Saccharomyces cerevisiae. Non-conventional yeast systems have several advantageous characteristics that are not easily engineered such as ethanol tolerance, low pH tolerance, thermotolerance, inhibitor tolerance, genetic diversity and so forth. Currently, synthetic biology is still in its initial steps for studies in non-conventional yeasts such as Yarrowia lipolytica, Kluyveromyces marxianus, Issatchenkia orientalis and Pichia pastoris. Therefore, the development and application of advanced synthetic engineering tools must also focus on these underexploited, non-conventional yeast species. Herein, we review the basic synthetic biology tools that can be applied to the standard S. cerevisiae model strain, as well as those that have been developed for non-conventional yeasts. In addition, we will discuss the recent advances employed to develop non-conventional yeast strains that are efficient for the production of a variety of chemicals through the use of metabolic engineering and synthetic biology.Microorganisms are effective platforms for the production of a variety of chemicals including biofuels, commodity chemicals, polymers and other natural products. However, deep cellular understanding is required for improvement of current biofuel cell factories to truly transform the Bioeconomy. Modifications in microbial metabolic pathways and increased resistance to various types of stress caused by the production of these chemicals are crucial in the generation of robust and efficient production hosts. Recent advances in systems and synthetic biology provide new tools for metabolic engineering to design strategies and construct optimal biocatalysts for the sustainable production of desired chemicals, especially in the case of ethanol and fatty acid production. Yeast is an efficient producer of bioethanol and most of the available synthetic biology tools have been developed for the industrial yeast  The production of biofuels and chemical bioproducts utilizing live cells and thus their enzymatic conversion pathways, requires less energy than other conversion processes. Therefore, microbial factories are increasingly being developed through the engineering of metabolic pathways and the redirection of carbon towards desired products . The maiOptimizing native gene expression and expression of non-native genes and pathways enables the engineering of microbes with new catalytic activities or improvements of native phenotypes such as tolerance to stress conditions ,3. CurreS. cerevisiae has long been the favorite organism used as a cell factory in the commercial production of biologically-based chemicals, mainly due to its ancient use in classical industrial applications (wine and beer production) and our vast physiological and genetic knowledge of this organism [With more than a thousand unique species already identified, yeasts are one of the most widely studied microorganisms. For thousands of years, these species have been associated with the production of fermented drinks . The budorganism . A schemIts success as a model organism is mainly due to the high degree of conservation of important cellular processes  and the availability of genetic engineering tools. In addition to its classic application in the production of wine and beer, its use has also extended to the production of bioethanol, aimed at the production of transport fuels. In 2017, the United States produced more than 60 billion liters, followed by Brazil with production of about 30 billion liters, with the rest of the world producing about 15 billion liters [S. cerevisiae has disadvantages, such as the inability to consume more economical and sustainable substrates such as xylose, arabinose and glycerol. Additionally, S. cerevisiae is not able to tolerate high temperatures (>34 °C) and low external pH (<3) [Yarrowia lipolytica, Kluyveromyces marxianus, Issatchenkia orientalis and Pichia pastoris have many physiological, metabolic and regulatory advantages that could overcome these limitations [Despite the ease of engineering,  pH <3) , so its  , so itsitations ,11.S. cerevisiae [S. cerevisiae and their adaptation for use in non-model yeast systems to further improve the range and scale of industries for the biological production of chemicals using yeast platforms.While most reviews in the literature addressing the engineering and characteristics of yeast for biofuel production focus on the model industrial yeast revisiae ,14,15,16revisiae ,17,18,19S. cerevisiae has been widely utilized as a model organism for basic science as well as for the production of biochemicals and heterologous expression of proteins [http://www.yeastgenome.org/), is a major scientific database that gathers genetic information from the yeast S. cerevisiae. This tool has developed several new features that allow to compare and integrate detailed information about individual genes throughout the genome, allowing to connect genetic groups with common characteristics [As the first eukaryote to have its genome sequenced  and the proteins ,22. Systproteins ,24. The eristics . This aleristics ,24.S. cerevisiae as a versatile host for metabolic engineering has been successfully demonstrated for the synthesis of a variety of molecules [The potential of olecules ,26. For olecules , as wellolecules .S. cerevisiae is its inability to tolerate important stress factors during the production of second-generation biofuels. In S. cerevisiae is also the lack of several metabolites of short-chain acyl-CoA that are necessary in the biosynthesis of economically important compounds, such as organic acids and linear alcohols [Myxobacterium xanthus, as well as the first report of production of n-butyryl-CoA and n-hexanoyl-CoA in S. cerevisiae.As mentioned above, one of the main disadvantages of alcohols . These calcohols . Bioalkaalcohols . Foo et alcohols  expressealcohols  introducS. cerevisiae as a cell factory has attracted great interest, mainly because they are currently chemically synthesized, which adds high cost to their production. This important class of secondary metabolites has attractive industrial applications, such as flavor and fragrance additives in the food and cosmetics industry, medicines and biofuels. The biosynthesis of terpenoids in S. cerevisiae uses two C5 precursors: isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). In yeasts and mammals, acetyl-CoA is converted into mevalonic acid leading to the production of IPP. Pre-transferase enzymes act on IPP synthesizing building blocks such as geranyl pyrophosphate (GPP); farnesyl pyrophosphate (FPP); and geranyl pyrophosphate (GGPP). FPP is usually the most common and desired product of the pathway that is used for sterol biosynthesis [The production of terpenoids and isoprenoids using ynthesis ,55.S. cerevisiae but its level of natural biosynthesis in yeast S. cerevisiae is very low. By blocking all competing unproductive pathways by gene deletions and expressing the cytosolic pathway of isobutanol, it was possible to produce 57 times more isobutanol when compared to the studied parent strain. Using isobutanol production pathways located in mitochondria, the respiratory response induced by xylose can be better used in engineered yeasts to use this substrate. Lane et al. [Isobutanol is also a promising chemical production platform for e et al.  employedS. cerevisiae is the most industrially applied and most studied in the field of yeast engineering, its efficient performance in the production of ethanol occurs under aerobic growth, which defines it as “Crabtree-positive” [Y. lipolytica is not able to produce ethanol from the moment glucose is supplied to respiring cells, which defines it as “Crabtree-negative” [Y. lipolytica produces various types of lipases that can be used as biocatalysts and is able to accumulate lipids (more than 80% cell dry weight) that can be used as starting materials for the production of biodiesel [Y. lipolytica research so far has been to elucidate its metabolism involving the biosynthesis of fatty acids, use of hydrophobic substrate and lipid degradation, such as β-oxidation [Although the yeast ositive” . Howeverositive” . The nonegative” ,61. Thisegative” . Strainsegative” . Y. lipoiodiesel . The maixidation .Y. lipolytica, a compound used mainly in variable mixtures of gasoline and aviation fuel. They used a soy lipoxygenase enzyme to separate linoleic acid into pentane and a tridecadienic acid as a by-product, producing pentane at ~5 mg/L. Y. lipolytrica has also been engineered to produce α-Farnesene, a terpenoid compound that can be utilized as a precursor to aviation fuel at 260 mg/L [In addition to biodiesel as a product, Blazeck et al.  designed260 mg/L .Y. lipolytica has shown great compatibility in large-scale bioprocesses, facilitating its use in the production of biofuels (biodiesel and bio-oil) [Y. lipolytica cultures leads to increased citrate concentrations [bio-oil) . Despitetrations , resultitrations . Metaboltrations . In additrations .K. marxianus is also defined as a Crabtree negative (respiring) yeast and has advantages over other yeast species, mainly because it has greater thermal stability (>40 °C), low pH tolerance [K. marxianus also has a high secretory capacity in relation to S. cerevisiae, due to properties such as appropriate glycosylation and strong signal peptides [olerance , a relatolerance . Fermentolerance . K. marxpeptides .S. cerevisiae in 2nd generation ethanol processes, as it is able to naturally assimilate a variety of sugars in addition to glucose, such as pentose, hexose, arabinose, cellobiose, lactose and xylose, as well as some toxic compounds present in some sources of lignocellulosic biomass [K. marxianus. Substrates such as lactose, glucose, inulin, xylose were evaluated. The modeling results suggested that the aeration control can optimize the product yield and distribution of the metabolic flow in K. marxianus. Gao et al. [K. marxianus, conducted experiments analyzed through RNA-seq, which allowed the identification of genes associated with ethanol metabolism. It was observed through the regulation of global expression that the increased expression of inulinase and the low activity of the gene related to autophagy, ATG8, guaranteed fast and ideal fermentation processes.This strain has been viewed as an alternative to  biomass ,75. Pent biomass  applied o et al. , aiming K. marxianus can effectively transport and metabolize pentoses via the aldose reductase pathway, it is still necessary to modify the metabolism of wild type strains for the production of ethanol from pentoses. The strategies used in the development of K. marxianus strains to improve their ability to ferment pentoses are generally mirrored in studies carried out on S. cerevisiae [Although revisiae . One of revisiae .K. marxianus grows well, replicating more quickly in aerobic conditions without fermentation to ethanol, reaching production of 5.1 g/L of ethanol from 10 g/L of glucose and a growth rate of 0.58–0.63 h−1 even at a temperature of 45 °C [It is known that of 45 °C . Due to of 45 °C ,82.K. marxianus in relation to glycolysis and the tricarboxylic acid (TCA) cycle and the dynamics of the corresponding genetic controls are still underexplored [K. marxianus has been genetically manipulated in order to increase its ethanol production efficiencies. Despite having excellent characteristics for bioprocesses, its capacity to resist ethanol is relatively low, being able to support only a maximum of 6% (v/v) of ethanol. This low ethanol tolerance has the consequence of low ethanol yield, being the main obstacle to practical application of this yeast in the industry [K. marxianus transcriptomes in adaptive evolution in 6% (v/v) ethanol using lactose as a carbon source. As a result, they obtained a decrease in the central metabolic flow and the biosynthesis of fatty acids. Li and colleagues examined random mutagenesis library of the TATA-binding protein of K. marxianus, Spt15, which facilitates the differential expression of hundreds of genes that act as an interconnected network for the ethanol tolerance phenotype. In this study, a strain with a maximum tolerance of 5% (v/v) of ethanol was obtained, while the original wild type strain had a tolerance of only 2% (v/v). With a metabolism similar to S. cerevisiae, the ethanol tolerance of K. marxianus also has a genetic and metabolic complexity that makes efficient strain engineering difficult, thus requiring greater efforts to understand its molecular base involved in resistance mechanisms to factors of stress [The general metabolism of explored . K. marxindustry ,84. Dinif stress . In a stf stress  fermentaK. marxianus in industrial applications is the development of improved genetic tools to more quickly and effectively manipulate the genome. There are efforts to develop these tools in K. marxianus, mainly aiming at the modification of metabolic pathways [The key element for using pathways ,87.I. orientalis—also known as Pichia kudriavzevii [I. orientalis also has shown great potential in the production of succinic acid and bioethanol [First described as a wine yeast, the yeast iavzevii —has seveoethanol ,90.I. orientalis can produce ethanol under various stress conditions including high temperatures (42 °C) and high concentrations of Na2SO4 at pH 2.0. Production of d-xylonate or succinic acid have also been demonstrated at a low pH of 3 [I. orientalis SD108. The projected strain achieved a production of 11.63 g/L of succinic acid, with a yield of 0.12 g·g−1 and productivity of 0.11 g/L. The production of succinic acid using I. orientalis has also been demonstrated industrially by companies such as BioAmber, DSM and Roquete. Bioamber designed a strain capable of producing 89 g/L of succinate at a production rate of 0.93 g/L/h. DSM and Roquette through a partnership sell Biosuccinum through bioprocesses with the strain SUC-297 that produces 43 g/L of succinic acid [ pH of 3 ,91,92,93 pH of 3 . Xiao et pH of 3  improvednic acid .I. orientalis to build a path for the functional use of xylose. The CEN-L sequence identified through functional screening was able to improve the expression of the target genes. The development of this plasmid enabled a stable CRISPR/Cas9 system for introduction of multiplexed gene deletions. These technologies are fundamental for future metabolic engineering of I. orientalis [The genetic manipulation of this organism presents the same difficulties in relation to other non-conventional yeasts due to the lack of efficient genetic editing tools specific to this organism . Cao et ientalis .P. pastoris is a Crabtree-negative methylotrophic organism used industrially as a host to produce recombinant proteins in the pharmaceutical industry and in the production of higher alcohols and corresponding acetate esters [P. pastoris for heterologous expression of cellulolytic and hemicellulolytic enzymes makes it a really attractive yeast for industrial bioprocesses [e esters ,99. The rocesses .P. pastoris has been used as a cell biocatalyst for the production of biodiesel due to superficial display of lipases. Another advantage is that its cultivation can be carried out at high cell densities in a medium containing crude glycerol as a carbon source, a by-product of biodiesel production [P. pastoris is capable of metabolizing this compound [oduction . Crude goduction . For moscompound .Streptomyces sp. can be expressed heterologously in P. pastoris and this system can be applied for the production of biodiesel by adding a methanol stage in systems without the use of solvent [P. pastoris as a whole cell biocatalyst (WCB) with overexpression for improved thermostability. The optimum conditions studied led to a fatty acid methyl ester (FAME) yield of 60.7%, with a 47.3% yield after 4 batch cycles without glycerol generation. This indicates that the catalyst system used is promising and potentially economical for the production of biodiesel.Enzymes such as lipases have been studied in catalytic processes in the production of biodiesel . The use solvent . Liu et  solvent  used P. P. pastoris. Li et al. [Rhizomucor miehei-derived β-mannanase (mRmMan5A), reaching a high level of expression . This expression rate makes the process highly economical for industrial applications. MRmMan5A was used to hydrolyze a substrate for the production of manno-oligosaccharide. Approximately 80% of mannan in the substrate was converted into manno-oligosaccharides. These results suggest an extremely effective β-mannanase for the bioconversion of biomass rich in mannan.Expression and secretion of enzymes that aid sugar release and utilization are also of interest to improve production processes. Glucuronoesterase (GE) is a carbohydrate esterase that is able to cleave alkaline bonds of lignin-carbohydrate complexes under acidic pH conditions . The remi et al.  expresseP. pastoris. With the advancement of new genome engineering technologies including CRISPR/Cas9 genome editing to optimize strains, the versatility and competitiveness of this species can be further improved in the expression of enzymes for the production of second-generation ethanol [Promoter engineering has increased its expression capacity in addition to improving the ability to metabolically engineer  ethanol  and otheThe data obtained through systems biology and synthetic biology applied to the development of microorganisms can decrease the cost of producing desired chemicals simply by reducing the number of experiments necessary to optimize the system through the utilization of omics data to guide design to control expression, redox and flux to increase titers towards the chemical of interest ,111. In Beyond standard homologous recombination, next generation genetic manipulation tools such as zinc finger nucleases (ZFNs) and transcription activator effector nucleases (TALENS) were some of the first approaches used for accurate genome editing. Zinc finger nucleases (ZFNs) are hybrid restriction enzymes comprised of a customizable zinc-finger protein DNA-binding domain, which is fused to the FokI endonuclease cleavage domain. ZFN induce double-strand breaks that through cellular DNA repair processes have their genes replaced and targeted at high frequencies. Many different sequences can be attacked allowing new assemblies of ZFs. For this reason, dimerization becomes an advantage where cleavage does not occur at single binding sites and requires that the fingers have adequate specificity .S. cerevisiae for the overproduction of fatty acids [Y. lipolytica [TALENs are also customizable restriction enzymes as well as zinc finger nucleases (ZNFs), consisting of a fused DNA-binding domain to a non-specific nuclease domain. Its DNA binding domain contains several highly conserved repeating sequences consisting of up to 35 amino acids that bind to a single DNA base with specificity for binding to two unconserved amino acids. A challenge in using this approach is the need to build plasmids capable of encoding long repeating arrays . TALENs ty acids  and havepolytica , demonstThese methodologies were developed to specifically cleave DNA sequences; however, they are not advantageous for editing multiple targets and involve a more laborious and time-consuming construction of DNA-binding proteins . MethodoS. cerevisiae by perturbing 25 regulatory genes with 43,020 mutations [The CRISPR-Cas based editing systems can be utilized for high throughput, multiplex genome editing technologies for genotype-phenotype discovery. Array-based oligo technologies enable creating hundreds of thousands of cassettes containing a donor template and a genome-targeting gRNA and a pooled plasmid library generates designed mutations in parallel. After CRISPR/Cas9 genome editing, each plasmid can be served as a genetic barcode and the frequency of each designed mutation can be tracked by next-generation sequencing (NGS). This approach was applied to improve ethanol tolerance and production in utations . Also, gutations . A uniquutations . Furtherutations . If the S. cerevisiae systems. Since little information on CRISPR/Cas9 toolbox such as RNA polymerase III promoter for Cas9/gRNA expression and autonomously replicating sequence (ARS) for plasmid expression in host strains is available for several yeasts, the implementation of CRISPR/Cas9 is closely related to the development of optimized expression system [PEX10 targeting efficiency up to 92% [K. marxianus. Among the design of native and hybrid RNA polymerase III promoters for gRNA expression, the RPR1-tRNAGly hybrid promoter achieved the highest knockout efficiencies [S. cerevisiae was functional for plasmid replication in I. orientalis and the resulting episomal plasmid with a hybrid RPR’1-tRNALeu promoter for gRNA expression could attain greater than 97% gene disruption efficiency for various gene targets. Also, the platform was expanded to double and triple gene knockouts, resulting in disruption efficiencies of 90% and 47%, respectively [P. pastoris. They tested 95 combinations where they varied codon optimized Cas9 variants, gRNA sequences and ribozyme-coupled RNA Polymerase III and RNA Polymerase II promoters for expression of gRNAs and RNA Polymerase II promoters for Cas9 expression. Only 6 of 95 constructs were functional for efficient genome editing, suggesting that optimal conditions for efficient CRISPR/Cas9 function for P. pastoris had a very narrow range [The CRISPR/Cas9 technology has been applied to non-model yeasts, based on the foundation of n system ,120,121.p to 92% . Similarciencies . Recentlow range .Another key tool in synthetic biology are biosensors to enable tunable control of systems or to act as a selection for key metabolites. Based on RNA aptamers or proteins linked to small molecules, biosensors are capable of eliciting a desired transcriptional or allosteric response. Its applications focus on notification of a certain metabolic state or even heterogeneity in the cellular response when coupled with regulation. Several biosensors have been successfully developed to regulate optimal gene expression, reducing inhibitory compounds or toxic proteins without the need for inducers. Its application has also been extended to control of pathways in response to metabolic switches .S. cerevisiae aiming at the production of isobutanol and isopentanol. A transcriptional regulator of BCAA biosynthesis (Leu3p) was used to produce isobutanol. With some modifications it was possible to implement Leu3p in a different biosensor for isopentanol, another product of interest derived from BCAA. Also, light is an attractive source for the biosensor systems to control gene expression in yeast. A light-sensitive transcription factor from Erythrobacter litoralis (EL222) and its corresponding C120 promoter (PC120) were used to construct bidirectional gene circuits to either induce or repress genes of interest with light for microbial chemical production. The optogenetic regulation of metabolic pathways enabled optimizing engineered pathways and fermentation conditions using periodic light pulses [The development of biosensor systems depends on the addition of sensor elements to achieve their functionality. For example, genes that encode extracellular receptors are controlled by an inducible promoter and coupled to an easily detectable output, such as fluorescence, luminescence, colorimetry or growth rate . As examt pulses .S. cerevisiae have boosted the application of CRISPR-Cas9 systems in various non-conventional yeasts, however it is necessary to overcome difficulties such as lack of stable plasmids, lack of native RNA promoters, limitation of the Cas9 NGG PAM motif and low homologous recombination (HR) efficiency [S. cerevisiae, which has a high native capacity to perform homologous recombination [Successful experiments with ficiency . Homologbination ,130.K. marxianus and efficiently targeted metabolic regulation increased the ethyl acetate titer by 3.8 times [Y. lipolytica to positively regulate two β-glucosidases (BGL1 and BGL2), enabling this yeast to use cellobioses as the only carbon source.CRISPR-Cas9 systems can help improve these yeasts as cell factories and increase the availability of bioproducts. Thus, a CRISPR multiple interference system (CRISPRi) was developed to redirect carbon flow from the central pathways in .8 times . Applyin.8 times  used theCRISPR-Cas9 systems have been applied to overcome the challenges associated with previous methodologies. Still, the most significant issue required to improve the utility of CRISPR-Cas9 systems further is off-target effects. Potential off-target cleavage activity can bring unwanted sequence changes in the genome, resulting in genomic instability . To reduSystems biology has become the key tool for efforts in bioprocesses, bringing innovation in the understanding and development of biological pathways or processes in order to achieve the objectives of production on an industrial scale ,140. TheBioinformatics has complemented systems biology with new computational and rational strategies, allowing for a comprehensive view of living information from the cellular environment as a result of omics analyses . Unlike As a systems biology tool, genome scale metabolic models (GEMs) are characterized by connecting the omics sciences  making possible metabolic and phenotypic predictions and representing valuable specific microorganism databases. In relation to the identification of targets (up and down regulation) of gene amplification, algorithms based on analysis of flow response have been used for improving production. For example, OptReg and Optforce identify strategies combined in a comparative way between deletions and super-down expressions, for the optimization of hosts as MILP (Mixed-Integer Linear Programming) solutions ,149,150.S. cerevisiae and non-conventional yeasts has been the main approach adopted in metabolic engineering for fermentation of sugar in ethanol in relation to second generation ethanol. The goal is to create or improve ways to use five-carbon sugars and ethanol synthesis in the same organism [S. cerevisiae is an ideal host used in industrial fermentations, this yeast has no metabolic capacity for xylose or arabinose. S. cerevisiae has a putative xylose metabolic pathway; however, its level of expression is generally very low for using xylose as the sole carbon source. Therefore, this yeast needs further studies for effective genetic engineering of these pathways for the use of xylose and arabinose [The genetic modification of organism . Althougrabinose .S. cerevisiae strains to ferment with xylose has focused on the xylose reductase (XR) pathway (dependent on a cofactor) or xylose isomerase (XI) pathway (independent of cofactor). The XR route offers an advantage in terms of productivity of ethanol as a product, whereas the XI route in terms of product yield [The engineering of ct yield . Controlct yield .The optimization of roads can have several unexpected consequences, unbalancing the cellular system. These consequences generally lead to the accumulation of often toxic intermediates, formation of secondary metabolites, alteration in the metabolic load and, still, low yield of the product. In the last few years, new tools for optimization of pathways have become more economically viable in relation to commercial synthesis of DNA and genome editing systems in a multiplexed way. These technologies have allowed the use of libraries in which road elements are simultaneously diversified, facilitating combinatorial optimization of roads .Synthetic biology has made it possible to control several cellular processes in order to obtain biofuels, the demand for which has increased in recent years. However, the metabolic engineering of cell factories still faces many limitations. The libraries of promoters and terminators, for example, still need to achieve greater amounts of sequence and functional diversity. Computational tools have driven the development of new hosts, facilitating the experimental design of new metabolic pathways and the understanding of genomic data.New strategies and tools are continually progressing for engineering non-microbes, so that they can be optimized for real industrial applications. Such natural microbial genetic obstacles have been overcome with tools such as CRISPR/Cas systems and DNA assembly, improved computer models, automation and design by computer systems that quickly build and characterize the cellular genome. The vast availability of generated biological data has more rapidly developed data science and artificial intelligence strategies to suggest better strategies for genetic and metabolic engineering of cellular systems ,159.S. cerevisiae genome, in comparison to other yeasts, make this yeast the model organism even more used in research for biofuels. However, the development of new techniques of systems biology and synthetic biology to generate high cellular performance, has also made possible the genetic engineering of non-conventional yeasts, whose phenotypes of cellular resistance are of extreme interest in the fuel industry.The simplicity in terms of regulation and the availability of excellent databases of the


Clostridium ljungdahlii  is natively endowed producing acetic acid, 2,3-butandiol, and ethanol consuming gas mixtures of CO2, CO, and H2 (syngas). Here, we present the syngas-based isobutanol formation using C. ljungdahlii harboring the recombinant amplification of the “Ehrlich” pathway that converts intracellular KIV to isobutanol. Autotrophic isobutanol production was studied analyzing two different strains in 3-L gassed and stirred bioreactors. Physiological characterization was thoroughly applied together with metabolic profiling and flux balance analysis. Thereof, KIV and pyruvate supply were identified as key “bottlenecking” precursors limiting preliminary isobutanol formation in CLJU[KAIA] to 0.02 g L–1. Additional blocking of valine synthesis in CLJU[KAIA]:ilvE increased isobutanol production by factor 6.5 finally reaching 0.13 g L–1. Future metabolic engineering should focus on debottlenecking NADPH availability, whereas NADH supply is already equilibrated in the current generation of strains. The WLP is energy-limited as only one ATP may be generated by the conversion of acetyl-CoA to acetate. This, in turn, is needed to reduce CO2 in the methyl branch, leaving no net ATP formation via substrate-level phosphorylation. Hence, a proton gradient coupled to an H+-translocating ATPase is decisive for the energy provision in C. ljungdahlii. In this context, the membrane-bound ferredoxin:NAD oxidoreductase (Rnf complex) plays a crucial role as it couples the electron transfer from reduced ferredoxin (Fdred) to NAD+ to a simultaneous translocation of protons through the cell membrane (via carbon monoxide dehydrogenase (CODH) or H2 using a bifurcating hydrogenase (Hyd) reaction . Thereof, promising metabolic engineering targets were derived finally yielding CLJU[KAIA]:ilvE which achieved 130 mg of isobutanol/L.Isobutanol is an important commodity in the chemical, food, and pharmaceutical industries with rising global market size . Furthereedstock . Severaleedstock . Thereof lactate , 2011. Iarticles . Figure membrane . The reqreaction . An electh Fdred . Consequd so far . Furtherineering . Weitz eA complete description of all methods below can be found in the appendix.Clostridium ljungdahlii DSM 13528 . Details of strain construction are described elsewhere SM 13528  was obta–1. The gas composition was 55% CO, 30% H2, 5% CO2, and 10% Ar. To set anaerobic conditions, the medium-containing bioreactor was sparged with nitrogen with a gassing rate of 60 L h–1 applied for 2 h. Off-gas measurements guaranteed that oxygen concentrations were always below 0.01% (vv–1). Afterward, the medium was equilibrated with the substrate gas for 5 h. Two hours prior to inoculation of the bioreactor, sterile reducing agent was added  with an operational volume of 1.5 L. The detailed reactor equipment was previously described in as added . To obseAll fermentations showed very similar growth and substrate uptake kinetics. Only product formation differed with respect to isobutanol production.via a UV/Visible spectrophotometer at 600 nm. A detailed description is found in Cell density was determined offline + column. Measuring parameters and sample preparation are described in The extracellular formation of ethanol, acetate, 2,3-butanediol, lactate, and isobutanol was observed using an isocratic high-performance liquid chromatography (HPLC) equipped with a RI detector and a Rezex ROA-Organic Acid HCDW–1] were quantified using an HPLC system coupled to a triple quadrupole tandem mass spectrometer (QQQ-MS/MS) equipped with an electrospray ion source. Therefore, 5 mL cell suspension each were taken periodically as triplicates in the course of the exponential growth phases of the batch cultures. Extraction and quantification of non-derivatized polar metabolites was described earlier (–1) formic acid stock solution and 18 μL of acetonitrile. Gxy was considered to monitor instrumental fluctuations and the standard mix was needed for the absolute quantification of the respective α-keto acids. Based on previous measurements, the composition of the standard mix was set to 12 μM pyruvate, 1.6 μM OAA, 2 μM KIV, and 6 μM aKG. By means of different water to standard mix ratios during sample preparation internal calibration curves with four levels for each metabolite were achieved. Thus, internal calibration curves resulted from a standard quadruple addition of defined amounts of the respective metabolite standards directly to the sample.Intracellular metabolites’ concentrations in  in the first (approximately 20 – 80 h) and μexp = 0.01 ± 0.001 h–1 in the following period (approximately 90 – 120 h). After approximately 140 h, the final CDW = 0.85 ± 0.06 g L–1 was reached representing 2.3 ± 0.6% of totally captured carbon. Furthermore, the two growth periods were characterized by different substrate uptake patterns. During the first phase, CO uptake accompanied by proportional CO2 formation occurred. Maximum volumetric and biomass specific rates were rCO = 16.3 ± 4.1 mmol (L∗h)–1 i.e., qCO = 38.8 ± 4.1 mmol (gCDW∗h)–1 and rCO2 = 11.3 ± 1.4 mmol (L∗h)–1 i.e., qCO2 = 27.4 ± 3.4 mmol (gCDW∗h)–1, respectively. Subsequently, both rates decreased to rCO = 12.8 ± 2.7 mmol (L∗h)–1 i.e., qCO = 16.1 ± 3.4 mmol (gCDW∗h)–1 and rCO2 = 8.7 mmol ± 1.0 (L∗h)–1 i.e., qCO2 = 11.00 mmol ± 1.3 (gCDW∗h)–1. Only very low H2 uptake even revealing large deviations between the two biological replicates was observed. On average, rH2 = 0.2 ± 0.03 mmol (L∗h)–1 and rH2 = −0.01 ± 0.4 mmol (L∗h)–1 were measured for the first and second period, respectively. Acetate patterns showed similar trends in biological duplicates with acetate formation during early first phase, reaching a maximum until the beginning of the second period when consumption started. The residual of 0.38 ± 0.08 g L–1 represented only 0.9 ± 0.02% of totally captured carbon. On the contrary, the formation of the reduced products ethanol, 2.3-butanediol, and lactate started after the initiation of acetate formation showing a steady rise with final concentrations of 5.9 ± 0.9 g L–1 for ethanol, 3.5 ± 0.6 g L–1 for 2.3-butanediol, and 0.01 ± 0.03 g L–1 for lactate. Accordingly, C ljungdahlii converted approximately 30% of consumed carbon into reduced products. The total free Gibbs reaction energy ΔGR of the process was – 33.55 ± 1.92 kJ C-mole–1.Before investigating a recombinant isobutanol formation based on syngas, a reference process (REF) was used to analyze growth, product formation, and substrate uptake of the LJU[WT]) . TherefoCDW–1 for KIV, oxaloacetate (OAA), and 2-ketoglutarate (αKG) to 8 μmol gCDW–1 for alanine. Whereas most pool sizes remained constant throughout the process, pyruvate and serine depleted during the second growth phase. In contrast, remarkably large pool sizes were found for valine, glutamate, aspartate, alanine, and lysine.To further characterize the physiological state of the cells 32 intracellular metabolite pools were analyzed  at repreCDW–1 during the second period. A similar trend was found for AEC starting at 0.46 and ending at 0.28. On contrary, AMP and ADP rather remained at about 0.25 μmolgCDW–1.Intracellular patterns of ATP, ADP, AMP, and the respective adenylate energy charge (AEC) were studied to evaluate whether or not non-wanted energy shortage might exist growing on syngas . ATP poovia recombinant Ehrlich pathway –1 i.e., qCO = 34.6 mmol (gCDW∗h)–1 and rCO2 = 13.8 mmol (L∗h)–1 i.e., qCO2 = 27.0 mmol (gCDW∗h)–1. During growth period II, biomass specific rates for CO uptake qCO = 25.2 mmol (gCDW∗h)–1 and CO2 formation qCO2 = 18.9 mmol (gCDW∗h)–1 decreased. Low H2 uptake was observed in each phase with rH2,I = 0.2 mmol (L∗h)–1 followed by rH2,II = 0.4 mmol (L∗h)–1. The product spectrum was similar to the REF with the final concentrations of 0.22 g L–1 of acetate, 5.25 g L–1 of ethanol, 2.38 g L–1 of 2.3-butanediol, 0.25 g L–1 of lactate, and 0.02 g L–1 of isobutanol. The total free Gibbs reaction energy ΔGR of the process was – 36.15 kJ C-mole–1  and μexp,II = 0.011 h–1 (90 – 140 h) reaching CDW = 0.89 g L–1 which represents 2.7% of CO captured in biomass. Despite a suspected valine auxotrophy of CLJU[KAIA]:ilvE there was no reduction of growth compared to the wildtype. This indicates that the valine concentration provided by the 0.5 g L−1 yeast extract in the medium is sufficient. During phase I, maximum volumetric and specific rates were rCO = 16.2 mmol (L∗h)–1 i.e., qCO = 33.8 mmol (gCDW∗h)–1 and rCO2 = 11.9 mmol (L∗h)–1 i.e., qCO2 = 24.9 mmol (gCDW∗h)–1. The mean volumetric H2 uptake rate was 0.2 mmol (L∗h)–1. In phase II, biomass specific CO uptake and CO2 formation rates decreased to qCO = 23.5 mmol (gCDW∗h)–1 and qCO2 = 16.1 mmol (gCDW∗h)–1. Volumetric H2 uptake rate slightly increased to 0.3 mmol (L∗h)–1. At the end of the process, 0.83 g L–1 acetate, 5.90 g L–1 ethanol, 3.42 g L–1 2.3-butanediol, 0.09 g L–1 lactate, and 0.13 g L–1 isobutanol were determined. The total free Gibbs reaction energy ΔGR of the process was – 37.89 kJ C-mole–1  and of the formate-hydrogen reaction rate enable this phenotype. Nevertheless, strongly decreasing NADPH yields of 65 – 70% were determined in the second growth phase for each cultivation. On the contrary, NADH availabilities almost remained constant during phases I and II, only showing a slight 20% decrease for CLJU[KAIA]:ilvE.For determination of NADH and NADPH availabilities in the strains, FBA were performed. Intracellular flux patterns of the “pseudo-steady states” in phases I and II were studied using the stoichiometric metabolic model “modified rSMM” , that waectively . The oveof Fdred . Utilizirogenase . Our simanogenum . ApparenC. ljungdahlii shows a biphasic growth behavior, that is characterized by growth reduction with simultaneously increased formation of reduced products. This coincidence was also observed in a two-stage process cultivating C. ljungdahlii on syngas –1 and CO2 production rates of 13 – 40 mmol (gCDW∗h)–1 during a batch-cultivation of C. ljungdahlii with continuous gas supply. These values fit fairly well to our study. In contrast to our process, however, they identified a simultaneous and equivalent uptake of CO and H2. The simultaneous utilization of these gases is also shown in further studies describing continuous cultivations performed in chemostat mode installing 0.04 h–1 dilution rate . H2 uptake and the product spectrum depend on the H2/CO ratio (2 consumption (2/CO affects the product portfolio strongly.Metabolizing syngas during a batch cultivation n syngas . Previouh medium . In thise-system . Furtherion rate , 2018. OCO ratio  which waCO ratio , but CO CO ratio . Moreovesumption . Hence, C. ljungdahlii’s capacity converting L-serine to pyruvate via L-serine dehydratase to refill the pyruvate pool (Intracellular metabolites pattern during the process reveals a clear depletion of pyruvate accompanied by a strong accumulation of alanine in the second growth phase. Pyruvate serves as key precursor for 2,3-butanediol and lactate, which are mainly formed in the second phase of the process. In this regard, pyruvate depletion may limit the formation of 2.3-butanediol and isobutanol. Notably, the decreasing L-serine pool might hint to ate pool .C. ljungdahlii is driven by its energy and redox management (C. ljungdahlii needs to produce more ethanol and 2.3-butanediol during the phase of retarded growth (C. ljungdahlii is not regulated at the proteome level but rather by thermodynamics. Therefore, we hypothesize that the intracellular accumulation of alanine, valine, and glutamate enables C. ljungdahlii to react flexibly on increasing demands for pyruvate, the “doorman” metabolite for getting rid of “surplus” electrons. This hypothesis is supported by the intracellular AEC and AXP patterns. Despite a reduction of AEC and the ATP concentration in the first growth phase, both values remained constant in the following period.Alanine may be synthesized by decarboxylation of aspartate and by transamination of valine, glutamate, and pyruvate . Accordinagement . To cound growth . In addiC. ljungdahlii to keep its ATP pool constant during the second period. This may be advantageous for the recombinant production of isobutanol. CDW–1 ATP, 0.09 μmolgCDW–1 ADP, and 0.95 μmolgCDW–1 AMP in CO-consuming, steadily growing C. autoethanogenum, a close relative of C. ljungdahlii. Those values lead to the AEC of 0.13 which fit to the observations of this study. Interestingly, AXP levels and AEC are much lower than the so-called “physiological” levels that range from 0.80 to 0.95 (The initial decrease of the ATP pools may be associated with anabolic ATP needs considering that acetogens are living at the edge of thermodynamic feasibility. Retarded growth, consumption of acetate, and enhanced formation of reducing products enable  to 0.95 . However to 0.95 .R was even lower than in REF (ilvE should be achievable by replacing the cofactor dependency on NADPH by NADH. Using a NADH-dependent variant of the ketol-acid reductoisomerase C. ljungdahli applying a H2/CO ratio of approximately 0.5 (C. ljungdahlii was not successful. Thus, further approaches need to be performed (2 may also increase the isobutanol formation. To check the hypothesis processes with higher H2/CO ratios and/or higher reactor pressures during the solventogenic phase could be performed.Syngas-based recombinant isobutanol formation by CLJU[KAIA] was successfully achieved, although at a low level, still. Given that the total free Gibbs reaction energy ΔGn in REF , no enern in REF  althoughtely 0.5 . Furthertely 0.5 . Howevererformed . AdditioClostridium ljungdahlii is well equipped to convert syngas mixtures with H2:CO ratios of 0.5 into reduced products. 30% of totally consumed carbon were used to produce mostly ethanol and 2.3-butanediol. Predominately, reducing equivalents originate from CO. However, the additional H2 uptake, even if low, enables C. ljungdahlii to adapt simultaneous CO2 reduction flexibly to NADPH needs. Intracellular pyruvate availability turned out to be a carbon bottleneck of alcohol formation. The implementation of the Ehrlich-pathway partly alleviated the carbon shortage. However, only the additional block of valine formation enabled to harvest the available carbon which resulted in a 6.5-fold increase of isobutanol formation. Analyzing the redox condition in CLJU[KAIA]:ilvE gives rise to the conclusion that a novel metabolic engineering target should be addressed next: The increased supply of NADPH. Targeting this goal should be in the focus of future autotrophic isobutanol formation with engineering C. ljungdahlii.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.ilvE. FB and PD supervised the strain reconstruction and advised the study. RT conceived the study and corrected the manuscript. MH, AT, SW, FB, PD, and RT read and approved the final manuscript. All authors contributed to the article and approved the submitted version.MH designed the study, conducted the bioreactor experiments, reconstructed the stoichiometric model, performed flux balance analyses, analyzed the datasets, drafted the manuscript, and supported the laboratory conversion. AT and MH designed and performed the metabolomics analysis. AF designed and set up the laboratory for the performance of synthesis gas bioreactor studies. AN advised the network reconstruction and flux balance analysis. SW constructed the recombinant strains CLJU[KAIA] and CLJU[KAIA]:The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Ogataea polymorpha is a thermotolerant, methylotrophic yeast with significant industrial applications. While previously mainly used for protein synthesis, it also holds promise for producing platform chemicals. O. polymorpha has the distinct advantage of using methanol as a substrate, which could be potentially derived from carbon capture and utilization streams. Full development of the organism into a production strain and estimation of the metabolic capabilities require additional strain design, guided by metabolic modeling with a genome-scale metabolic model. However, to date, no genome-scale metabolic model is available for O. polymorpha.Komagataella phaffii as a reference and corrected reactions based on KEGG and MGOB annotation. Additionally, we conducted phenotype microarray experiments to test the suitability of 190 substrates as carbon sources. Over three-quarter of the substrate use was correctly reproduced by the model and 27 new substrates were added, that were not present in the K. phaffii reference model.To overcome this limitation, we used a published reconstruction of the closely related yeast O. polymorpha will support the engineering of synthetic metabolic capabilities and enable the optimization of production processes, thereby supporting a sustainable future methanol economy.The developed genome-scale metabolic model of The online version contains supplementary material available at (10.1186/s12896-021-00675-w). Ogataea polymorpha  is a widely used yeast for biotechnological applications. It is environmentally ubiquitous and has been isolated, among others, from orange juice, maize meal, and insect guts [O. polymorpha has some unique features among methylotrophic yeasts such as high growth rate, temperature tolerance, and nitrate fixation [ect guts . The abiect guts . O. polyfixation  and has fixation . A partifixation , 5. Furtfixation .Komagataella phaffii, O. polymorpha, and Candida arabinofermentans [Ogataea strains most frequently mentioned in the literature are DL-1 (ATCC 26012), CBS4732  and NCYC 495  [O. polymorpha DL-1 and K. phaffii showed that they share three-quarters of enzymes whereas the non-overlapping proteome contains mostly hypothetical, uncharacterized proteins, indicating their close relationship and overlapping metabolic features [Methylotrophy in yeast is restricted to a single multigenus clade, with its most prominent members being rmentans . The phyrmentans . The Oga Y-1798) . The gen Y-1798) . A genomfeatures .K. phaffii is one of the few yeasts that have the ability to use methanol as the only carbon source for energy production. K. phaffii was developed into an effective producer of recombinant proteins based on the strength of the native methanol-responsive promoter expression system. The wealth of knowledge has lead to the development of metabolic models on the genome level (GSMM) for simulation and strain engineering [O. polymorpha is desirable.The methylotrophic model organism ineering , 12. Theineering , 14. BasO. polymorpha and K. phaffii share many similarities especially regarding their methanol metabolism. However, several metabolic features are distinctive. O. polymorpha is capable of growth at 50∘C and is therefore one of the most thermotolerant eukaryotic microorganisms [K. phaffii, O. polymorpha can ferment xylose to ethanol [O. polymorpha. In K. phaffii, activity of AOX strictly depends on the presence of methanol [rganisms . In cont ethanol  and can  ethanol . Moreovemethanol .O. polymorpha NCYC 495. The model is based on existing models for K. phaffii, extended by species-specific substrate utilization identified in phenotype microarrays. Model predictions of growth rates were found to represent experimental growth in different conditions. We tested the performance for overproduction of lactate, and succinate with methanol and glucose as substrates for biotechnological applications and identified potential targets for amplification of reaction activities.Here, we present iUL909 as the first GSMM of the biotechnological relevant methylotrophic yeast O. polymorpha named iUL909. iUL909 was generated on the basis of an existing metabolic model of K. phaffii iMT1026v3 [K. phaffii in iMT1026v3 were replaced by those of homologs from O. polymorpha. The model was validated against physiological data from substrate utilization tests with phenotype microarrays and shake flask experiments, as well as growth rates from the literature. We further simulated the production of industrially relevant molecules to identify future biotechnological applications. The validity of the SBML model was tested with Memote [Here, we report on the construction of a genome-scale metabolic model of the methylotrophic yeast MT1026v3 , 14. Genh Memote .O. polymorpha and K. phaffii [K. phaffii to O. polymorpha using a homolog-search  as well as the biomass reaction [O. polymorpha  was still satisfied for 62 genes, but 22 gene products were sole catalysts for the associated reactions  in iMT1026 for which neither KEGG nor the model itself would allow metabolite flux. However, thirteen reactions were retained because they were metabolically well connected, and their deletion would result in reaction gaps. For example, the identities and the location of all non-proton pumping mitochondrial NADH dehydrogenase system is currently not sufficiently well known. We decided to retain dehydrogenase reactions from the reference model iMT1026. More experiments and data will guide improvements regarding the actual electron and proton homeostasis.Notwithstanding the high degree of similarity between K. phaffii iMT1026 with the actual growth phenotype of O. polymorpha. Biolog’s Phenotype Microarray™ plates test was used to analyze the metabolic utilization of 190 carbon substrates . In eight cases no growth was experimentally measured, whereas it was predicted by the simulation. Substrates of this class comprised mainly organic acids associated with the TCA cycle  would be present. We corrected the equation of over 30 reactions, for example fatty acid synthesis and uridine kinase reactions were corrected to maintain proton balance. There are still 59 unbalanced reactions due to multiple allowed charges in BIGG database and complex interrelations. Overall, the mass and charge balance is correct for more than 96% of all reactions as testified by the Memote report (see GitHub repository) and allows for faithful simulations.New metabolic features were added and existing reactions corrected. All genes in the reconstruction iUL909 were checked in the methylotroph gene order browser (MGOB)  and KEGGO. polymorpha [O. polymorpha. These enzymes are essential for the alcoholic fermentation of xylose [The largest connected path we added was connected with the metabolism of erythritol. iUL909 contains four successive reactions that represent the further processing of erythritol. Erythritol is phosphorylated by a kinase to D-erythritol-1-phosphate, which in turn is converted to L-erythrulose-1-phosphate by means of a dehydrogenase and an epimerase. The L-erythrulose-1-phosphate is then split into dihydroxyacetone phosphate and formaldehyde. These cytosolic reactions were necessary to enable the observed growth on the sugar alcohol erythritol. The hydrolysis of many sugars with growth in the phenotype assays is catalyzed in our reconstruction by the maltase enzyme (EC-number 3.2.1.20). Maltase is known to display broad substrate specificity, which was also shown explicitly for lymorpha . Two reaf xylose .Overproduction of important platform chemicals can be achieved with a limited number of genetic manipulations. We chose lactate and succinate as target compounds from methanol and glucose, and applied the FSEOF approach to identify reactions whose increased activity stimulates target production . The optK. phaffii with a score of only 24 %.iUL909 fulfills key standards for reconstructed models and was tested with Memote and the SBML validator , 31. TheO. polymorpha was constructed using homolog identification based on the existing GSMM of K. phaffii. The resulting GSMM iUL909 was further tailored for appropriate substrate utilization on the basis of phenotypic microarray and shake flask experiments. Although biomass composition and energetic parameters were adopted from K. phaffii, the resulting growth predictions were in good agreement with chemostat experiments for various industrially relevant substrates. The biotechnological applicability was explored by testing overproduction of lactate, succinate, showing that high productivity can be achieved with limited genetic manipulation. Thus, a metabolic model is now available for further strain engineering.A genome-scale metabolic model of ∘C and 150 rpm, growth and respiratory activity was monitored by measuring the optical density (OD) at 490 nm for dye reduction and 750 nm for biomass in a plate reader  at 0 h and 72 h.The substrate utilization tests were conducted with the Biolog’s Phenotype Microarray™ plates P1 and P2 with a total of 190 carbon sources. The medium was prepared with the manufacturer’s inoculation medium IFY-0 and the dye mix H according to the guideline. The organism was cultivated in the microarrays in the Growth Profiler  at 37To discriminate growth from non-growth, we considered the distribution of the OD increase for each plate at 490 nm and derived a suitable cut-off. The majority of the OD increases clustered in a normal distribution at the lower OD-end, followed by a long tail with larger OD increases. The tail of the distribution characterizes explicit substrate respiration, while the normal distribution contains metabolized as well as non-utilized substrates. To identify metabolized substrates within the normal distribution, we first separated the normal distribution from the tail with the clear positive substrates. This was achieved by removing all values above the arithmetic mean within a plate. Then, a normal distribution was fitted over the remaining measurements, and one standard deviation above the mean was used as the cut-off for metabolized substrates  relationships was conducted by identification of homologs among K. phaffii and O. polymorpha. We used the genome sequence of K. phaffii GS115, the foundation of iMT1026, extracted from UniProt. The homologs were identified using the software package ProteinOrtho using the default parameter settings for homolog detection [K. phaffii input protein sequence. K. phaffii GPRs were replaced by the O. polymorpha homolog, while maintaining isoenzyme and multi-protein complex GPRs, which was possible for 908 GPRs. In the case of failed homolog mappings, manual Blast searches were conducted, and for the reactions of the central carbon metabolism manual comparison of gene annotations were performed with the JGI linked KEGG database of O. polymorpha [The based on  for strabased on . The K.  iMT1026  was usedetection . Proteinlymorpha  and the lymorpha .K. phaffii MGOB-IDs of reactions in the central carbon metabolism were derived by querying the MGOB database with KEGG gene IDs. The MGOB-IDs were then used to locate the genes in their genomic context. The alignment with the homologs of all methylotrophic yeasts within MGOB provided information on annotation including subcellular localization. In this way, we could characterize 518 additional genes compared to UniProt. The model completeness and quality was tested with Memote [http://sbml.org/Facilities/Validator).The MGOB shows the local genomic organization among methylotrophic yeasts. h Memote . SBML Lehttps://github.com/iAMB-RWTH-Aachen/Opol-GSMM). Flux scanning based on enforced objective flux (FSEOF) [Simulations of the GSMM were performed with COBRAv3 on Matlab and with COBRApy. A Jupyter Notebook guide for the simulation of iUL909 and reproduction of experimental data as in Fig.  (FSEOF)  was perfAdditional file 1 Supplementary information.Additional file 2 Experimental data. Excel sheet with comparison of substrate utilization tests conducted in this paper and Suh et al. [™ plates test, namely (i) a list of substrates with abiotic activity, (ii) for plate PM1: OD490/600 and selection of metabolized substrates, (iii) for plate PM1: OD490/600 and selection of metabolized substrates, (iv) shake flask growth results for the eight substrates not grown in phenotype assay .h et al. . DetaileAdditional file 3 Genome scale metabolic model details. It contains a list of reactions with only one associated gene-protein-reaction association for which no homolog in O. polymorpha was identified. A list of all genes in iMT1026 for which no homologs were identified in O. polymorpha. Lists of new reactions and new genes added to the model. A table of literature values for growth rates and substrate uptake rates and finally the results of the FSEOF analysis.


Escherichia coli native enzymes. We identify the GED (Gnd–Entner–Doudoroff) cycle as the simplest pathway that can operate with high thermodynamic driving force. This autocatalytic route is based on reductive carboxylation of ribulose 5-phosphate (Ru5P) by 6-phosphogluconate dehydrogenase (Gnd), followed by reactions of the Entner–Doudoroff pathway, gluconeogenesis, and the pentose phosphate pathway. We demonstrate the in vivo feasibility of this new-to-nature pathway by constructing E. coli gene deletion strains whose growth on pentose sugars depends on the GED shunt, a linear variant of the GED cycle which does not require the regeneration of Ru5P. Several metabolic adaptations, most importantly the increased production of NADPH, assist in establishing sufficiently high flux to sustain this growth. Our study exemplifies a trajectory for the emergence of carbon fixation in a heterotrophic organism and demonstrates a synthetic pathway of biotechnological interest.Carbon fixation is one of the most important biochemical processes. Most natural carbon fixation pathways are thought to have emerged from enzymes that originally performed other metabolic tasks. Can we recreate the emergence of a carbon fixation pathway in a heterotrophic host by recruiting only endogenous enzymes? In this study, we address this question by systematically analyzing possible carbon fixation pathways composed only of  2-assimilation pathway in E. coli based only on endogenous enzymes and a reversible decarboxylase.Current efforts to establish synthetic carbon fixation in model heterotrophs rely on expression of heterologous enzymes. Here, the authors explore the presence and activity of a latent CO Six other carbon fixation pathways are known to operate in various bacterial and archaeal lineages4 and also contribute to primary production1. Recent studies have made considerable progress in establishing carbon fixation pathways in heterotrophic organisms, with the long-term goal of achieving synthetic autotrophy, which could pave the way towards sustainable bioproduction schemes rooted in CO2 and renewable energy6. Most notably, overexpression of phosphoribulokinase and Rubisco, followed by long-term evolution, enabled the industrial hosts Escherichia coli8 and Pichia pastoris9 to synthesize all biomass from CO2 via the RuBP cycle. Also, overexpression of enzymes of the 3-hydroxypropionate bicycle established the activity of different modules of this carbon fixation pathway in E. coli10. While such studies help us to gain a deeper understanding of the physiological changes required to adapt a heterotrophic organism to autotrophic growth, they do not, however, shed light on the origin of the carbon fixation pathways themselves.The ability to assimilate inorganic carbon into biomass sets a clear distinction between autotrophic primary producers and the heterotrophs depending on them for the supply of organic carbon. Most primary production occurs via the ribulose bisphosphate (RuBP) cycle—better known as the Calvin–Benson cycle—used in bacteria, algae, and plants11—the other carbon fixation routes are thought to have evolved by recruiting enzymes from other metabolic pathways. For example, Rubisco—the carboxylating enzyme of the RuBP cycle—probably evolved from a non-CO2-fixing ancestral enzyme, thus emerging in a non-autotrophic context12. Similarly, acetyl-CoA carboxylase likely originated as a key component of fatty acid biosynthesis before being recruited into carbon fixation pathways in several prokaryotic lineages3. The limited number of natural carbon fixation pathways indicates that the recruitment of endogenous enzymes to support carbon fixation is a rather exceptional event. To understand this process better we aimed to recreate it in a heterotrophic bacterium.Besides the reductive acetyl-CoA pathway and the reductive TCA cycle—both of which are believed to have originated early in the evolution of metabolismE. coli enzymes. We identify a promising candidate route—the GED cycle—that is expected to enable carbon fixation with minimal reactions and with a high thermodynamic driving force. This synthetic route combines reductive carboxylation of ribulose 5-phosphate (Ru5P) with the Entner–Doudoroff (ED) pathway, gluconeogenesis, and the pentose phosphate pathway. We demonstrate that overexpression of key pathway enzymes together with small modifications of the endogenous metabolic network enable growth via the GED shunt—a linear route that requires the key reactions of the GED cycle, including the carboxylation step, for the biosynthesis of  all biomass building blocks. Our findings indicate the feasibility of recruiting endogenous enzymes to establish a non-native carbon fixation pathway and pave the way for future establishment of synthetic autotrophy based on new-to-nature pathways.Here, we use a computational approach to comprehensively search for all thermodynamically feasible carbon fixation pathways that rely solely on endogenous E. coli enzymes13, we used the genome-scale metabolic model of this bacterium14. We assumed that all reactions are reversible and then used an algorithm to systematically uncover all possible combinations of enzymes, the net reaction of which use CO2 and cofactors H) as sole substrates to produce pyruvate, a reference product commonly used to compare carbon fixation pathways5 (see “Methods” section and Supplementary Method 15. We assumed an elevated CO2 concentration of 20% (200 mbar), which is easily attainable in microbial cultivation within an industrial context and further characterizes the natural habitats of E. coli, e.g., the mammalian gut17. The MDF criterion enabled us to discard thermodynamically infeasible routes (having MDF <0) and to compare the feasible pathways according to their energetic driving force, which directly affects their kinetics15.To identify possible carbon fixation pathways that can be established using only native E. coli enzymes (see Supplementary Table 15). This cycle is based on the reductive carboxylation of ribulose 5-phosphate (Ru5P) by 6-phosphogluconate (6PG) dehydrogenase (Gnd). The carboxylation product, 6PG, is then metabolized by the enzymes of the Entner–Doudoroff (ED) pathway—6PG dehydratase (Edd) and 2-keto-3-deoxygluconate 6-phosphate aldolase (Eda)—to produce glyceraldehyde-3-phosphate (GAP) and pyruvate .The GED cycle mirrors the structure of the canonical RuBP cycle, where phosphoribulokinase and Rubisco are replaced with Gnd, Edd, Eda, and gluconeogenic enzymes Fig. . Similar2: ΔrG′m ≈ −1.5 kJ/mol in the oxidative decarboxylation direction . Indeed, similar oxidative decarboxylation enzymes are known to support reductive carboxylation, for example, the malic enzyme28 and isocitrate dehydrogenase30. While sporadic studies have reported that some Gnd variants support the reductive carboxylation of Ru5P in vitro35, a comprehensive kinetic characterization of this activity in bacterial Gnd variants is lacking. More importantly, it remains unclear whether this reaction could operate under physiological conditions, where the concentrations of substrates and products are constrained; that is, substrate concentrations are not necessarily saturating and product concentrations are non-negligible.All enzymes of the GED cycle are known to carry flux in the direction required for pathway activity, with the exception of Gnd. The Gibbs energy of the Gnd reaction indicates that it should be fully reversible under elevated COE. coli Gnd. We found Gnd to have a rather high kcat in the reductive carboxylation direction, approaching 6 s−1 . Notably, these kinetic parameters are substantially better than those previously reported for a eukaryotic Gnd variant (kcat ~ 1 s−1 and KM(CO2) ≥ 15 mM32).First, we measured the kinetics of To check how prevalent the potential of carbon fixation via the GED cycle is, we performed a phylogenetic analysis to identify bacteria that harbor its key enzymes (see “Methods” section). We found that the enzymes of the GED cycle are ubiquitous in alpha-proteobacteria (962 species), gamma-proteobacteria (1684), and actinobacteria (312) Fig. . The speE. coli for autotrophic growth via the GED cycle would be a challenging task requiring considerable metabolic adaptation of the host; for example, establishing a delicate balance between the metabolic fluxes within the cycle and those diverging out of the cycle (as found in previous efforts to establish the RuBP cycle in E. coli8). Hence, to check the feasibility of the cycle, we focused on establishing growth via the GED shunt, representing a segment of the full cycle which consists of reductive carboxylation by Gnd and the subsequent ED pathway . As we show below, growth via this linear shunt requires the activity of most enzymes of the GED cycle but relies on a pentose substrate rather than the regeneration of Ru5P.Engineering E. coli strain deleted in the gene encoding for ribulose 5-phosphate 3-epimerase (Δrpe). This strain cannot grow on ribose as a sole carbon source, as ribose-5-phosphate (R5P) cannot be converted to xylulose 5-phosphate, thus blocking the pentose phosphate pathway  or of Edd and Eda alone (pED) did not support growth  enabled the growth of the Δ13C-labeling experiment. We cultivated the Δrpe + pGED strain with unlabeled ribose and 13CO2, and measured the labeling pattern of five proteinogenic amino acids—serine, glycine, alanine, valine, and histidine. The results confirm the activity of the GED shunt  pyruvate is generated both from GAP and directly from Eda activity .To confirm that growth indeed proceeds via the GED shunt, we performed a unt Fig. : (i) sinity Fig. , such thThese results confirm that, upon overexpression of Gnd, Edd, and Eda, the GED shunt is sufficiently active to provide the cell with almost all cellular building blocks as well as energy (by complete oxidation of pyruvate via the TCA cycle). As mentioned above, the growth of this strain requires the simultaneous activity of most enzymes on the GED cycle, including those of glycolysis and the pentose phosphate pathway; for example, net production of erythrose 4-phosphate (E4P) from ribose requires the combined activity of Gnd, the ED pathway, and enzymes of the pentose phosphate pathway.tktAB). This strain, in which the non-oxidative pentose phosphate pathway is effectively abolished, cannot grow when provided with a pentose as the sole carbon source41. Furthermore, as E4P cannot be synthesized in this strain, small amounts of essential cellular components, the biosynthesis of which is E4P-dependent, need to be added to the media41: phenylalanine, tyrosine, tryptophan, shikimate, pyridoxine, 4-aminobenzoate, 4-hydroxybenzoate, and 2,3-dihydroxybenzoate .To check whether the operation of the GED shunt is robust, we decided to use another metabolic background to select for its activity. We deleted the genes encoding for both isozymes of transketolase   Fig. . HowevertktAB context, we sought to harness adaptive evolution to provide us with information on further cellular adaptations required for the activity of the synthetic route. Toward this aim, we inoculated the ΔtktAB+pGED strain into multiple test-tubes with xylose and E4P supplements and incubated them for an extended period of time at 37 °C and 20% CO2. After ~2 weeks, the culture in several of these test-tubes showed apparent growth. When the cells from the growing cultures were transferred to a fresh selective medium, we observed immediate growth, indicating that genetic adaptation had occurred. Yet, only one of these cultures showed robust growth on the selective medium, whereas that of the others was less reproducible and highly sensitive to the exact conditions, preculture, and inoculation. An isolated single clone from the robust culture grew, under elevated CO2 concentration, with a doubling time of 5.3 h  with three previously characterized constitutive promoters: weak (W-pntAB), medium (M-pntAB), and strong (S-pntAB), with relative strengths of 1:10:20, respectively48. We found that while the weak promoter failed to support growth : the mobile element IS5ins Fig. . The contktAB + pGED strain. We tested the deletion of the gene encoding for the soluble transhydrogenase (Δsth), as this enzyme is known to provide a strong sink for NADPH45. Yet, the ΔtktAB Δsth+pGED strain did not grow on xylose even at an elevated CO2 concentration , the first enzyme of the oxidative pentose phosphate pathway, has been reported to increase under conditions of high NADPH demand 50. Hence, we wondered whether the deletion of zwf could remove a barrier for reductive carboxylation by Gnd and thus assist the activity of the GED shunt. We found that this is indeed the case, where the ΔtktAB Δzwf+pGED strain was able to grow under elevated CO2 concentrations with a doubling time of 5.4 h  and transketolase (Tkt) are essential for the operation of the full GED cycle, that is, for the regeneration of Ru5P from GAP. To address this problem, we aimed to construct a strain which keeps all necessary enzymes of the GED cycle intact, while still allowing to select for the activity of the GED shunt, i.e., preventing utilization of a pentose substrate as sole carbon source via the canonical pentose phosphate pathway. Such a strain would enable a smooth transition from GED shunt-dependent growth on a pentose substrate towards autotrophic growth via the GED cycle.While the ΔpfkAB ΔfsaAB ΔfruK) or channel it into the oxidative pentose phosphate pathway (Δzwf). The latter gene deletion should also support the activity of the GED shunt, as was shown above within the ΔtktAB context. The strain containing all of these deletions, which we term ΔPZF, establishes a uni-directional block within the pentose phosphate pathway. That is, growth on a pentose substrate is not possible due to the accumulation of F6P that prevents further conversion of pentose phosphates into GAP , directly or indirectly, into a downstream glycolytic intermediate  was able to grow on a xylose minimal medium, but was accompanied by the secretion of a substantial amount of glucose (34% of consumed xylose)51. Such secretion of a dephosphorylated sugar could relieve the inhibitory accumulation of F6P and thus theoretically enable the growth of the ∆PZF strain even without the activity of the GED shunt. However, the growth of the ∆PZF mutants we identified cannot be explained by such a phenomenon since (i) growth at ambient CO2 was not observed . We found that this strain displayed a labeling pattern almost identical to that of the ΔtktAB Δzwf + pGED strain described above, thus confirming growth via the GED shunt  and, in most colonies, avtA, encoding for valine-pyruvate aminotransferase, had mutated. While the exact contribution of these mutations to the growth phenotype remains elusive, the isolated strains provide a promising starting point for the evolution of the full GED cycle.To provide unequivocal confirmation that the xylose assimilation in the ∆PZF strain mutants proceeds via the GED shunt, we conducted E. coli enzymes. A key factor for the success of this analysis was to ignore the rather arbitrary dichotomic classification of reactions as reversible or irreversible as suggested by metabolic models. Instead, we first identified potential pathways based on pure stoichiometric analysis and then calculated the thermodynamic feasibility and driving force of each of the candidate routes. This enabled us to uncover potential carbon fixation pathways that were not identified before13. Indeed, the GED cycle itself was previously ignored as Gnd was considered to be an irreversible decarboxylating enzyme. As we have shown here, however, Gnd can catalyze the carboxylation reaction quite efficiently, with a kcat almost double that of a typical plant Rubisco36. This finding is similar to a recent study that found that citrate synthase—which is usually thought to be irreversible—can catalyze citrate cleavage, thus enabling carbon fixation via a unique variant of the reductive TCA cycle53. These examples indicate that we should revise our dogmatic interpretation of enzyme reversibility and instead adopt a more quantitative approach to understand reaction directionality.Our computational analysis identified multiple carbon fixation pathways that are based solely on endogenous 55. The most advanced of these pathways is the CETCH cycle55 that combines segments of the 3-hydroxypropionate/4-hydroxybutyrate cycle56 and the ethylmalonyl-CoA pathway57. The CETCH cycle was assembled in vitro using enzymes from nine organisms and optimized in several rounds of enzyme engineering58. However, the in vivo implementation of this synthetic pathway, as well as of other previously suggested routes, is highly challenging due to its complexity and requirement for the considerable rewiring of central metabolic fluxes. The GED cycle provides a favorable alternative to these routes, as the fluxes it requires mostly correspond to native gluconeogenesis and the pentose phosphate pathway. Hence, the establishment of carbon fixation via the GED cycle might be less demanding and more likely to succeed.Previous studies have suggested multiple synthetic carbon fixation pathways that could surpass the natural routes in terms of resource use efficiency, thermodynamics, and/or kineticsrpe strain but not of a ∆tktAB strain or a ∆PZF strain, even though in all of them the pentose phosphate pathway is disrupted. Despite this, a short-term adaptation was able to restore the growth of the latter two strains. Within the ∆tktAB strain, we demonstrated that either an increase in the supply of the substrate  or a decrease in the availability of the product  is sufficient to enable Gnd-dependent carboxylation and growth via the GED shunt.We demonstrated the feasibility of carbon fixation via the GED cycle by establishing growth via the GED shunt—a linear pathway variant that requires the key pathway reactions to provide  all biomass building blocks and cellular energy without regenerating the substrate Ru5P Figs. a and 3a.2 can increase production yields from common feedstocks such as sugars61. This is attributed to the fact that the biosynthesis of certain compounds from sugars results in the production of excess reducing power, which can be utilized to fix CO2 and thereby generate more product. Such assimilation of CO2 can also serve to compensate for the carbon released during the oxidation of pyruvate to acetyl-CoA, thus addressing a common challenge in the production of value-added chemicals derived from acetyl-CoA64. Indeed, we applied flux balance analysis to simulate production in non-growing cells (see “Methods” section) and found that rerouting the utilization of sugar substrates via the GED shunt is expected to increase the yield of various commercially interesting products, such as acetate, pyruvate, acetone, citrate, and itaconate  could compensate for the low affinity towards CO2 . As some variants of similar reductive carboxylation enzymes—isocitrate dehydrogenase and the malic enzyme—incorporate CO2 with kcat surpassing 100 s−173, identifying a Gnd variant supporting such a high carboxylation rate might be feasible. Engineering such an optimized GED cycle into crop plants could boost agricultural productivity, thus addressing one of our key societal challenges67.The GED cycle could be used to replace the RuBP cycle in plants, algae, and bacteriaE. coli endogenous enzymes, we used an approach similar to the one we have previously developed54. In this previous study, all reactions found in the KEGG database74 (https://www.kegg.jp/), denoted the universal stoichiometric matrix, were used to design potential CO2 fixating pathways using a Mixed-Integer Linear Programming (MILP) approach. Here, we focused only on enzymes present in the most recent genome-scale metabolic reconstruction of E. coli: iML151514. We further added thermodynamic constraints in order to exclude infeasible pathways and to rank the feasible ones based on their MDF15.In order to find all possible carbon fixation cycles using 75. First, we removed all exchange and transport reactions and kept only the strictly cytoplasmic ones. Then, we added reactions that allow the free flow of electrons and energy  as well as inorganic compounds . Finally, we defined an optimization problem, where the set of reactions in each pathway should overall convert 3 moles of CO2 to one mole of pyruvate. The objective function of this optimization is a combination of the MDF15 and the minimum number of reactions in each pathway. The standard approach for multi-objective optimization is the maximization of a linear combination of the two functions. Here, we maximized the MDF (in units of RT) minus the number of reactions. We note that changing the relative weight between these two objectives did not change our main result, namely that the GED pathway is Pareto-optimal. A more detailed list of changes to the model and the formal description of the optimization problem can be found in Supplementary Method We used the COBRApy package (version 0.17) to identify carbon fixation pathwaysWe used the framework described above to iterate the space of possible solutions, i.e., all pathways that have a positive MDF. This requirement ensures that the pathway is thermodynamically feasible given our constraints, i.e., all of its reactions can operate simultaneously without violating the second law of thermodynamics. Covering this space exhaustively would require many days of computer time and is not useful since most of these solutions are unnecessarily complicated. The Pareto-optimal solutions are those that are not dominated by any other pathway in both objectives simultaneously . We found only two such pathways: the GED cycle and a pathway that uses the reverse glycine cleavage system as its carboxylating mechanism 76. AnnoTree generates a phylogenetic tree and highlights genomes that include all of the KEGG orthologies74 selected in the query. Since most annotations are homology based, and therefore not always precise, we restricted our search to the enzymes that are most indicative of the GED pathway and cannot be easily replaced by a metabolic bypass :K00033—6-phosphogluconate dehydrogenase (EC:1.1.1.44 1.1.1.343): the enzymatic keystone of the GED cycle.K01690—6-phosphogluconate dehydratase [EC:4.2.1.12]: indicative of the Entner–Doudoroff pathway.K00615—transketolase [EC:2.2.1.1]: indicative of the pentose phosphate pathway.K00927—phosphoglycerate kinase [EC:2.7.2.3]: indicative of gluconeogenesis .In order to assess how many bacterial genomes contain the genes necessary for GED, we used AnnoTree—a web tool for visualization of genome annotations in prokaryotes  transhydrogenase (THD2pp) translocates one proton instead of two77; (ii) homoserine dehydrogenase (HSDy) produces homoserine from aspartate-semialdehyde irreversibly instead of reversibly78; (iii) GLYCK (glycerate-3P producing glycerate kinase) and POR5 (pyruvate synthase) were removed from the model as their existence in E. coli is highly disputable; (iv) the exchange reactions of Fe2+, H2S, methionine, and cysteine were removed to prevent respiration with inorganic Fe or S as electron acceptors in simulated anaerobic conditions; (v) the threonine cleavage routes were blocked by deleting threonine aldolase (THRA), threonine dehydrogenase (THRD), and glycine C-acetyltransferase (GLYAT); (vi) two unrealistic pathways for the conversion of pentoses to acetyl-CoA were removed by deleting reactions DRPA and PAI2T; (vii) the pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase (GLCDpp) was removed from the model; (viii) pyruvate formate lyase (PFL) and 2-oxobutanoate formate lyase (OBTFL) were deactivated only under aerobic conditions. The default value of the model for the non-growth-associated ATP maintenance reaction was used  to predict maximal theoretical yields in non-growing cells.Theoretical yields were estimated via flux balance analysis, conducted in Python with COBRApyThe Gnd reaction was changed to be reversible as part of the GED cycle/shunt; phosphoribulokinase (PRK) and Rubisco (RBPC) reactions were added to create the RuBP cycle/shunt. In order to use hydrogen as a proxy electron donor, hydrogen dehydrogenase reaction  was added to the model. We used the model with these modifications as a “wild-type” reference.2  and unconstrained hydrogen (when noted). The uptake rates for xylose and glucose were set to experimentally determined values for anaerobic, fermentatively growing E. coli cultures 79. The full code, including changes to the model and the new reactions of each production route, can be found at https://gitlab.com/elad.noor/ged-cycle/-/tree/master/FBA.To analyze yields of the GED shunt and RuBP shunt, these linear pathway variants were created by blocking the reactions of phosphofructokinase (PFK), S7P-reacting phosphofructokinase (PFK_3), fructose 6-phosphate aldolase (F6PA), glucose 6-phosphate dehydrogenase (G6PDH2r), and fructose-bisphosphatase (FBP). Then, xylose or glucose was assumed as a constrained carbon source together with unconstrained COE. coli SIJ48880, a strain derived from wild-type E.coli MG1655. The SIJ488 strain contains inducible genes for λ-Red recombineering (Red-recombinase and flippase) integrated into its genome to increase ease-of-use for multiple genomic modifications80. Gene deletion strains were either taken from previous studies (Table 81. Strains from the Keio collection carrying single gene deletions with a kanamycin-resistance gene (KmR) as a selective marker were used as donor strains82. Strains that had acquired the desired deletion were selected by plating on appropriate antibiotics  and confirmed by determining the size of the respective genomic locus via PCR  by a weak (pgi#1), moderate (pgi#10), or strong (pgi#20) constitutive promoter84 and a medium-strength ribosome binding site (RBS “C”: AAGTTAAGAGGCAAGA85), downstream of a CmR cassette for selection  and confirmed by Sanger sequencing. Linear dsDNA donors for λ-Red recombineering were generated by amplification with primers pntA-Prom1 and pntA-Prom2. DNA (400 ng) was transformed into the desired strains by electroporation after fresh culturing to OD600 ~ 0.3 and induction of recombinase enzymes by the addition of 15 mM l-Arabinose for 45 minutes. Confirmation of the engineered promoter region, ancestral background deletions, and removal of the selective marker was performed as described above for the P1 transduction method. The pntAB promoter locus was additionally verified by Sanger sequencing  after amplification with pntA-V1 and pntA-V2.For genomic overexpression of the rong pgi#0 constitE. coli DH5α. The native E.coli genes encoding 6-phosphogluconate dehydrogenase , KHG/KDPG aldolase , and phosphogluconate dehydratase  were amplified from E. coli MG1655 genomic DNA with high-fidelity Phusion Polymerase  using primers listed in Supplementary Table gnd (C292T to remove a PstI site) and eda (G196A to remove a PvuI site and C202T to remove a PstI site). Assembly of synthetic operons and expression plasmids was performed as described before85. In brief, genes were first inserted individually into a pNivC vector85 downstream of a ribosomal binding site . For synthetic operons, multiple genes were assembled in pNivC vectors using BioBrick restriction enzymes . The generated operons were excised from the pNivC vector by restriction with EcoRI and NheI  and inserted into a pZ-ASS vector48 . The order of genes in the operons was gnd, eda, edd for pGED; and eda, edd for pED. Constructed vectors were confirmed by Sanger sequencing . The software Geneious 8  was used for in silico cloning and sequence analysis.Cloning was carried out in E. coli strains, LB medium was used . Antibiotics were added when appropriate at the following concentrations: Kanamycin 50 µg/mL; Chloramphenicol 30 µg/mL; Ampicillin 100 µg/mL; Streptomycin 100 µg/mL. Growth assays were performed in M9 minimal medium , supplemented with trace elements . Carbon sources were added as described in the text at a concentration of 20 mM. No antibiotics were used in growth experiments, except in precultures. When elevated CO2 was required, cultures were grown in an orbital shaker set to maintain 37 °C and an atmosphere of 20% CO2 mixed with air. Growth on strain deleted in tktAB required further supplementation of E4P Supplements41: 1 mM shikimic acid, 1 μM pyridoxine, 250 μM tyrosine, 500 μM phenylalanine, 200 μM tryptophan, 6 μM 4-aminobenzoic acid, 6 μM 4-hydroxybenzoic acid, and 50 μM 2,3-dihydroxybenzoic acid.For routine culturing of 600 of 0.02 into M9 media with the final carbon sources as detailed in the text. 96-well plates  were filled with 150 µL culture and covered with 50 µL mineral oil  to avoid evaporation while allowing gas exchange. Aerobic growth was monitored in technical duplicates, triplicates, or quadruplicates at 37 °C in a BioTek Epoch 2 Microplate Spectrophotometer  by absorbance measurements (600 nm) of each well every ~10 min with intermittent orbital and linear shaking. Blank measurements were subtracted and OD600 measurements were converted to cuvette OD600 values by multiplying with a factor of 4.35, as previously established empirically for the instruments. When elevated CO2 was required, the atmosphere was maintained at 20% CO2 mixed with 80% air by placing the plate reader inside a Kuhner ISF1-X incubator shaker .Precultures for growth experiments were generally grown in M9 medium with 20 mM gluconate as carbon source (relaxing conditions). Antibiotics were added to the precultures if appropriate but omitted for growth experiments. Cells from the preculture were washed three times in M9 medium without carbon source and inoculated to a starting OD600 of 0.05 (~1.5 × 107 cells) and monitored during prolonged incubation at 37 °C and 20% CO2 (up to 3 weeks). Several cultures reached OD600 values above 1.0 after ~2 weeks. Single colonies were isolated from these cultures by dilution streak from liquid cultures onto LB medium with chloramphenicol (to maintain the pGED plasmid). Individual clones were then re-assayed for immediate growth (observable OD600 increase within 48 h) on selective liquid minimal medium (M9 + E4Ps + Xylose + 20% CO2).Tube cultures (batch growth) of 4 mL selective minimal medium (M9 + E4P supplements + 20 mM xylose) were inoculated to an OD9 cells of overnight culture in LB medium supplied with chloramphenicol (to maintain the pGED plasmid). Construction of PCR-free libraries for single-nucleotide variant detection and generation of 150 bp paired-end reads on an Illumina HiSeq 3000 platform were performed by the Max-Planck Genome Centre . Reads were mapped to the reference genome of E.coli MG165586 (GenBank accession no. U00096.3) using the software Geneious 8 . Using algorithms supplied by the software package, we identified single-nucleotide variants  and searched for regions with coverage deviating more than 2 standard deviations from the global median coverage. Confirmation of the pntA promoter locus in the ∆tkt + pGED mutant was performed by Sanger Sequencing of a PCR product from amplification of the respective locus with high-fidelity Phusion Polymerase . Sanger sequencing was performed by LGC Genomics .Genomic DNA was extracted using the GeneJET genomic DNA purification Kit  from 2 × 10600 0.5–0.6) on M9 minimal medium with 20 mM carbon source  in presence of 20% CO2. Total RNA was purified with the RNeasy Mini Kit  as instructed by the manufacturer. In brief, ~5 × 108 cells (1 ml of OD600 0.5) were mixed with 2 volumes of RNAprotect Bacteria Reagent  and pelleted, followed by enzymatic lysis, on-column removal of genomic DNA with RNase-free DNase  and spin-column-based purification of RNA. Integrity and concentration of the isolated RNA were determined by NanoDrop and gel electrophoresis. Reverse transcription to synthesize cDNA was performed on 1 µg RNA with the qScript cDNA Synthesis Kit . Quantitative real-time PCR was performed in technical triplicates using the Maxima SYBR Green/ROX qPCR Master Mix . An input corresponding to 3.125 ng total RNA was used per reaction. Non-specific amplification products were excluded by melting curve analysis. The gene encoding 16S rRNA (rrsA) was chosen as a well-established reference transcript for expression normalization87. Two alternative primer pairs for amplification of pntA were tested and the primer pair showing highest specificity and amplification efficiency was chosen . All cultures were inoculated to an OD600 0.02 and grown at 37 °C until the stationary phase. Then, ~109 cells (1 mL of culture with OD600 = 1) were pelleted, washed once with ddH2O, and hydrolyzed in 1 mL hydrochloric acid (6 M) at 95 °C for a duration of 24 h. Subsequently, the acid was evaporated by heating at 95 °C and the hydrolyzed biomass was re-suspended in ddH2O.For isotope tracing, cells were cultured in a 3 mL M9 medium supplied with the labeled/unlabeled carbon sources described in the main text. For 90. Mass spectra were acquired using an Exactive mass spectrometer . Data analysis was performed using Xcalibur . Prior to analysis, amino-acid standards  were analyzed under the same conditions in order to determine typical retention times.Hydrolyzed amino acids were separated using ultra-performance liquid chromatography  using a C18-reversed-phase column E. coli BL21-AI strains (Invitrogen) carrying appropriate plasmids for expression of E. coli Gnd or E.coli RpiA (ribose-5-phosphate isomerase), which were taken from the ASKA collection91. Expression was induced overnight at 30 °C in TB medium  by addition of 0.5 mM IPTG and 2.5 mM arabinose upon reaching an OD600 of 1. Cells were lysed in 500 mM NaCl, 20 mM Tris-HCl pH 6.9 by sonication. After centrifugation , proteins were purified on an ÄKTA start system  by HisTrap Purification  as instructed by the manufacturer, using a wash step with 18% Buffer B . Desalting was performed in 100 mM NaCl, 20 mM Tris-HCl pH 6.9, and enzymes were stored at −20 °C in desalting buffer with 20% glycerol.Proteins were expressed from ε340 nm = 6.2 cm−1 mM−1). The reductive carboxylation parameters for Gnd were determined with assays containing 2.4 mM NADPH, 16 mM ribose-5-phosphate, 1.5 M KHCO3, and 140 µM RpiA (with varying concentrations of the substrate under investigation). The assays were preincubated for 2 min and started with the addition of 750 nM of freshly diluted Gnd. Carbonic anhydrase was used to confirm that CO2 equilibration was not rate-limiting in these assays. Isomerization of ribose-5-phosphate to ribulose 5-phosphate was confirmed not to be rate-limiting. The concentration of ribulose 5-phosphate was calculated from the equilibrium constant of the isomerization reaction: Keq = 0.458 . The kinetic parameters of the oxidative decarboxylation were determined with assays containing either 800 µM NADP+ (for 6-phosphogluconate parameters) or 200 µM 6-phosphogluconate (for NADP + parameters). Assays were started with the addition of 7.5 nM Gnd. All points were measured in triplicates and each Michaelis–Menten curve was determined using at least 15 measurements.Kinetic assays were carried out on a Carry-60 UV-vis spectrometer  at 30 °C using a 1 mm quartz cuvette (Hellma). All assays were carried out in 100 mM Tris-HCl buffer at pH 8 following consumption or production, respectively, of NADPH at 340 nm (600 = 0.4–0.8) and in early stationary phase (OD600 ~ 1.1), centrifuged for 3 min at 20.000 g and supernatants frozen at −20 °C for later use. A standard curve with varying xylose concentrations confirmed negligible background signal from the xylose contained in the medium  glucose). The lower detection limit for glucose in the supernatant was thus assumed to be 0.17 mM, i.e., such concentrations or higher would be detected even in the case of complete consumption of all xylose in the media by the growing cells . Glucose standards were prepared in triplicate in the growth medium at the following concentrations (mg/mL): 0, 0.02, 0.04, and 0.08. Assays were performed by mixing 400 µL of standard or supernatant sample with 800 µL reagent mix, prepared following the manufacturer’s instructions, and incubated at 37 °C for 30 min. The reaction was stopped by adding 800 µL of sulfuric acid (6 M). Absorbance was measured at 540 nm and sample glucose concentrations determined by means of a standard curve. No glucose signal above background was detected in any culture sample.Glucose concentrations in supernatants of ΔPZF mutant cultures were determined using a commercial glucose oxidase-based assay kit following the manufacturer’s instructions . In brief, three independent cultures each of ΔPZF mutant “B” and “C” were grown in 3 mL M9 medium with 20 mM xylose, and samples were taken in exponential phase (ODFurther information on research design is available in the Supplementary InformationPeer Review FileReporting SummaryDescription of Additional Supplementary FilesSupplementary Dataset 1Supplementary Dataset 2