Physiological characterization of recombinant Saccharomyces cerevisiae expressing the Aspergillus nidulans phosphoketolase pathway: validation of activity through 13C-based metabolic flux analysis

Several bacterial species and filamentous fungi utilize the phosphoketolase pathway (PHK) for glucose dissimilation as an alternative to the Embden-Meyerhof-Parnas pathway. In Aspergillus nidulans, the utilization of this metabolic pathway leads to increased carbon flow towards acetate and acetyl CoA. In the first step of the PHK, the pentose phosphate pathway intermediate xylulose-5-phosphate is converted into acetylphosphate and glyceraldehyde-3-phosphate through the action of xylulose-5-phosphate phosphoketolase, and successively acetylphosphate is converted into acetate by the action of acetate kinase. In the present work, we describe a metabolic engineering strategy used to express the fungal genes of the phosphoketolase pathway in Saccharomyces cerevisiae and the effects of the expression of this recombinant route in yeast. The phenotype of the engineered yeast strain MP003 was studied during batch and chemostat cultivations, showing a reduced biomass yield and an increased acetate yield during batch cultures. To establish whether the observed effects in the recombinant strain MP003 were due directly or indirectly to the expression of the phosphoketolase pathway, we resolved the intracellular flux distribution based on (13)C labeling during chemostat cultivations. From flux analysis it is possible to conclude that yeast is able to use the recombinant pathway. Our work indicates that the utilization of the phosphoketolase pathway does not interfere with glucose assimilation through the Embden-Meyerhof-Parnas pathway and that the expression of this route can contribute to increase the acetyl CoA supply, therefore holding potential for future metabolic engineering strategies having acetyl CoA as precursor for the biosynthesis of industrially relevant compounds.     

Unlocking the biosynthesis of sesquiterpenoids from methane via the methylerythritol phosphate pathway in methanotrophic bacteria,using α-humulene as a model compound

Isoprenoids are an abundant and diverse class of natural products with various applications in the pharmaceutical, cosmetics and biofuel industries. A methanotroph-based biorefinery is an attractive scenario for the production of a variety of value-added compounds from methane, because methane is a promising alternative feedstock for industrial biomanufacturing. In this study, we metabolically engineered Methylotuvimicrobium alcaliphilum 20Z for de novo synthesis of a sesquiterpenoid from methane, using α-humulene as a model compound, via optimization of the native methylerythritol phosphate (MEP) pathway. Expression of codon-optimized α-humulene synthase from Zingiber zerumbet in M. alcaliphilum 20Z resulted in an initial yield of 0.04 mg/g dry cell weight. Overexpressing key enzymes (IspA, IspG, and Dxs) for debottlenecking of the MEP pathway increased α-humulene production 5.2-fold compared with the initial strain. Subsequently, redirecting the carbon flux through the Embden–Meyerhof–Parnas pathway resulted in an additional 3-fold increase in α-humulene production. Additionally, a genome-scale model using flux scanning based on enforced objective flux method was used to identify potential overexpression targets to increase flux towards isoprenoid production. Several target reactions from cofactor synthesis pathways were probed and evaluated for their effects on α-humulene synthesis, resulting in α-humulene yield up to 0.75 mg/g DCW with 18.8-fold enhancement from initial yield. This study first demonstrates production of a sesquiterpenoid from methane using methanotrophs as the biocatalyst and proposes potential strategies to enhance production of sesquiterpenoid and related isoprenoid products in engineered methanotrophic bacteria.     

Enhanced isoamyl acetate production upon manipulation of the acetyl-CoA node in Escherichia coli

Coenzyme A (CoA) and its thioester derivative acetyl-Coenzyme A (acetyl-CoA) participate in over 100 different reactions in intermediary metabolism of microorganisms. Earlier results indicated that overexpression of upstream rate-limiting enzyme pantothenate kinase with simultaneous supplementation of precursor pantothenic acid to the culture media increased intracellular CoA levels significantly ( approximately 10-fold). The acetyl-CoA levels also increased ( approximately 5-fold) but not as much as that of CoA, showing that the carbon flux from the pyruvate node is rate-limiting upon an increase in CoA levels. In this study, pyruvate dehydrogenase was overexpressed under elevated CoA levels to increase carbon flux from pyruvate to acetyl-CoA. This coexpression did not increase intracellular acetyl-CoA levels but increased the accumulation of extracellular acetate. The production of isoamyl acetate, an industrially useful compound derived from acetyl-CoA, was used as a model reporter system to signify the beneficial effects of this metabolic engineering strategy. In addition, a strain was created in which the acetate production pathway was inactivated to relieve competition at the acetyl-CoA node and to efficiently channel the enhanced carbon flux to the ester production pathway. The synergistic effect of cofactor CoA manipulation and pyruvate dehydrogenase overexpression in the acetate pathway deletion mutant led to a 5-fold increase in isoamyl acetate production. Under normal growth conditions the acetate pathway deletion mutant strains accumulate intracellular pyruvate, leading to excretion of pyruvate. However, upon enhancing the carbon flux from pyruvate to acetyl-CoA, the excretion of pyruvate was significantly reduced.     

Auxin-mediated induction of GAL promoters by conditional degradation of Mig1p improves sesquiterpene production in Saccharomyces cerevisiae with engineered acetyl-CoA synthesis

The yeast Saccharomyces cerevisiae uses the pyruvate dehydrogenase-bypass for acetyl-CoA biosynthesis. This relatively inefficient pathway limits production potential for acetyl-CoA-derived biochemical due to carbon loss and the cost of two high-energy phosphate bonds per molecule of acetyl-CoA. Here, we attempted to improve acetyl-CoA production efficiency by introducing heterologous acetylating aldehyde dehydrogenase and phosphoketolase pathways for acetyl-CoA synthesis to enhance production of the sesquiterpene trans-nerolidol. In addition, we introduced auxin-mediated degradation of the glucose-dependent repressor Mig1p to allow induced expression of GAL promoters on glucose so that production potential on glucose could be examined. The novel genes that we used to reconstruct the heterologous acetyl-CoA pathways did not sufficiently complement the loss of endogenous acetyl-CoA pathways, indicating that superior heterologous enzymes are necessary to establish fully functional synthetic acetyl-CoA pathways and properly explore their potential for nerolidol synthesis. Notwithstanding this, nerolidol production was improved twofold to a titre of ˜ 900 mg l⁻¹ in flask cultivation using a combination of heterologous acetyl-CoA pathways and Mig1p degradation. Conditional Mig1p depletion is presented as a valuable strategy to improve the productivities in the strains engineered with GAL promoters-controlled pathways when growing on glucose.     

Acetate kinase: not just a bacterial enzyme

The bacterial enzymes acetate kinase (AK) and phosphotransacetylase (PTA) form a key pathway for synthesis of the central metabolic intermediate acetyl coenzyme A (acetyl-CoA) from acetate or for generation of ATP from excess acetyl-CoA. Putative AK genes have now been identified in some eukaryotic microbes. In Chlamydomonas reinhardtii and Phytophthora species, AK forms a pathway with PTA. AK has also been identified in non-yeast fungi but these fungi do not have PTA. Instead, AK forms a pathway with D-xylulose 5-phosphate phosphoketolase (XFP), a pathway that was also previously found only in bacteria. In Entamoeba histolytica, neither PTA nor XFP was found as a partner for AK. Thus, eukaryotic microbes seem to have incorporated the 'bacterial' enzyme AK into at least three different metabolic pathways.     

Survival and Recovery of Methanotrophic Bacteria Starved under Oxic and Anoxic Conditions

The effects of carbon deprivation on survival of methanotrophic bacteria were compared in cultures incubated in the presence and absence of oxygen in the starvation medium. Survival and recovery of the examined methanotrophs were generally highest for cultures starved under anoxic conditions as indicated by poststarvation measurements of methane oxidation, tetrazolium salt reduction, plate counts, and protein synthesis. Methylosinus trichosporium OB3b survived up to 6 weeks of carbon deprivation under anoxic conditions while maintaining a physiological state that allowed relatively rapid (hours) methane oxidation after substrate addition. A small fraction of cells starved under oxic and anoxic conditions (4 and 10%, respectively) survived more than 10 weeks but required several days for recovery on plates and in liquid medium. A non-spore-forming methanotroph, strain WP 12, displayed 36 to 118% of its initial methane oxidation capacity after 5 days of carbon deprivation. Oxidation rates varied with growth history prior to the experiments as well as with starvation conditions. Strain WP 12 starved under anoxic conditions showed up to 90% higher methane oxidation activity and 46% higher protein production after starvation than did cultures starved under oxic conditions. Only minor changes in biomass and morphology were seen for methanotrophic bacteria starved under anoxic conditions. In contrast, starvation under oxic conditions resulted in morphology changes and an initial 28 to 35% loss of cell protein. These data suggest that methanotrophic bacteria can survive carbon deprivation under anoxic conditions by using maintenance energy derived solely from an anaerobic endogenous metabolism. This capability could partly explain a significant potential for methane oxidation in environments not continuously supporting aerobic methanotrophic growth.     

Induction of xylulose-5-phosphate phosphoketolase in a variety of yeasts grown ond-xylose: the key to efficient xylose metabolism

Activity of a pentulose (xylulose 5-phosphate) phosphoketolase was detected in 20 out of 25 yeasts examined. No significant activity was detected in any yeast grown with glucose, and the enzyme was induced by up to 70-fold when the yeasts were grown on xylose as sole carbon source. Biomass yields from xylose were greater than, or approximately equal to, those from glucose in 15 of the 19 yeasts which possessed phosphoketolase activity. The molar yield of C₂ units from xylose, by metabolism via the pentose phosphate pathway, can be calculated to be insufficient to account for the high yields of biomass and ethanol obtained from xylose. We have shown that the presence of a phosphoketolase system can account for such yields by producing 2 mol C₂ from 1 mol C₅. This pathway must therefore be regarded as a major route of pentose dissimilation in such yeasts.     

Evidence,through C13-labelling analysis,of phosphoketolase activity in fungi

Phosphoketolase is a well characterised enzyme in Bifidobacterium species. However, limited information about this enzyme is available in higher organisms. Physiological characterisation of the filamentous fungus Penicillium chrysogenum, using ¹³C-labelling analysis, revealed that an unrecognised pathway was involved in formation of cytosolic acetyl-CoA. Comparison of the labelling pattern of pyruvate and acetyl-CoA showed that the ¹³C-content of pyruvate was higher than the corresponding labelling of acetyl-CoA, indicating that a less labelled source was contributing to the acetyl-CoA pool. Examination of previously published ¹³C data from Aspergillus nidulans showed the same trend. It was speculated that phosphoketolase activity could be contributing to the formation of acetyl-CoA in these fungi. Since the traditional methods for measuring phosphoketolase activity are tedious, two new enzyme assays were developed and used for identification of phosphoketolase activity. Furthermore, using genomic sequencing information, targets for creating an entire pathway from xylulose 5-phosphate to acetate was identified in A. nidulans.     

Promiscuous phosphoketolase and metabolic rewiring enables novel non-oxidative glycolysis in yeast for high-yield production of acetyl-CoA derived products

Carbon-conserving pathways have the potential of increasing product yields in biotechnological processes. The aim of this project was to investigate the functionality of a novel carbon-conserving pathway that produces 3 mol of acetyl-CoA from fructose-6-phosphate without carbon loss in the yeast Saccharomyces cerevisiae. This cyclic pathway relies on a generalist phosphoketolase (Xfspk), which can convert xylulose-5-phosphate, fructose-6-phosphate and sedoheptulose-7-phosphate (S7P) to acetyl phosphate. This cycle is proposed to overcome bottlenecks from the previously reported non-oxidative glycolysis (NOG) cycle. Here, in silico simulations showed accumulation of S7P in the NOG cycle, which was resolved by blocking the non-oxidative pentose phosphate pathway and introducing Xfspk and part of the riboneogenesis pathway. To implement this, a transketolase and transaldolase deficient S. cerevisiae was generated and a cyclic pathway, the Glycolysis AlTernative High Carbon Yield Cycle (GATHCYC), was enabled through xfspk expression and sedoheptulose bisphosphatase (SHB17) overexpression. Flux through the GATHCYC was demonstrated in vitro with a phosphoketolase assay on crude cell free extracts, and in vivo by constructing a strain that was dependent on a functional pathway to survive. Finally, we showed that introducing the GATHCYC as a carbon-conserving route for 3-hydroxypropionic acid (3-HP) production resulted in a 109% increase in 3-HP titers when the glucose was exhausted compared to the phosphoketolase route only.     

Design and construction of acetyl-CoA overproducing Saccharomyces cerevisiae strains

Saccharomyces cerevisiae has increasingly been engineered as a cell factory for efficient and economic production of fuels and chemicals from renewable resources. Notably, a wide variety of industrially important products are derived from the same precursor metabolite, acetyl-CoA. However, the limited supply of acetyl-CoA in the cytosol, where biosynthesis generally happens, often leads to low titer and yield of the desired products in yeast. In the present work, combined strategies of disrupting competing pathways and introducing heterologous biosynthetic pathways were carried out to increase acetyl-CoA levels by using the CoA-dependent n-butanol production as a reporter. By inactivating ADH1 and ADH4 for ethanol formation and GPD1 and GPD2 for glycerol production, the glycolytic flux was redirected towards acetyl-CoA, resulting in 4-fold improvement in n-butanol production. Subsequent introduction of heterologous acetyl-CoA biosynthetic pathways, including pyruvate dehydrogenase (PDH), ATP-dependent citrate lyase (ACL), and PDH-bypass, further increased n-butanol production. Recombinant PDHs localized in the cytosol (cytoPDHs) were found to be the most efficient, which increased n-butanol production by additional 3 fold. In total, n-butanol titer and acetyl-CoA concentration were increased more than 12 fold and 3 fold, respectively. By combining the most effective and complementary acetyl-CoA pathways, more than 100 mg/L n-butanol could be produced using high cell density fermentation, which represents the highest titer ever reported in yeast using the clostridial CoA-dependent pathway.     

Enhanced lycopene productivity by manipulation of carbon flow to isopentenyl diphosphate in Escherichia coli

Lycopene is a useful phytochemical that holds great commercial value. In our study the lycopene production pathway in E. coli originating from the precursor isopentenyl diphosphate (IPP) of the non-mevalonate pathway was reconstructed. This engineered strain of E. coli accumulated lycopene intracellularly under aerobic conditions. As a next step, the production of lycopene was enhanced through metabolic engineering methodologies. Various competing pathways at the pyruvate and acetyl-CoA nodes were inactivated to divert more carbon flux to IPP and subsequently to lycopene. It was found that the ackA-pta, nuo mutant produced a higher amount of lycopene compared to the parent strain. To further enhance lycopene production, a novel mevalonate pathway, in addition to the already existing non-mevalonate pathway, was engineered. This pathway utilizes acetyl-CoA as precursor, condensing it to form acetoacetyl-CoA and subsequently leading to formation of IPP. Upon the introduction of this new pathway, lycopene production increased by over 2-fold compared to the ackA-pta, nuo mutant strain.     

微生物固碳的电子供给策略研究进展北大核心CSCD

随着生物化工技术的不断发展成熟,通过改造微生物已可以实现二氧化碳、甲烷等温室气体的固定、转化和利用,而电子传递及能量供给对微生物固碳效率起着决定性的作用。本文首先分析了好氧性嗜甲烷菌、化能自养微生物等天然微生物细胞内外的直接、间接电子传递系统。在此基础上,围绕微生物固碳细胞工厂的构建,进一步介绍了基于光能、电能的人工电子供给策略及其对固碳过程中代谢通量、合成路径和供能效率的影响。最后针对微生物固碳的关键共性技术难点,简要展望了可行性的解决方案及相关应用前景。     

Sustainable biosynthesis of chemicals from methane and glycerol via reconstruction of multi-carbon utilizing pathway in obligate methanotrophic bacteria

Obligate methanotrophic bacteria can utilize methane, an inexpensive carbon feedstock, as a sole energy and carbon substrate, thus are considered as the only nature-provided biocatalyst for sustainable biomanufacturing of fuels and chemicals from methane. To address the limitation of native C1 metabolism of obligate type I methanotrophs, we proposed a novel platform strain that can utilize methane and multi-carbon substrates, such as glycerol, simultaneously to boost growth rates and chemical production in Methylotuvimicrobium alcaliphilum 20Z. To demonstrate the uses of this concept, we reconstructed a 2,3-butanediol biosynthetic pathway and achieved a fourfold higher titer of 2,3-butanediol production by co-utilizing methane and glycerol compared with that of methanotrophic growth. In addition, we reported the creation of a methanotrophic biocatalyst for one-step bioconversion of methane to methanol in which glycerol was used for cell growth, and methane was mainly used for methanol production. After the deletion of genes encoding methanol dehydrogenase (MDH), 11.6 mM methanol was obtained after 72 h using living cells in the absence of any chemical inhibitors of MDH and exogenous NADH source. A further improvement of this bioconversion was attained by using resting cells with a significantly increased titre of 76 mM methanol after 3.5 h with the supply of 40 mM formate. The work presented here provides a novel framework for a variety of approaches in methane-based biomanufacturing.     

Metabolic Engineering of a Phosphoketolase Pathway for Pentose Catabolism in Saccharomyces cerevisiae

Low ethanol yields on xylose hamper economically viable ethanol production from hemicellulose-rich plant material with Saccharomyces cerevisiae. A major obstacle is the limited capacity of yeast for anaerobic reoxidation of NADH. Net reoxidation of NADH could potentially be achieved by channeling carbon fluxes through a recombinant phosphoketolase pathway. By heterologous expression of phosphotransacetylase and acetaldehyde dehydrogenase in combination with the native phosphoketolase, we installed a functional phosphoketolase pathway in the xylose-fermenting Saccharomyces cerevisiae strain TMB3001c. Consequently the ethanol yield was increased by 25% because less of the by-product xylitol was formed. The flux through the recombinant phosphoketolase pathway was about 30% of the optimum flux that would be required to completely eliminate xylitol and glycerol accumulation. Further overexpression of phosphoketolase, however, increased acetate accumulation and reduced the fermentation rate. By combining the phosphoketolase pathway with the ald6 mutation, which reduced acetate formation, a strain with an ethanol yield 20% higher and a xylose fermentation rate 40% higher than those of its parent was engineered.     

Improving cellular malonyl-CoA level in Escherichia coli via metabolic engineering

Escherichia coli only maintains a small amount of cellular malonyl-CoA, impeding its utility for overproducing natural products such as polyketides and flavonoids. Here, we report the use of various metabolic engineering strategies to redirect the carbon flux inside E. coli to pathways responsible for the generation of malonyl-CoA. Overexpression of acetyl-CoA carboxylase (Acc) resulted in 3-fold increase in cellular malonyl-CoA concentration. More importantly, overexpression of Acc showed a synergistic effect with increased acetyl-CoA availability, which was achieved by deletion of competing pathways leading to the byproducts acetate and ethanol as well as overexpression of an acetate assimilation enzyme. These engineering efforts led to the creation of an E. coli strain with 15-fold elevated cellular malonyl-CoA level. To demonstrate its utility, this engineered E. coli strain was used to produce an important polyketide, phloroglucinol, and showed near 4-fold higher titer compared with wild-type E. coli, despite the toxicity of phloroglucinol to cell growth. This engineered E. coli strain with elevated cellular malonyl-CoA level should be highly useful for improved production of important natural products where the cellular malonyl-CoA level is rate-limiting.     

Assimilation of methane and inorganic carbon by microbial communities mediating the anaerobic oxidation of methane

The anaerobic oxidation of methane (AOM) is a major sink for methane on Earth and is performed by consortia of methanotrophic archaea (ANME) and sulfate-reducing bacteria (SRB). Here we present a comparative study using in vitro stable isotope probing to examine methane and carbon dioxide assimilation into microbial biomass. Three sediment types comprising different methane-oxidizing communities (ANME-1 and -2 mixture from the Black Sea, ANME-2a from Hydrate Ridge and ANME-2c from the Gullfaks oil field) were incubated in replicate flow-through systems with methane-enriched anaerobic seawater medium for 5–6 months amended with either ¹³CH₄ or H¹³CO₃^-. In all three sediment types methane was anaerobically oxidized in a 1:1 stoichiometric ratio compared with sulfate reduction. Similar amounts of ¹³CH₄ or ¹³CO₂ were assimilated into characteristic archaeal lipids, indicating a direct assimilation of both carbon sources into ANME biomass. Specific bacterial fatty acids assigned to the partner SRB were almost exclusively labelled by ¹³CO₂, but only in the presence of methane as energy source and not during control incubations without methane. This indicates an autotrophic growth of the ANME-associated SRB and supports previous hypotheses of an electron shuttle between the consortium partners. Carbon assimilation efficiencies of the methanotrophic consortia were low, with only 0.25–1.3 mol% of the methane oxidized.     

Carbon isotope analysis of acetaldehyde emitted from leaves following mechanical stress and anoxia

Although the emission of acetaldehyde from plants into the atmosphere following biotic and abiotic stresses may significantly impact air quality and climate, its metabolic origin(s) remains uncertain. We investigated the pathway(s) responsible for the production of acetaldehyde in plants by studying variations in the stable carbon isotope composition of acetaldehyde emitted during leaf anoxia or following mechanical stress. Under an anoxic environment, C3 leaves produced acetaldehyde during ethanolic fermentation with a similar carbon isotopic composition to C3 bulk biomass. In contrast, the initial emission burst following mechanical wounding was 5–12‰ more depleted in ¹³C than emissions under anoxia. Due to a large kinetic isotope effect during pyruvate decarboxylation catalysed by pyruvate dehydrogenase, acetyl-CoA and its biosynthetic products such as fatty acids are also depleted in ¹³C relative to bulk biomass. It is well known that leaf wounding stimulates the release of large quantities of fatty acids from membranes, as well as the accumulation of reactive oxygen species (ROS). We suggest that, following leaf wounding, acetaldehyde depleted in ¹³C is produced from fatty acid peroxidation reactions initiated by the accumulation of ROS. However, a variety of other pathways could also explain our results, including the conversion of acetyl-CoA to acetaldehyde by the esterase activity of aldehyde dehydrogenase.     

Engineering cofactor and transport mechanisms in Saccharomyces cerevisiae for enhanced acetyl-CoA and polyketide biosynthesis

Synthesis of polyketides at high titer and yield is important for producing pharmaceuticals and biorenewable chemical precursors. In this work, we engineered cofactor and transport pathways in Saccharomyces cerevisiae to increase acetyl-CoA, an important polyketide building block. The highly regulated yeast pyruvate dehydrogenase bypass pathway was supplemented by overexpressing a modified Escherichia coli pyruvate dehydrogenase complex (PDHm) that accepts NADP⁺ for acetyl-CoA production. After 24 h of cultivation, a 3.7-fold increase in NADPH/NADP⁺ ratio was observed relative to the base strain, and a 2.2-fold increase relative to introduction of the native E. coli PDH. Both E. coli pathways increased acetyl-CoA levels approximately 2-fold relative to the yeast base strain. Combining PDHm with a ZWF1 deletion to block the major yeast NADPH biosynthesis pathway resulted in a 12-fold NADPH boost and a 2.2-fold increase in acetyl-CoA. At 48 h, only this coupled approach showed increased acetyl-CoA levels, 3.0-fold higher than that of the base strain. The impact on polyketide synthesis was evaluated in a S. cerevisiae strain expressing the Gerbera hybrida 2-pyrone synthase (2-PS) for the production of the polyketide triacetic acid lactone (TAL). Titers of TAL relative to the base strain improved only 30% with the native E. coli PDH, but 3.0-fold with PDHm and 4.4-fold with PDHm in the Δzwf1 strain. Carbon was further routed toward TAL production by reducing mitochondrial transport of pyruvate and acetyl-CoA; deletions in genes POR2, MPC2, PDA1, or YAT2 each increased titer 2–3-fold over the base strain (up to 0.8 g/L), and in combination to 1.4 g/L. Combining the two approaches (NADPH-generating acetyl-CoA pathway plus reduced metabolite flux into the mitochondria) resulted in a final TAL titer of 1.6 g/L, a 6.4-fold increase over the non-engineered yeast strain, and 35% of theoretical yield (0.16 g/g glucose), the highest reported to date. These biological driving forces present new avenues for improving high-yield production of acetyl-CoA derived compounds.     

Computation of metabolic fluxes and efficiencies for biological carbon dioxide fixation

With rising energy prices and concern over the environmental impact of fossil fuel consumption, the push to develop biomass derived fuels has increased significantly. Although most global carbon fixation occurs via the Calvin Benson Bassham cycle, there are currently five other known pathways for carbon fixation; the goal of this study was to determine the thermodynamic efficiencies of all six carbon fixation pathways for the production of biomass using flux balance analysis. The three chemotrophic pathways, the reductive acetyl-CoA pathway, the 3-hydroxypropionate/4-hydroxybutyrate cycle and the dicarboxylate/4-hydroxybutyrate cycle, were found to be more efficient than photoautotrophic carbon fixation pathways. However, as hydrogen is not freely available, the energetic cost of hydrogen production from sunlight was calculated and included in the overall energy demand, which results in a 5 fold increase in the energy demand of chemoautotrophic carbon fixation. Therefore, when the cost of hydrogen production is included, photoautotrophic pathways are more efficient. However, the energetic cost for the production of 12 metabolic precursors was found to vary widely across the different carbon fixation pathways; therefore, different pathways may be more efficient at producing products from a single precursor than others. The results of this study have significant impact on the selection or design of autotrophic organisms for biofuel or biochemical production. Overall biomass production from solar energy is most efficient in organisms using the reductive TCA cycle, however, products derived from one metabolic precursor may be more efficiently produced using other carbon fixation pathways.