Engineering the cellulolytic bacterium, Clostridium thermocellum, to co-utilize hemicellulose.

Consolidated bioprocessing (CBP) of lignocellulosic biomass holds promise to realize economic production of second-generation biofuels/chemicals, and Clostridium thermocellum is a leading candidate for CBP due to it being one of the fastest degraders of crystalline cellulose and lignocellulosic biomass. However, CBP by C. thermocellum is approached with co-cultures, because C. thermocellum does not utilize hemicellulose. When compared with a single-species fermentation, the co-culture system introduces unnecessary process complexity that may compromise process robustness. In this study, we engineered C. thermocellum to co-utilize hemicellulose without the need for co-culture. By evolving our previously engineered xylose-utilizing strain in xylose, an evolved clonal isolate (KJC19-9) was obtained and showed improved specific growth rate on xylose by ∼3-fold and displayed comparable growth to a minimally engineered strain grown on the bacteria's naturally preferred substrate, cellobiose. To enable full xylan deconstruction to xylose, we recombinantly expressed three different ß-xylosidase enzymes originating from Thermoanaerobacterium saccharolyticum into KJC19-9 and demonstrated growth on xylan with one of the enzymes. This recombinant strain was capable of co-utilizing cellulose and xylan simultaneously, and we integrated the ß-xylosidase gene into the KJC19-9 genome, creating the KJCBXint strain. The strain, KJC19-9, consumed monomeric xylose but accumulated xylobiose when grown on pretreated corn stover, whereas the final KJCBXint strain showed significantly greater deconstruction of xylan and xylobiose. This is the first reported C. thermocellum strain capable of degrading and assimilating hemicellulose polysaccharide while retaining its cellulolytic capabilities, unlocking significant potential for CBP in advancing the bioeconomy.

Engineering improved ethylene production: Leveraging systems biology and adaptive laboratory evolution.

Ethylene is a small hydrocarbon gas widely used in the chemical industry. Annual worldwide production currently exceeds 150 million tons, producing considerable amounts of CO2 contributing to climate change. The need for a sustainable alternative is therefore imperative. Ethylene is natively produced by several different microorganisms, including Pseudomonas syringae pv. phaseolicola via a process catalyzed by the ethylene-forming enzyme (EFE), subsequent heterologous expression of EFE has led to ethylene production in non-native bacterial hosts including Escherichia coli and cyanobacteria. However, solubility of EFE and substrate availability remain rate-limiting steps in biological ethylene production. We employed a combination of genome-scale metabolic modelling, continuous fermentation, and protein evolution to enable the accelerated development of a high efficiency ethylene producing E. coli strain, yielding a 49-fold increase in production, the most significant improvement reported to date. Furthermore, we have clearly demonstrated that this increased yield resulted from metabolic adaptations that were uniquely linked to EFE (wild type versus mutant). Our findings provide a novel solution to deregulate metabolic bottlenecks in key pathways, which can be readily applied to address other engineering challenges.

Inactivation of the uptake hydrogenase in the purple non-sulfur photosynthetic bacterium Rubrivivax gelatinosus CBS enables a biological water-gas shift platform for H2 production.

Biological H2 production has potential to address energy security and environmental concerns if produced from renewable or waste sources. The purple non-sulfur photosynthetic bacterium Rubrivivax gelatinosus CBS produces H2 while oxidizing CO, a component of synthesis gas (Syngas). CO-linked H2 production is facilitated by an energy-converting hydrogenase (Ech), while a subsequent H2 oxidation reaction is catalyzed by a membrane-bound hydrogenase (MBH). Both hydrogenases contain [NiFe] active sites requiring 6 maturation factors (HypA-F) for assembly, but it is unclear which of the two annotated sets of hyp genes are required for each in R. gelatinosus CBS. Herein, we report correlated expression of hyp1 genes with Ech genes and hyp2 expression with MBH genes. Moreover, we find that while Ech H2 evolving activity is only delayed when hyp1 is deleted, hyp2 deletion completely disrupts MBH H2 uptake, providing a platform for a biologically driven water-gas shift reaction to produce H2 from CO.

CO2-fixing one-carbon metabolism in a cellulose-degrading bacterium Clostridium thermocellum.

Clostridium thermocellum can ferment cellulosic biomass to formate and other end products, including CO2 This organism lacks formate dehydrogenase (Fdh), which catalyzes the reduction of CO2 to formate. However, feeding the bacterium 13C-bicarbonate and cellobiose followed by NMR analysis showed the production of 13C-formate in C. thermocellum culture, indicating the presence of an uncharacterized pathway capable of converting CO2 to formate. Combining genomic and experimental data, we demonstrated that the conversion of CO2 to formate serves as a CO2 entry point into the reductive one-carbon (C1) metabolism, and internalizes CO2 via two biochemical reactions: the reversed pyruvate:ferredoxin oxidoreductase (rPFOR), which incorporates CO2 using acetyl-CoA as a substrate and generates pyruvate, and pyruvate-formate lyase (PFL) converting pyruvate to formate and acetyl-CoA. We analyzed the labeling patterns of proteinogenic amino acids in individual deletions of all five putative PFOR mutants and in a PFL deletion mutant. We identified two enzymes acting as rPFOR, confirmed the dual activities of rPFOR and PFL crucial for CO2 uptake, and provided physical evidence of a distinct in vivo "rPFOR-PFL shunt" to reduce CO2 to formate while circumventing the lack of Fdh. Such a pathway precedes CO2 fixation via the reductive C1 metabolic pathway in C. thermocellum These findings demonstrated the metabolic versatility of C. thermocellum, which is thought of as primarily a cellulosic heterotroph but is shown here to be endowed with the ability to fix CO2 as well.

Engineering cellulolytic bacterium Clostridium thermocellum to co-ferment cellulose- and hemicellulose-derived sugars simultaneously.

Cellulose and hemicellulose are the most abundant components in plant biomass. A preferred Consolidated Bioprocessing (CBP) system is one which can directly convert both cellulose and hemicellulose into target products without adding the costly hydrolytic enzyme cocktail. In this work, the thermophilic, cellulolytic, and anaerobic bacterium, Clostridium thermocellum DSM 1313, was engineered to grow on xylose in addition to cellulose. Both xylA (encoding for xylose isomerase) and xylB (encoding for xylulokinase) genes from the thermophilic anaerobic bacterium Thermoanaerobacter ethanolicus were introduced to enable xylose utilization while still retaining its inherent ability to grow on 6-carbon substrates. Targeted integration of xylAB into C. thermocellum genome realized simultaneous fermentation of xylose with glucose, with cellobiose (glucose dimer), and with cellulose, respectively, without carbon catabolite repression. We also showed that the respective H2 and ethanol production were twice as much when both xylose and cellulose were consumed simultaneously than when consuming cellulose alone. Moreover, the engineered xylose consumer can also utilize xylo-oligomers (with degree of polymerization of 2-7) in the presence of xylose. Isotopic tracer studies also revealed that the engineered xylose catabolism contributed to the production of ethanol from xylan which is a model hemicellulose in mixed sugar fermentation, demonstrating immense potential of this enhanced CBP strain in co-utilizing both cellulose and hemicellulose for the production of fuels and chemicals.

Unification of [FeFe]-hydrogenases into three structural and functional groups.

BACKGROUND: [FeFe]-hydrogenases (Hyd) are structurally diverse enzymes that catalyze the reversible oxidation of hydrogen (H2). Recent biochemical data demonstrate new functional roles for these enzymes, including those that function in electron bifurcation where an exergonic reaction is coupled with an endergonic reaction to drive the reversible oxidation/production of H2. METHODS: To identify the structural determinants that underpin differences in enzyme functionality, a total of 714 homologous sequences of the catalytic subunit, HydA, were compiled. Bioinformatics approaches informed by biochemical data were then used to characterize differences in inferred quaternary structure, HydA active site protein environment, accessory iron-sulfur clusters in HydA, and regulatory proteins encoded in HydA gene neighborhoods. RESULTS: HydA homologs were clustered into one of three classification groups, Group 1 (G1), Group 2 (G2), and Group 3 (G3). G1 enzymes were predicted to be monomeric while those in G2 and G3 were predicted to be multimeric and include HydB, HydC (G2/G3) and HydD (G3) subunits. Variation in the HydA active site and accessory iron-sulfur clusters did not vary by group type. Group-specific regulatory genes were identified in the gene neighborhoods of both G2 and G3 Hyd. Analyses of purified G2 and G3 enzymes by mass spectrometry strongly suggest that they are post-translationally modified by phosphorylation. CONCLUSIONS: These results suggest that bifurcation capability is dictated primarily by the presence of both HydB and HydC in Hyd complexes, rather than by variation in HydA. GENERAL SIGNIFICANCE: This classification scheme provides a framework for future biochemical and mutagenesis studies to elucidate the functional role of Hyd enzymes.

Microbial Photoelectrosynthesis for Self-Sustaining Hydrogen Generation.

Current artificial photosynthesis (APS) systems are promising for the storage of solar energy via transportable and storable fuels, but the anodic half-reaction of water oxidation is an energy intensive process which in many cases poorly couples with the cathodic half-reaction. Here we demonstrate a self-sustaining microbial photoelectrosynthesis (MPES) system that pairs microbial electrochemical oxidation with photoelectrochemical water reduction for energy efficient H2 generation. MPES reduces the overall energy requirements thereby greatly expanding the range of semiconductors that can be utilized in APS. Due to the recovery of chemical energy from waste organics by the mild microbial process and utilization of cost-effective and stable catalyst/electrode materials, our MPES system produced a stable current of 0.4 mA/cm2 for 24 h without any external bias and ∼10 mA/cm2 with a modest bias under one sun illumination. This system also showed other merits, such as creating benefits of wastewater treatment and facile preparation and scalability.

Engineered xylose utilization enhances bio-products productivity in the cyanobacterium Synechocystis sp. PCC 6803.

Hydrolysis of plant biomass generates a mixture of simple sugars that is particularly rich in glucose and xylose. Fermentation of the released sugars emits CO2 as byproduct due to metabolic inefficiencies. Therefore, the ability of a microbe to simultaneously convert biomass sugars and photosynthetically fix CO2 into target products is very desirable. In this work, the cyanobacterium, Synechocystis 6803, was engineered to grow on xylose in addition to glucose. Both the xylA (xylose isomerase) and xylB (xylulokinase) genes from Escherichia coli were required to confer xylose utilization, but a xylose-specific transporter was not required. Introduction of xylAB into an ethylene-producing strain increased the rate of ethylene production in the presence of xylose. Additionally, introduction of xylAB into a glycogen-synthesis mutant enhanced production of keto acids. Isotopic tracer studies found that nearly half of the carbon in the excreted keto acids was derived from the engineered xylose metabolism, while the remainder was derived from CO2 fixation.

Genetic analysis of the Hox hydrogenase in the cyanobacterium Synechocystis sp. PCC 6803 reveals subunit roles in association, assembly, maturation, and function.

Hydrogenases are metalloenzymes that catalyze 2H(+) + 2e(-) ↔ H(2). A multisubunit, bidirectional [NiFe]-hydrogenase has been identified and characterized in a number of bacteria, including cyanobacteria, where it is hypothesized to function as an electron valve, balancing reductant in the cell. In cyanobacteria, this Hox hydrogenase consists of five proteins in two functional moieties: a hydrogenase moiety (HoxYH) with homology to heterodimeric [NiFe]-hydrogenases and a diaphorase moiety (HoxEFU) with homology to NuoEFG of respiratory Complex I, linking NAD(P)H ↔ NAD(P)(+) as a source/sink for electrons. Here, we present an extensive study of Hox hydrogenase in the cyanobacterium Synechocystis sp. PCC 6803. We identify the presence of HoxEFUYH, HoxFUYH, HoxEFU, HoxFU, and HoxYH subcomplexes as well as association of the immature, unprocessed large subunit (HoxH) with other Hox subunits and unidentified factors, providing a basis for understanding Hox maturation and assembly. The analysis of mutants containing individual and combined hox gene deletions in a common parental strain reveals apparent alterations in subunit abundance and highlights an essential role for HoxF and HoxU in complex/subcomplex association. In addition, analysis of individual and combined hox mutant phenotypes in a single strain background provides a clear view of the function of each subunit in hydrogenase activity and presents evidence that its physiological function is more complicated than previously reported, with no outward defects apparent in growth or photosynthesis under various growth conditions.

Draft genome sequence of Rubrivivax gelatinosus CBS.

Rubrivivax gelatinosus CBS, a purple nonsulfur photosynthetic bacterium, can grow photosynthetically using CO and N(2) as the sole carbon and nitrogen nutrients, respectively. R. gelatinosus CBS is of particular interest due to its ability to metabolize CO and yield H(2). We present the 5-Mb draft genome sequence of R. gelatinosus CBS with the goal of providing genetic insight into the metabolic properties of this bacterium.

Hydrogenases and hydrogen photoproduction in oxygenic photosynthetic organisms.

The photobiological production of H2 gas, using water as the only electron donor, is a property of two types of photosynthetic microorganisms: green algae and cyanobacteria. In these organisms, photosynthetic water splitting is functionally linked to H(2) production by the activity of hydrogenase enzymes. Interestingly, each of these organisms contains only one of two major types of hydrogenases, [FeFe] or [NiFe] enzymes, which are phylogenetically distinct but perform the same catalytic reaction, suggesting convergent evolution. This idea is supported by the observation that each of the two classes of hydrogenases has a different metallo-cluster, is encoded by entirely different sets of genes (apparently under the control of different promoter elements), and exhibits different maturation pathways. The genetics, biosynthesis, structure, function, and O2 sensitivity of these enzymes have been the focus of extensive research in recent years. Some of this effort is clearly driven by the potential for using these enzymes in future biological or biohybrid systems to produce renewable fuel or in fuel cell applications.

Characterization of genes responsible for the CO-linked hydrogen production pathway in Rubrivivax gelatinosus.

Upon exposure to carbon monoxide, the purple nonsulfur photosynthetic bacterium Rubrivivax gelatinosus produces hydrogen concomitantly with the oxidation of CO according to the equation CO + H(2)O <--> CO(2) + H(2). Yet little is known about the genetic elements encoding this reaction in this organism. In the present study, we use transposon mutagenesis and functional complementation to uncover three clustered genes, cooL, cooX, and cooH, in Rubrivivax gelatinosus putatively encoding part of a membrane-bound, multisubunit NiFe-hydrogenase. We present the complete amino acid sequences for the large catalytic subunit and its electron-relaying small subunit, encoded by cooH and cooL, respectively. Sequence alignment reveals a conserved region in the large subunit coordinating a binuclear [NiFe] center and a conserved region in the small subunit coordinating a [4Fe-4S] cluster. Protein purification experiments show that a protein fraction of 58 kDa molecular mass could function in H(2) evolution mediated by reduced methyl viologen. Western blotting experiments show that the two hydrogenase subunits are detectable and accumulate only when cells are exposed to CO. The cooX gene encodes a putative Fe-S protein mediating electron transfer to the hydrogenase small subunit. We conclude that these three Rubrivivax proteins encompass part of a membrane-bound, multisubunit NiFe-hydrogenase belonging to the energy-converting hydrogenase (Ech) type, which has been found among diverse microbes with a common feature in coupling H(2) production with proton pumping for energy generation.

The Metabolism of Clostridium ljungdahlii in Phosphotransacetylase Negative Strains and Development of an Ethanologenic Strain.

The sustainable production of chemicals from non-petrochemical sources is one of the greatest challenges of our time. CO2 release from industrial activity is not environmentally friendly yet provides an inexpensive feedstock for chemical production. One means of addressing this problem is using acetogenic bacteria to produce chemicals from CO2, waste streams, or renewable resources. Acetogens are attractive hosts for chemical production for many reasons: they can utilize a variety of feedstocks that are renewable or currently waste streams, can capture waste carbon sources and covert them to products, and can produce a variety of chemicals with greater carbon efficiency over traditional fermentation technologies. Here we investigated the metabolism of Clostridium ljungdahlii, a model acetogen, to probe carbon and electron partitioning and understand what mechanisms drive product formation in this organism. We utilized CRISPR/Cas9 and an inducible riboswitch to target enzymes involved in fermentation product formation. We focused on the genes encoding phosphotransacetylase (pta), aldehyde ferredoxin oxidoreductases (aor1 and aor2), and bifunctional alcohol/aldehyde dehydrogenases (adhE1 and adhE2) and performed growth studies under a variety of conditions to probe the role of those enzymes in the metabolism. Finally, we demonstrated a switch from acetogenic to ethanologenic metabolism by these manipulations, providing an engineered bacterium with greater application potential in biorefinery industry.

Covalent organic framework-supported platinum nanoparticles as efficient electrocatalysts for water reduction.

The hydrogen evolution reaction (HER) is one of the most effective and sustainable ways to produce hydrogen gas as an alternative clean fuel. The rate of this electrocatalytic reaction is highly dependent on the properties (dispersity and stability) of electrocatalysts. Herein, we developed well-dispersed and highly stable platinum nanoparticles (PtNPs) supported on a covalent organic framework (COF-bpyTPP), which exhibit excellent catalytic activities toward HER as well as the hydride reduction reaction. The nanoparticles have an average size of 2.95 nm and show superior catalytic performance compared to the commercially available Pt/C under the same alkaline conditions, producing 13 times more hydrogen with a far more positive onset potential (-0.13 V vs.-0.63 V) and ca. 100% faradaic efficiency. The reaction rate of the hydride reduction of 4-nitrophenol was also 10 times faster in the case of PtNPs@COF compared to the commercial Pt/C under the same loading and conditions. More importantly, the PtNPs@COF are highly stable under the aqueous reactions conditions and can be reused without showing noticeable aggregation and activity degradation.

Transcriptomic analysis of a Clostridium thermocellum strain engineered to utilize xylose: responses to xylose versus cellobiose feeding.

Clostridium (Ruminiclostridium) thermocellum is recognized for its ability to ferment cellulosic biomass directly, but it cannot naturally grow on xylose. Recently, C. thermocellum (KJC335) was engineered to utilize xylose through expressing a heterologous xylose catabolizing pathway. Here, we compared KJC335's transcriptomic responses to xylose versus cellobiose as the primary carbon source and assessed how the bacteria adapted to utilize xylose. Our analyses revealed 417 differentially expressed genes (DEGs) with log2 fold change (FC) >|1| and 106 highly DEGs (log2 FC >|2|). Among the DEGs, two putative sugar transporters, cbpC and cbpD, were up-regulated, suggesting their contribution to xylose transport and assimilation. Moreover, the up-regulation of specific transketolase genes (tktAB) suggests the importance of this enzyme for xylose metabolism. Results also showed remarkable up-regulation of chemotaxis and motility associated genes responding to xylose feeding, as well as widely varying gene expression in those encoding cellulosomal enzymes. For the down-regulated genes, several were categorized in gene ontology terms oxidation-reduction processes, ATP binding and ATPase activity, and integral components of the membrane. This study informs potentially critical, enabling mechanisms to realize the conceptually attractive Next-Generation Consolidated BioProcessing approach where a single species is sufficient for the co-fermentation of cellulose and hemicellulose.

Integrated thermodynamic analysis of electron bifurcating [FeFe]-hydrogenase to inform anaerobic metabolism and H2 production.

Electron bifurcating, [FeFe]-hydrogenases are recently described members of the hydrogenase family and catalyze a combination of exergonic and endergonic electron exchanges between three carriers (2 ferredoxinred- + NAD(P)H + 3 H+ = 2 ferredoxinox + NAD(P)+ + 2 H2). A thermodynamic analysis of the bifurcating, [FeFe]-hydrogenase reaction, using electron path-independent variables, quantified potential biological roles of the reaction without requiring enzyme details. The bifurcating [FeFe]-hydrogenase reaction, like all bifurcating reactions, can be written as a sum of two non-bifurcating reactions. Therefore, the thermodynamic properties of the bifurcating reaction can never exceed the properties of the individual, non-bifurcating, reactions. The bifurcating [FeFe]-hydrogenase reaction has three competitive properties: 1) enabling NAD(P)H-driven proton reduction at pH2 higher than the concurrent operation of the two, non-bifurcating reactions, 2) oxidation of NAD(P)H and ferredoxin simultaneously in a 1:1 ratio, both are produced during typical glucose fermentations, and 3) enhanced energy conservation (~10 kJ mol-1 H2) relative to concurrent operation of the two, non-bifurcating reactions. Our analysis demonstrated ferredoxin E°' largely determines the sensitivity of the bifurcating reaction to pH2, modulation of the reduced/oxidized electron carrier ratios contributed less to equilibria shifts. Hydrogenase thermodynamics data were integrated with typical and non-typical glycolysis pathways to evaluate achieving the 'Thauer limit' (4 H2 per glucose) as a function of temperature and pH2. For instance, the bifurcating [FeFe]-hydrogenase reaction permits the Thauer limit at 60 °C if pH 2 ≤ ~10 mbar. The results also predict Archaea, expressing a non-typical glycolysis pathway, would not benefit from a bifurcating [FeFe]-hydrogenase reaction; interestingly, no Archaea have been observed experimentally with a [FeFe]-hydrogenase enzyme.

Developing Riboswitch-Mediated Gene Regulatory Controls in Thermophilic Bacteria.

Thermophilic bacteria are attractive hosts to produce bio-based chemicals. While various genetic manipulations have been employed in the metabolic engineering of thermophiles, a robust means to regulate gene expression in these bacteria (∼55 °C) is missing. Our bioinformatic search for various riboswitches in thermophilic bacteria revealed that major classes of riboswitches are present, suggesting riboswitches' regulatory roles in these bacteria. By building synthetic constructs incorporating natural and engineered purine riboswitch sequences originated from foreign species, we quantified respective riboswitches activities in repressing and up-regulating gene expression in Geobacillus thermoglucosidasius using a green fluorescence protein. The elicited regulatory response was ligand-concentration-dependent. We further demonstrated that riboswitch-mediated gene expression of adhE (responsible for ethanol production) in Clostridium thermocellum can modulate ethanol production, redirect metabolites, and control cell growth in the adhE knockout mutant. This work has made tunable gene expression feasible across different thermophiles for broad applications including biofuels production and gene-to-trait mapping.

Membrane-Inlet Mass Spectrometry Enables a Quantitative Understanding of Inorganic Carbon Uptake Flux and Carbon Concentrating Mechanisms in Metabolically Engineered Cyanobacteria.

Photosynthesis uses solar energy to drive inorganic carbon (Ci) uptake, fixation, and biomass formation. In cyanobacteria, Ci uptake is assisted by carbon concentrating mechanisms (CCM), and CO2 fixation is catalyzed by RubisCO in the Calvin-Benson-Bassham (CBB) cycle. Understanding the regulation that governs CCM and CBB cycle activities in natural and engineered strains requires methods and parameters that quantify these activities. Here, we used membrane-inlet mass spectrometry (MIMS) to simultaneously quantify Ci concentrating and fixation processes in the cyanobacterium Synechocystis 6803. By comparing cultures acclimated to ambient air conditions to cultures transitioning to high Ci conditions, we show that acclimation to high Ci involves a concurrent decline of Ci uptake and fixation parameters. By varying light input, we show that both CCM and CBB reactions become energy limited under low light conditions. A strain over-expressing the gene for the CBB cycle enzyme fructose-bisphosphate aldolase showed higher CCM and carbon fixation capabilities, suggesting a regulatory link between CBB metabolites and CCM capacity. While the engineering of an ethanol production pathway had no effect on CCM or carbon fixation parameters, additional fructose-bisphosphate aldolase gene over-expression enhanced both activities while simultaneously increasing ethanol productivity. These observations show that MIMS can be a useful tool to study the extracellular Ci flux and how CBB metabolites regulate Ci uptake and fixation.

A Genetic Toolbox for Modulating the Expression of Heterologous Genes in the Cyanobacterium Synechocystis sp. PCC 6803.

Cyanobacteria, genetic models for photosynthesis research for decades, have recently become attractive hosts for producing renewable fuels and chemicals, owing to their genetic tractability, relatively fast growth, and their ability to utilize sunlight, fix carbon dioxide, and in some cases, fix nitrogen. Despite significant advances, there is still an urgent demand for synthetic biology tools in order to effectively manipulate genetic circuits in cyanobacteria. In this study, we have compared a total of 17 natural and chimeric promoters, focusing on expression of the ethylene-forming enzyme (EFE) in the cyanobacterium Synechocystis sp. PCC 6803. We report the finding that the E. coli σ70 promoter Ptrc is superior compared to the previously reported strong promoters, such as PcpcB and PpsbA, for the expression of EFE. In addition, we found that the EFE expression level was very sensitive to the 5'-untranslated region upstream of the open reading frame. A library of ribosome binding sites (RBSs) was rationally designed and was built and systematically characterized. We demonstrate a strategy complementary to the RBS prediction software to facilitate the rational design of an RBS library to optimize the gene expression in cyanobacteria. Our results show that the EFE expression level is dramatically enhanced through these synthetic biology tools and is no longer the rate-limiting step for cyanobacterial ethylene production. These systematically characterized promoters and the RBS design strategy can serve as useful tools to tune gene expression levels and to identify and mitigate metabolic bottlenecks in cyanobacteria.

Overcoming substrate limitations for improved production of ethylene in E. coli.

BACKGROUND: Ethylene is an important industrial compound for the production of a wide variety of plastics and chemicals. At present, ethylene production involves steam cracking of a fossil-based feedstock, representing the highest CO2-emitting process in the chemical industry. Biological ethylene production can be achieved via expression of a single protein, the ethylene-forming enzyme (EFE), found in some bacteria and fungi; it has the potential to provide a sustainable alternative to steam cracking, provided that significant increases in productivity can be achieved. A key barrier is determining factors that influence the availability of substrates for the EFE reaction in potential microbial hosts. In the presence of O2, EFE catalyzes ethylene formation from the substrates α-ketoglutarate (AKG) and arginine. The concentrations of AKG, a key TCA cycle intermediate, and arginine are tightly controlled by an intricate regulatory system that coordinates carbon and nitrogen metabolism. Therefore, reliably predicting which genetic changes will ultimately lead to increased AKG and arginine availability is challenging. RESULTS: We systematically explored the effects of media composition (rich versus defined), gene copy number, and the addition of exogenous substrates and other metabolites on the formation of ethylene in Escherichia coli expressing EFE. Guided by these results, we tested a number of genetic modifications predicted to improve substrate supply and ethylene production, including knockout of competing pathways and overexpression of key enzymes. Several such modifications led to higher AKG levels and higher ethylene productivity, with the best performing strain more than doubling ethylene productivity (from 81 ± 3 to 188 ± 13 nmol/OD600/mL). CONCLUSIONS: Both EFE activity and substrate supply can be limiting factors in ethylene production. Targeted modifications in central carbon metabolism, such as overexpression of isocitrate dehydrogenase, and deletion of glutamate synthase or the transcription regulator ArgR, can effectively enhance substrate supply and ethylene productivity. These results not only provide insight into the intricate regulatory network of the TCA cycle, but also guide future pathway and genome-scale engineering efforts to further boost ethylene productivity.