CO\u3csub\u3e2\u3c/sub\u3e-Fixing One-Carbon Metabolism in a Cellulose-Degrading Bacterium \u3cem\u3eClostridium thermocellum\u3c/em\u3e

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

Heterologous Expression of Alteromonas macleodii and Thiocapsa roseopersicina [NiFe] Hydrogenases in Synechococcus elongatus

Oxygen-tolerant [NiFe] hydrogenases may be used in future photobiological hydrogen production systems once the enzymes can be heterologously expressed in host organisms of interest. To achieve heterologous expression of [NiFe] hydrogenases in cyanobacteria, the two hydrogenase structural genes from Alteromonas macleodii Deep ecotype (AltDE), hynS and hynL, along with the surrounding genes in the gene operon of HynSL were cloned in a vector with an IPTG-inducible promoter and introduced into Synechococcus elongatus PCC7942. The hydrogenase protein was expressed at the correct size upon induction with IPTG. The heterologously-expressed HynSL hydrogenase was active when tested by in vitro H2 evolution assay, indicating the correct assembly of the catalytic center in the cyanobacterial host. Using a similar expression system, the hydrogenase structural genes from Thiocapsa roseopersicina (hynSL) and the entire set of known accessory genes were transferred to S. elongatus. A protein of the correct size was expressed but had no activity. However, when the 11 accessory genes from AltDE were co-expressed with hynSL, the T. roseopersicina hydrogenase was found to be active by in vitro assay. This is the first report of active, heterologously-expressed [NiFe] hydrogenases in cyanobacteria

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 E. 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 the EFE enzyme (WT vs mutant). Our findings provide a novel solution to deregulate metabolic bottlenecks in key pathways, which can be readily applied to address other engineering challenges

Characterization of the Oxygen Tolerance of a Hydrogenase Linked to a Carbon Monoxide Oxidation Pathway in Rubrivivax gelatinosus

A hydrogenase linked to the carbon monoxide oxidation pathway in Rubrivivax gelatinosus displays tolerance to O(2). When either whole-cell or membrane-free partially purified hydrogenase was stirred in full air (21% O(2), 79% N(2)), its H(2) evolution activity exhibited a half-life of 20 or 6 h, respectively, as determined by an anaerobic assay using reduced methyl viologen. When the partially purified hydrogenase was stirred in an atmosphere containing either 3.3 or 13% O(2) for 15 min and evaluated by a hydrogen-deuterium (H-D) exchange assay, nearly 80 or 60% of its isotopic exchange rate was retained, respectively. When this enzyme suspension was subsequently returned to an anaerobic atmosphere, more than 90% of the H-D exchange activity was recovered, reflecting the reversibility of this hydrogenase toward O(2) inactivation. Like most hydrogenases, the CO-linked hydrogenase was extremely sensitive to CO, with 50% inhibition occurring at 3.9 μM dissolved CO. Hydrogen production from the CO-linked hydrogenase was detected when ferredoxins of a prokaryotic source were the immediate electron mediator, provided they were photoreduced by spinach thylakoid membranes containing active water-splitting activity. Based on its appreciable tolerance to O(2), potential applications of this hydrogenase are discussed

A Genetic Toolbox for Modulating the Expression of Heterologous Genes in the Cyanobacterium <i>Synechocystis</i> 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 <i>Synechocystis</i> sp. PCC 6803. We report the finding that the <i>E. coli</i> 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