hence, our proposed cycles have a higher potential for implementation in microalgae and plants. The fact that none of the proposed pathways massively outperform the CBB cycle (at high CO 2 ) also highlights the fact that carbon fixation cycles are not exclusively limited by carboxylation activity and that Rubisco, despite its relatively slow catalysis and oxygenation side activity, still leads to relatively high pathway activity when considered in its metabolic context 59 . Still, an open question remains for the field: why is the CBB ubiquitous, if stoichiometrically and/or kinetically superior pathways are potentially possible? One possible explanation for this is the high degree of overlap between the CBB cycle and the pentose phosphate pathway, which exists in all living organisms. In fact, only Rubisco and PRK are necessary on top of the standard pentose phosphate pathway genes, as demonstrated by the engineering of E. coli to grow autotrophically via the CBB cycle, and it is common in nature for these two genes to be expressed from a plasmid, which can easily be horizontally transferred, thus spreading the CBB cycle across phyla. Furthermore, several essential biomass precursors are formed as part of the cycle, which would make a loss of activity detrimental. At least in the short term, an organism that acquired all the necessary genes to support an alternative carbon fixation cycle pathway would have to support both cycles at the same time, likely with competing if not outright incompatible fluxes and metabolic profiles. Even though the proposed pathways likely did not emerge naturally, the well-demonstrated metabolic engineering approach to rewire metabolism using auxotrophies and bypasses to force metabolic rearrangements may allow their realization 60 . This strategy has proven highly reliable in engineering a diverse array of pathways and will likely be just as useful in the future 38 , 61 – 64 . Overall, our study expands the solution space for carbon fixation by providing realistic and biochemically feasible avenues for enzymatic transformations based on known enzyme mechanisms and substrate promiscuity. Notably, we identified two potential novel carboxylases that could lead to entirely new pathway architectures once implemented. New carbon fixation pathways with better yield and insensitivity to oxygen could lead to improved carbon fixation in algae or plants, potentially leading to more efficient renewable production of chemicals from CO 2 and increases in crop yields. Author Contributions VR conceptualized the research, performed all computational analyses, and wrote the manuscript. SDA and NJC acquired funding, supervised the research, and edited the manuscript. Competing Interest Statement The authors declare no competing interests. Methods All scripts and input files are available on gitlab ( https://git.wur.nl/vittorio.rainaldi/lvl4 ), along with all MDF and ECM results. All pathways were computationally analyzed using flux balance analysis 65 (FBA) with cobrapy 66 (version 0.29.1) using the E. coli iML1515 12 genome scale-metabolic model. The base model was modified to change membrane-bound transhydrogenase (TDH2pp) to translocate one proton rather than two 67 . Anaerobic reactions such as PFL, OBTFL, and POR5 were set to zero. Non-growth associated maintenance (ATPM) was set to zero. Additional modifications were added for each individual pathway. FBA returns a flux value in gCDW/mol substrate. In this case, since CO 2 is freely available in the atmosphere, it was left unconstrained, however an energy source is required to run CO 2 fixation cycles, and this was used as a constraint for all pathways. For this purpose, we added a dummy reaction which produces NADPH, which is the main source of reducing power in photosynthetic organisms such as algae and plants. This results in slightly higher values compared to using NADH as an energy source, since NADH to NADPH conversion requires the translocation of one proton, which amounts to roughly one-third of an ATP 67 . Max-min driving force 11 and enzyme cost 13 were computed with a custom script modified from previous work 42 , 68 using equilibrator (version 0.6.0). Metabolite concentrations were constrained to the physiologically relevant range between 10 µM and 10 mM. Ambient CO 2 is constrained between 10 and 20 µM. To model the CBB cycle, we assumed a 20% rate of Rubisco oxygenation at ambient CO 2 , and no oxygenation at high CO 2 (3.4 mM, corresponding to a partial pressure of 10%, where photorespiration is virtually absent). ECM was computed using the following parameters: algorithm ECM, version 3, kcat_source fwd, denominator CM, regularization volume, p_h 7.5, ionic_strength 0.3 molar, p_mg 3, dg_confidence 0.95. Kinetics values were taken from Brenda 69 or previous work 7 . In the case of new-to-nature enzymatic activities, the kinetics were estimated by taking values from similar enzymes. For example, kinetic values for α-ketobutyrate carboxylase are not available, so values for pyruvate carboxylase were used instead. Since this enzyme catalyzes the same reaction on a slightly smaller substrate, it is reasonable to assume that an engineered enzyme could reach similar kinetic properties. The ECM algorithm returns a list of enzyme costs in moles of enzyme per liter based on a total flux through the pathway for 1 mM/s of product formed. These values were multiplied by the molecular weight of the respective enzyme to obtain the enzyme cost in g/l. The sum of all the values is the enzyme cost (EC) for the pathway. To obtain the pathway activity we performed the following calculations: Acknowledgements The authors would like to thank Dr. Beau Dronsella for helpful comments on the manuscript. VR is funded by the VLAG Graduate School Wageningen University, NJC acknowledges funding from the ERC StG FASTFIX by the European Research Council and a Vidi grant (VI.Vidi.233.193) from the Dutch Science Organisation (NWO). 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