RESUMO
Elevated bacterial sialidase activity in the female genital tract is strongly associated with poor health outcomes including preterm birth and bacterial vaginosis (BV). These negative effects may arise from sialidase-mediated degradation of the protective mucus layer in the cervicovaginal environment. Prior biochemical studies of vaginal bacterial sialidases have focused solely on the BV-associated organism Gardnerella vaginalis. Despite their implications for sexual and reproductive health, sialidases from other vaginal bacteria have not been characterized. Here, we show that vaginal Prevotella species produce sialidases that possess variable activity toward mucin substrates. The sequences of sialidase genes and their presence are largely conserved across clades of Prevotella from different geographies, hinting at their importance globally. Finally, we find that Prevotella sialidase genes and transcripts, including those encoding mucin-degrading sialidases from Prevotella timonensis, are highly prevalent and abundant in human vaginal genomes and transcriptomes. Together, our results identify Prevotella as a critical source of sialidases in the vaginal microbiome, improving our understanding of this detrimental bacterial activity.
Assuntos
Microbiota , Neuraminidase , Prevotella , Vagina , Humanos , Prevotella/enzimologia , Prevotella/genética , Prevotella/isolamento & purificação , Neuraminidase/metabolismo , Neuraminidase/genética , Feminino , Vagina/microbiologia , Mucinas/metabolismo , Vaginose Bacteriana/microbiologia , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genéticaRESUMO
Acetogenic Clostridia are obligate anaerobes that have emerged as promising microbes for the renewable production of biochemicals owing to their ability to efficiently metabolize sustainable single-carbon feedstocks. Additionally, Clostridia are increasingly recognized for their biosynthetic potential, with recent discoveries of diverse secondary metabolites ranging from antibiotics to pigments to modulators of the human gut microbiota. Lack of efficient methods for genomic integration and expression of large heterologous DNA constructs remains a major challenge in studying biosynthesis in Clostridia and using them for metabolic engineering applications. To overcome this problem, we harnessed chassis-independent recombinase-assisted genome engineering (CRAGE) to develop a workflow for facile integration of large gene clusters (>10 kb) into the human gut acetogen Eubacterium limosum. We then integrated a non-ribosomal peptide synthetase gene cluster from the gut anaerobe Clostridium leptum, which previously produced no detectable product in traditional heterologous hosts. Chromosomal expression in E. limosum without further optimization led to production of phevalin at 2.4 mg/L. These results further expand the molecular toolkit for a highly tractable member of the Clostridia, paving the way for sophisticated pathway engineering efforts, and highlighting the potential of E. limosum as a Clostridial chassis for exploration of anaerobic natural product biosynthesis.
RESUMO
Eubacterium limosum is a Clostridial acetogen that efficiently utilizes a wide range of single-carbon substrates and contributes to metabolism of health-associated compounds in the human gut microbiota. These traits have led to interest in developing it as a platform for sustainable CO2-based biofuel production to combat carbon emissions, and for exploring the importance of the microbiota in human health. However, synthetic biology and metabolic engineering in E. limosum have been hindered by the inability to rapidly make precise genomic modifications. Here, we screened a diverse library of recombinase proteins to develop a highly efficient oligonucleotide-based recombineering system based on the viral recombinase RecT. Following optimization, the system is capable of catalyzing ssDNA recombination at an efficiency of up to 2%. Addition of a Cas9 counterselection system eliminated unrecombined cells, with up to 100% of viable cells encoding the desired mutation, enabling creation of genomic point mutations in a scarless and markerless manner. We deployed this system to create a clean knockout of the extracellular polymeric substance (EPS) gene cluster, generating a strain incapable of biofilm formation. This approach is rapid and simple, not requiring laborious homology arm cloning, and can readily be retargeted to almost any genomic locus. This work overcomes a major bottleneck in E. limosum genetic engineering by enabling precise genomic modifications, and provides both a roadmap and associated recombinase plasmid library for developing similar systems in other Clostridia of interest.
Assuntos
Sistemas CRISPR-Cas , Eubacterium , Eubacterium/genética , Sistemas CRISPR-Cas/genética , Engenharia Metabólica/métodos , Recombinação Genética/genética , Genoma Bacteriano/genética , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Recombinases/genética , Recombinases/metabolismo , Engenharia Genética/métodos , Edição de Genes/métodos , Família MultigênicaRESUMO
In oxygen (O2)-controlled cell culture, an indispensable tool in biological research, it is presumed that the incubator setpoint equals the O2 tension experienced by cells (i.e., pericellular O2). However, it is discovered that physioxic (5% O2) and hypoxic (1% O2) setpoints regularly induce anoxic (0% O2) pericellular tensions in both adherent and suspension cell cultures. Electron transport chain inhibition ablates this effect, indicating that cellular O2 consumption is the driving factor. RNA-seq analysis revealed that primary human hepatocytes cultured in physioxia experience ischemia-reperfusion injury due to cellular O2 consumption. A reaction-diffusion model is developed to predict pericellular O2 tension a priori, demonstrating that the effect of cellular O2 consumption has the greatest impact in smaller volume culture vessels. By controlling pericellular O2 tension in cell culture, it is found that hypoxia vs. anoxia induce distinct breast cancer transcriptomic and translational responses, including modulation of the hypoxia-inducible factor (HIF) pathway and metabolic reprogramming. Collectively, these findings indicate that breast cancer cells respond non-monotonically to low O2, suggesting that anoxic cell culture is not suitable for modeling hypoxia. Furthermore, it is shown that controlling atmospheric O2 tension in cell culture incubators is insufficient to regulate O2 in cell culture, thus introducing the concept of pericellular O2-controlled cell culture.
Assuntos
Neoplasias da Mama , Técnicas de Cultura de Células , Oxigênio , Humanos , Oxigênio/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/genética , Técnicas de Cultura de Células/métodos , Feminino , Hipóxia Celular/fisiologia , Consumo de OxigênioRESUMO
Hydrogen sulfide (H2S) is a gaseous microbial metabolite whose role in gut diseases is debated, with contradictory results stemming from experimental difficulties associated with accurate dosing and measuring H2S and the use of model systems that do not accurately represent the human gut environment. Here, we engineer Escherichia coli to titrate H2S across the physiological range in a gut microphysiological system (chip) supportive of the co-culture of microbes and host cells. The chip is engineered to maintain H2S gas tension and enables visualization of co-culture in real time with confocal microscopy. Engineered strains colonize the chip and are metabolically active for 2 days, during which they produce H2S across a 16-fold range and induce changes in host gene expression and metabolism in an H2S-concentration-dependent manner. These results validate a platform for studying the mechanisms underlying microbe-host interactions by enabling experiments that are infeasible with current animal and in vitro models.
Assuntos
Microbioma Gastrointestinal , Sulfeto de Hidrogênio , Animais , Humanos , Sulfeto de Hidrogênio/metabolismo , Sistemas Microfisiológicos , Bactérias/metabolismo , Interações entre Hospedeiro e Microrganismos , Escherichia coli/metabolismoRESUMO
Oxygen (O2) tension plays a key role in tissue function and pathophysiology. O2-controlled cell culture, in which the O2 concentration in an incubator's gas phase is controlled, is an indispensable tool to study the role of O2 in vivo. For this technique, it is presumed that the incubator setpoint is equal to the O2 tension that cells experience (i.e., pericellular O2). We discovered that physioxic (5% O2) and hypoxic (1% O2) setpoints regularly induce anoxic (0.0% O2) pericellular tensions in both adherent and suspension cell cultures. Electron transport chain inhibition ablates this effect, indicating that cellular O2 consumption is the driving factor. RNA-seq revealed that primary human hepatocytes cultured in physioxia experience ischemia-reperfusion injury due to anoxic exposure followed by rapid reoxygenation. To better understand the relationship between incubator gas phase and pericellular O2 tensions, we developed a reaction-diffusion model that predicts pericellular O2 tension a priori. This model revealed that the effect of cellular O2 consumption is greatest in smaller volume culture vessels (e.g., 96-well plate). By controlling pericellular O2 tension in cell culture, we discovered that MCF7 cells have stronger glycolytic and glutamine metabolism responses in anoxia vs. hypoxia. MCF7 also expressed higher levels of HIF2A, CD73, NDUFA4L2, etc. and lower levels of HIF1A, CA9, VEGFA, etc. in response to hypoxia vs. anoxia. Proteomics revealed that 4T1 cells had an upregulated epithelial-to-mesenchymal transition (EMT) response and downregulated reactive oxygen species (ROS) management, glycolysis, and fatty acid metabolism pathways in hypoxia vs. anoxia. Collectively, these results reveal that breast cancer cells respond non-monotonically to low O2, suggesting that anoxic cell culture is not suitable to model hypoxia. We demonstrate that controlling atmospheric O2 tension in cell culture incubators is insufficient to control O2 in cell culture and introduce the concept of pericellular O2-controlled cell culture.
RESUMO
The human vaginal microbiota is frequently dominated by lactobacilli and transition to a more diverse community of anaerobic microbes is associated with health risks. Glycogen released by lysed epithelial cells is believed to be an important nutrient source in the vagina. However, the mechanism by which vaginal bacteria metabolize glycogen is unclear, with evidence implicating both bacterial and human enzymes. Here we biochemically characterize six glycogen-degrading enzymes (GDEs), all of which are pullanases (PulA homologues), from vaginal bacteria that support the growth of amylase-deficient Lactobacillus crispatus on glycogen. We reveal variations in their pH tolerance, substrate preferences, breakdown products and susceptibility to inhibition. Analysis of vaginal microbiome datasets shows that these enzymes are expressed in all community state types. Finally, we confirm the presence and activity of bacterial and human GDEs in cervicovaginal fluid. This work establishes that bacterial GDEs can participate in the breakdown of glycogen, providing insight into metabolism that may shape the vaginal microbiota.
Assuntos
Amilases , Microbiota , Feminino , Humanos , Vagina/microbiologia , Bactérias/genética , Bactérias/metabolismo , Microbiota/fisiologia , Glicogênio/metabolismoRESUMO
Hydrogen sulfide (H2S) is a gaseous microbial metabolite whose role in gut diseases is debated, largely due to the difficulty in controlling its concentration and the use of non-representative model systems in previous work. Here, we engineered E. coli to titrate H2S controllably across the physiological range in a gut microphysiological system (chip) supportive of the co-culture of microbes and host cells. The chip was designed to maintain H2S gas tension and enable visualization of co-culture in real-time with confocal microscopy. Engineered strains colonized the chip and were metabolically active for two days, during which they produced H2S across a sixteen-fold range and induced changes in host gene expression and metabolism in an H2S concentration-dependent manner. These results validate a novel platform for studying the mechanisms underlying microbe-host interactions, by enabling experiments that are infeasible with current animal and in vitro models.
RESUMO
Gastrointestinal mucus plays essential roles in modulating interactions between intestinal lumen contents, including orally delivered drug carriers and the gut microbiome, and underlying epithelial and immune tissues and cells. This review is focused on the properties of and methods for studying native gastrointestinal mucus and its interactions with intestinal lumen contents, including drug delivery systems, drugs, and bacteria. The properties of gastrointestinal mucus important to consider in its analysis are first presented, followed by a discussion of different experimental setups used to study gastrointestinal mucus. Applications of native intestinal mucus are then described, including experimental methods used to study mucus as a barrier to drug delivery and interactions with intestinal lumen contents that impact barrier properties. Given the significance of the microbiota in health and disease, its impact on drug delivery and drug metabolism, and the use of probiotics and microbe-based delivery systems, analysis of interactions of bacteria with native intestinal mucus is then reviewed. Specifically, bacteria adhesion to, motility within, and degradation of mucus is discussed. Literature noted is focused largely on applications of native intestinal mucus models as opposed to isolated mucins or reconstituted mucin gels.
Assuntos
Aderência Bacteriana , Portadores de Fármacos , Humanos , Portadores de Fármacos/metabolismo , Intestinos , Mucinas/metabolismo , Muco/metabolismo , Bactérias/metabolismo , Mucosa Intestinal/metabolismoRESUMO
Eubacterium limosum is an acetogenic bacterium of potential industrial relevance for its ability to efficiently metabolize a range of single carbon compounds. However, extracellular polymeric substance (EPS) produced by the type strain ATCC 8486 is a serious impediment to bioprocessing and genetic engineering. To remove these barriers, here we bioinformatically identified genes involved in EPS biosynthesis, and targeted several of the most promising candidates for inactivation, using a homologous recombination-based approach. Deletion of a single genomic region encoding homologues for epsABC, ptkA, and tmkA resulted in a strain incapable of producing EPS. This strain is significantly easier to handle by pipetting and centrifugation, and retains important wild-type phenotypes including the ability to grow on methanol and carbon dioxide and limited oxygen tolerance. Additionally, this strain is also more genetically tractable with a 2-fold increase in transformation efficiency compared to the highest previous reports. This work advances a simple, rapid protocol for gene knockouts in E. limosum using only the native homologous recombination machinery. These results will hasten the development of this organism as a workhorse for valorization of single carbon substrates, as well as facilitate exploration of its role in the human gut microbiota.
Assuntos
Eubacterium , Matriz Extracelular de Substâncias Poliméricas , Humanos , Eubacterium/genética , Eubacterium/metabolismo , Engenharia GenéticaRESUMO
Replacing petroleum-based fuels in high-power sectors like aviation and rocketry is a major sustainability challenge. Polycyclopropanated hydrocarbons provide excellent fuel characteristics for these applications, but their synthesis is challenging. Cruz-Morales et al. demonstrated microbial production of a range of polycyclopropanated 'fuelimycins' based on an unusual iterative polyketide synthase (iPKS).
Assuntos
Bactérias , Hidrocarbonetos , Policetídeo SintasesRESUMO
Acetogenic bacteria are an increasingly popular choice for producing fuels and chemicals from single carbon (C1) substrates. Eubacterium limosum is a promising acetogen with several native advantages, including the ability to catabolize a wide repertoire of C1 feedstocks and the ability to grow well on agar plates. However, despite its promise as a strain for synthetic biology and metabolic engineering, there are insufficient engineering tools and molecular biology knowledge to leverage its native strengths for these applications. To capitalize on the natural advantages of this organism, here we extended its limited engineering toolbox. We evaluated the copy number of three common plasmid origins of replication and devised a method of controlling copy number and heterologous gene expression level by modulating antibiotic concentration. We further quantitatively assessed the strength and regulatory tightness of a panel of promoters, developing a series of well-characterized vectors for gene expression at varying levels. In addition, we developed a black/white colorimetric genetic reporter assay and leveraged the high oxygen tolerance of E. limosum to develop a simple and rapid transformation protocol that enables benchtop transformation. Finally, we developed two new antibiotic selection markers-doubling the number available for this organism. These developments will enable enhanced metabolic engineering and synthetic biology work with E. limosum.
Assuntos
Engenharia Genética , Engenharia Metabólica , Ágar , Antibacterianos , Carbono , Eubacterium , Engenharia Metabólica/métodos , OxigênioRESUMO
Vaginal microbiota composition affects many facets of reproductive health. Lactobacillus iners-dominated microbial communities are associated with poorer outcomes, including higher risk of bacterial vaginosis (BV), compared with vaginal microbiota rich in L. crispatus. Unfortunately, standard-of-care metronidazole therapy for BV typically results in dominance of L. iners, probably contributing to post-treatment relapse. Here we generate an L. iners isolate collection comprising 34 previously unreported isolates from 14 South African women with and without BV and 4 previously unreported isolates from 3 US women. We also report an associated genome catalogue comprising 1,218 vaginal Lactobacillus isolate genomes and metagenome-assembled genomes from >300 women across 4 continents. We show that, unlike L. crispatus, L. iners growth is dependent on L-cysteine in vitro and we trace this phenotype to the absence of canonical cysteine biosynthesis pathways and a restricted repertoire of cysteine-related transport mechanisms. We further show that cysteine concentrations in cervicovaginal lavage samples correlate with Lactobacillus abundance in vivo and that cystine uptake inhibitors selectively inhibit L. iners growth in vitro. Combining an inhibitor with metronidazole promotes L. crispatus dominance of defined BV-like communities in vitro by suppressing L. iners growth. Our findings enable a better understanding of L. iners biology and suggest candidate treatments to modulate the vaginal microbiota to improve reproductive health for women globally.
Assuntos
Microbiota , Vaginose Bacteriana , Cisteína/metabolismo , Feminino , Humanos , Lactobacillus/genética , Lactobacillus/metabolismo , Masculino , Metronidazol/metabolismo , Metronidazol/farmacologia , Metronidazol/uso terapêutico , Vagina/microbiologia , Vaginose Bacteriana/tratamento farmacológico , Vaginose Bacteriana/microbiologiaRESUMO
Single-carbon (C1, or one-carbon) substrates are promising feedstocks for sustainable biofuel and biochemical production. Crucial to the goal of engineering C1-utilizing strains for improved production is a quantitative understanding of the organization, regulation and rates of the reactions that underpin C1 metabolism. 13C Metabolic flux analysis (MFA) is a well-established platform for interrogating these questions with multi-carbon substrates, and uses the differential labeling of metabolites that results from feeding a substrate with position-specific incorporation of 13C in order to infer quantitative fluxes and pathway topology. Adapting isotopic tracer approaches to C1 metabolism, where position-specific substrate labeling is impossible, requires additional experimental considerations. Here we review recent studies that have developed isotopic tracer approaches to overcome the challenge of uniform metabolite labeling and provide quantitative insight into C1 metabolism.
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Análise do Fluxo Metabólico , Modelos Biológicos , Carbono , Isótopos de Carbono/metabolismo , Marcação por Isótopo/métodos , Análise do Fluxo Metabólico/métodosRESUMO
Single carbon (C1) substrates such as methanol are gaining increasing attention as cost-effective and environmentally friendly microbial feedstocks. Recent impressive metabolic engineering efforts to import C1 catabolic pathways into the non-methylotrophic bacterium Escherichia coli have led to synthetic strains growing on methanol as the sole carbon source. However, the growth rate and product yield in these strains remain inferior to native methylotrophs. Meanwhile, an ever-expanding genetic engineering toolbox is increasing the tractability of native C1 utilizers, raising the question of whether it is best to use an engineered strain or a native host for the microbial assimilation of C1 substrates. Here we provide perspective on this debate, using recent work in E. coli and the methylotrophic acetogen Eubacterium limosum as case studies.
Assuntos
Engenharia Metabólica , Metanol , Bactérias/metabolismo , Carbono/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Metanol/metabolismoRESUMO
Single carbon (C1) feedstocks are attractive for bioconversion, but native C1 assimilation pathways are difficult to engineer.Chou et al.developed a novel process for elongating the C1 compound formyl-CoA. Besides demonstrating a new approach for C1 bioconversion, this work paves the way for engineering synthetic methylotrophy into chassis organisms.
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Carbono , Liases , Álcoois Graxos , OxirreduçãoRESUMO
Synthetic methylotrophy, the modification of organisms such as E. coli to grow on methanol, is a longstanding goal of metabolic engineering and synthetic biology. The poor kinetic properties of NAD-dependent methanol dehydrogenase, the first enzyme in most methanol assimilation pathways, limit pathway flux and present a formidable challenge to synthetic methylotrophy. To address this bottleneck, we used a formaldehyde biosensor to develop a phage-assisted noncontinuous evolution (PANCE) selection for variants of Bacillus methanolicus methanol dehydrogenase 2 (Bm Mdh2). Using this selection, we evolved Mdh2 variants with up to 3.5-fold improved Vmax. The mutations responsible for enhanced activity map to the predicted active site region homologous to that of type III iron-dependent alcohol dehydrogenases, suggesting a new critical region for future methanol dehydrogenase engineering strategies. Evolved Mdh2 variants enable twice as much 13C-methanol assimilation into central metabolites than previously reported state-of-the-art methanol dehydrogenases. This work provides improved Mdh2 variants and establishes a laboratory evolution approach for metabolic pathways in bacterial cells.
Assuntos
Oxirredutases do Álcool/genética , Bacillus/genética , Proteínas de Bactérias/genética , Bacteriófagos/genética , Escherichia coli/genética , Formaldeído/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Engenharia Metabólica/métodos , Redes e Vias Metabólicas/genética , Metanol , NAD/genéticaRESUMO
Monoethylene glycol (MEG) is an important commodity chemical with applications in numerous industrial processes, primarily in the manufacture of polyethylene terephthalate (PET) polyester used in packaging applications. In the drive towards a sustainable chemical industry, bio-based production of MEG from renewable biomass has attracted growing interest. Recent attempts for bio-based MEG production have investigated metabolic network modifications in Escherichia coli, specifically rewiring the xylose assimilation pathways for the synthesis of MEG. In the present study, we examined the suitability of Saccharomyces cerevisiae, a preferred organism for industrial applications, as platform for MEG biosynthesis. Based on combined genetic, biochemical and fermentation studies, we report evidence for the existence of an endogenous biosynthetic route for MEG production from D-xylose in S. cerevisiae which consists of phosphofructokinase and fructose-bisphosphate aldolase, the two key enzymes in the glycolytic pathway. Further metabolic engineering and process optimization yielded a strain capable of producing up to 4.0â¯g/L MEG, which is the highest titer reported in yeast to-date.
Assuntos
Etilenoglicol/metabolismo , Glicólise/genética , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , DNA Fúngico/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Fermentação , Frutose-Bifosfato Aldolase/genética , Frutose-Bifosfato Aldolase/metabolismo , Engenharia Metabólica , Redes e Vias Metabólicas/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Xilose/metabolismoRESUMO
Synthesis gas (syngas) fermentation via the Wood-Ljungdahl pathway is receiving growing attention as a possible platform for the fixation of CO2 and renewable production of fuels and chemicals. However, the pathway operates near the thermodynamic limit of life, resulting in minimal adenosine triphosphate (ATP) production and long doubling times. This calls into question the feasibility of producing high-energy compounds at industrially relevant levels. In this study, we investigated the possibility of co-utilizing nitrate as an inexpensive additional electron acceptor to enhance ATP production during H2 -dependent growth of Clostridium ljungdahlii, Moorella thermoacetica, and Acetobacterium woodii. In contrast to other acetogens tested, growth rate and final biomass titer were improved for C. ljungdahlii growing on a mixture of H2 and CO2 when supplemented with nitrate. Transcriptomic analysis, 13CO2 labeling, and an electron balance were used to understand how electron flux was partitioned between CO2 and nitrate. We further show that, with nitrate supplementation, the ATP/adenosine diphosphate (ADP) ratio and acetyl-CoA pools were increased by fivefold and threefold, respectively, suggesting that this strategy could be useful for the production of ATP-intensive heterologous products from acetyl-CoA. Finally, we propose a pathway for enhanced ATP production from nitrate and use this as a basis to calculate theoretical yields for a variety of products. This study demonstrates a viable strategy for the decoupling of ATP production from carbon dioxide fixation, which will serve to significantly improve the CO2 fixation rate and the production metrics of other chemicals from CO2 and H2 in this host.
Assuntos
Acetobacterium/metabolismo , Dióxido de Carbono/metabolismo , Clostridium/metabolismo , Hidrogênio/metabolismo , Moorella/metabolismo , Nitratos/metabolismo , Acetobacterium/crescimento & desenvolvimento , Trifosfato de Adenosina/biossíntese , Ciclo do Carbono , Clostridium/crescimento & desenvolvimento , Análise do Fluxo Metabólico , Moorella/crescimento & desenvolvimentoRESUMO
Advanced bioproduct synthesis via reductive metabolism requires coordinating carbons, ATP and reducing agents, which are generated with varying efficiencies depending on metabolic pathways. Substrate mixtures with direct access to multiple pathways may optimally satisfy these biosynthetic requirements. However, native regulation favouring preferential use precludes cells from co-metabolizing multiple substrates. Here we explore mixed substrate metabolism and tailor pathway usage to synergistically stimulate carbon reduction. By controlled cofeeding of superior ATP and NADPH generators as 'dopant' substrates to cells primarily using inferior substrates, we circumvent catabolite repression and drive synergy in two divergent organisms. Glucose doping in Moorella thermoacetica stimulates CO2 reduction (2.3 g gCDW-1 h-1) into acetate by augmenting ATP synthesis via pyruvate kinase. Gluconate doping in Yarrowia lipolytica accelerates acetate-driven lipogenesis (0.046 g gCDW-1 h-1) by obligatory NADPH synthesis through the pentose cycle. Together, synergistic cofeeding produces CO2-derived lipids with 38% energy yield and demonstrates the potential to convert CO2 into advanced bioproducts. This work advances the systems-level control of metabolic networks and CO2 use, the most pressing and difficult reduction challenge.