Heterozygous deficiency of PHD2 restores tumor oxygenation and inhibits metastasis via endothelial normalization.

A key function of blood vessels, to supply oxygen, is impaired in tumors because of abnormalities in their endothelial lining. PHD proteins serve as oxygen sensors and may regulate oxygen delivery. We therefore studied the role of endothelial PHD2 in vessel shaping by implanting tumors in PHD2(+/-) mice. Haplodeficiency of PHD2 did not affect tumor vessel density or lumen size, but normalized the endothelial lining and vessel maturation. This resulted in improved tumor perfusion and oxygenation and inhibited tumor cell invasion, intravasation, and metastasis. Haplodeficiency of PHD2 redirected the specification of endothelial tip cells to a more quiescent cell type, lacking filopodia and arrayed in a phalanx formation. This transition relied on HIF-driven upregulation of (soluble) VEGFR-1 and VE-cadherin. Thus, decreased activity of an oxygen sensor in hypoxic conditions prompts endothelial cells to readjust their shape and phenotype to restore oxygen supply. Inhibition of PHD2 may offer alternative therapeutic opportunities for anticancer therapy.

The ethanol pathway from Thermoanaerobacterium saccharolyticum improves ethanol production in Clostridium thermocellum.

Clostridium thermocellum ferments cellulose, is a promising candidate for ethanol production from cellulosic biomass, and has been the focus of studies aimed at improving ethanol yield. Thermoanaerobacterium saccharolyticum ferments hemicellulose, but not cellulose, and has been engineered to produce ethanol at high yield and titer. Recent research has led to the identification of four genes in T. saccharolyticum involved in ethanol production: adhE, nfnA, nfnB and adhA. We introduced these genes into C. thermocellum and observed significant improvements to ethanol yield, titer, and productivity. The four genes alone, however, were insufficient to achieve in C. thermocellum the ethanol yields and titers observed in engineered T. saccharolyticum strains, even when combined with gene deletions targeting hydrogen production. This suggests that other parts of T. saccharolyticum metabolism may also be necessary to reproduce the high ethanol yield and titer phenotype in C. thermocellum.

Angiopoietin-2 secretion by endothelial cell exosomes: regulation by the phosphatidylinositol 3-kinase (PI3K)/Akt/endothelial nitric oxide synthase (eNOS) and syndecan-4/syntenin pathways.

Angiopoietin-2 (Ang2) is an extracellular protein and one of the principal ligands of Tie2 receptor that is involved in the regulation of vascular integrity, quiescence, and inflammation. The mode of secretion of Ang2 has never been established, however. Here, we provide evidence that Ang2 is secreted from endothelial cells via exosomes and that this process is inhibited by the PI3K/Akt/endothelial nitric oxide synthase (eNOS) signaling pathway, whereas it is positively regulated by the syndecan-4/syntenin pathway. Vascular defects in Akt1 null mice arise, in part, because of excessive Ang2 secretion and can be rescued by the syndecan-4 knock-out that reduces extracellular Ang2 levels. This novel mechanism connects three critical signaling pathways: angiopoietin/Tie2, PI3K/Akt/eNOS, and syndecan/syntenin, which play important roles in vascular growth and stabilization.

PTP1b is a physiologic regulator of vascular endothelial growth factor signaling in endothelial cells.

BACKGROUND: Regulation of vascular endothelial growth factor receptor-2 (VEGFR2) signaling is a control point that determines the extent of vascular tree formation. Recent studies demonstrated an important role played by VEGFR2 endothelial trafficking in control of its activity and suggested the involvement of a phosphotyrosine phosphatase 1b (PTP1b) in this process. This study was designed to define the role of PTP1b in endothelial VEGFR2 signaling and its role in regulation of angiogenesis and arteriogenesis. METHODS AND RESULTS: We generated mice carrying an endothelial-specific deletion of PTP1b and examined the effect of this knockout on VEGF signaling, angiogenesis, and arteriogenesis in vitro and in vivo. PTP1b knockout endothelial cells had increased VEGF-dependent activation of extracellular signal-regulated kinase signaling, sprouting, migration, and proliferation compared with controls. Endothelial PTP1b null mice had increased retinal and Matrigel implant angiogenesis and accelerated wound healing, pointing to enhanced angiogenesis. Increased arteriogenesis was demonstrated by observations of faster recovery of arterial blood flow and large numbers of newly formed arterioles in the hindlimb ischemia mouse model. PTP1b endothelial knockout also rescued impaired blood flow recovery after common femoral artery ligation in synectin null mice. CONCLUSIONS: PTP1b is a key regulator of endothelial VEGFR2 signaling and plays an important role in regulation of the extent of vascular tree formation.

Endothelial cell-dependent regulation of arteriogenesis.

RATIONALE: Arteriogenesis is the process of formation of arterial conduits. Its promotion is an attractive therapeutic strategy in occlusive atherosclerotic diseases. Despite the functional and clinical importance of arteriogenesis, the biology of the process is poorly understood. Synectin, a gene previously implicated in the regulation of vascular endothelial cell growth factor signaling, offers a unique opportunity to determine relative contributions of various cell types to arteriogenesis. OBJECTIVE: We investigated the cell-autonomous effects of a synectin knockout in arterial morphogenesis. METHODS AND RESULTS: A floxed synectin knockin mouse line was crossbred with endothelial-specific (Tie2, Cdh5, Pdgfb) and smooth muscle myosin heavy chain-specific Cre driver mouse lines to produce cell type-specific deletions. Ablation of synectin expression in endothelial, but not smooth muscle cells resulted in the presence of developmental arterial morphogenetic defects (smaller size of the arterial tree, reduced number of arterial branches and collaterals) and impaired arteriogenesis in adult mice. CONCLUSIONS: Synectin modulates developmental and adult arteriogenesis in an endothelial cell-autonomous fashion. These findings show for the first time that endothelial cells are central to both developmental and adult arteriogenesis and provide a model for future studies of factors involved in this process.

Endothelial nuclear factor-κB-dependent regulation of arteriogenesis and branching.

BACKGROUND: Arteriogenesis and collateral formation are complex processes requiring integration of multiple inputs to coordinate vessel branching, growth, maturation, and network size. Factors regulating these processes have not been determined. METHODS AND RESULTS: We used an inhibitor of NFκB activation (IκBαSR) under control of an endothelial-specific inducible promoter to selectively suppress endothelial nuclear factor-κB activation during development, in the adult vasculature, or in vitro. Inhibition of nuclear factor-κB activation resulted in formation of an excessively branched arterial network that was composed of immature vessels and provided poor distal tissue perfusion. Molecular analysis demonstrated reduced adhesion molecule expression leading to decreased monocyte influx, reduced hypoxia-inducible factor-1α levels, and a marked decrease in δ-like ligand 4 expression with a consequent decrease in Notch signaling. The latter was the principal cause of increased vascular branching as treatment with Jagged-1 peptide reduced the size of the arterial network to baseline levels. CONCLUSIONS: These findings identify nuclear factor-κB as a key regulator of adult and developmental arteriogenesis and collateral formation. Nuclear factor-κB achieves this by regulating hypoxia-inducible factor-1α-dependent expression of vascular endothelial growth factor-A and platelet-derived growth factor-BB, which are necessary for the development and maturation of the arterial collateral network, and by regulating δ-like ligand 4 expression, which in turn determines the size and complexity of the network.

Ethanol tolerance in engineered strains of Clostridium thermocellum.

Clostridium thermocellum is a natively cellulolytic bacterium that is promising candidate for cellulosic biofuel production, and can produce ethanol at high yields (75-80% of theoretical) but the ethanol titers produced thus far are too low for commercial application. In several strains of C. thermocellum engineered for increased ethanol yield, ethanol titer seems to be limited by ethanol tolerance. Previous work to improve ethanol tolerance has focused on the WT organism. In this work, we focused on understanding ethanol tolerance in several engineered strains of C. thermocellum. We observed a tradeoff between ethanol tolerance and production. Adaptation for increased ethanol tolerance decreases ethanol production. Second, we observed a consistent genetic response to ethanol stress involving mutations at the AdhE locus. These mutations typically reduced NADH-linked ADH activity. About half of the ethanol tolerance phenotype could be attributed to the elimination of NADH-linked activity based on a targeted deletion of adhE. Finally, we observed that rich growth medium increases ethanol tolerance, but this effect is eliminated in an adhE deletion strain. Together, these suggest that ethanol inhibits growth and metabolism via a redox-imbalance mechanism. The improved understanding of mechanisms of ethanol tolerance described here lays a foundation for developing strains of C. thermocellum with improved ethanol production.

Selective regulation of arterial branching morphogenesis by synectin.

Branching morphogenesis is a key process in the formation of vascular networks. To date, little is known regarding the molecular events regulating this process. We investigated the involvement of synectin in this process. In zebrafish embryos, synectin knockdown resulted in a hypoplastic dorsal aorta and hypobranched, stunted, and thin intersomitic vessels due to impaired migration and proliferation of angioblasts and arterial endothelial cells while not affecting venous development. Synectin(-/-) mice demonstrated decreased body and organ size, reduced numbers of arteries, and an altered pattern of arterial branching in multiple vascular beds while the venous system remained normal. Murine synectin(-/-) primary arterial, but not venous, endothelial cells showed decreased in vitro tube formation, migration, and proliferation and impaired polarization due to abnormal localization of activated Rac1. We conclude that synectin is involved in selective regulation of arterial, but not venous, growth and branching morphogenesis and that Rac1 plays an important role in this process.

Vascular endothelial growth factor and semaphorin induce neuropilin-1 endocytosis via separate pathways.

The neuropilin (Nrp)1 receptor is essential for both nervous and vascular system development. Nrp1 is unusually versatile, because it transmits both chemoattractive and repulsive signals in response to vascular endothelial growth factor (VEGF)-A and class 3 semaphorins, respectively. Both Nrp1 and VEGF receptor 2 undergo ligand-dependent endocytosis. We sought to establish the endocytic pathway of Nrp1 and to determine whether uptake is required for its signaling. Whereas Nrp1 underwent clathrin-dependent endocytosis in response to VEGFA(165) treatment, semaphorin 3C (sema3C) induced lipid raft-dependent endocytosis. The myosin VI PDZ (postsynaptic density 95, Disk large, Zona occludens-1) adaptor protein synectin was essential for Nrp1 trafficking. Sema3C failed to inhibit migration of synectin(-/-) endothelial cells, mirroring the lower migratory response of these cells to VEGFA(165). These results show that the endocytic pathway of Nrp1 is determined by its ligand and that the trafficking of Nrp1 is essential for its signaling.

Metabolic and evolutionary responses of Clostridium thermocellum to genetic interventions aimed at improving ethanol production.

BACKGROUND: Engineering efforts targeted at increasing ethanol by modifying the central fermentative metabolism of Clostridium thermocellum have been variably successful. Here, we aim to understand this variation by a multifaceted approach including genomic and transcriptomic analysis combined with chemostat cultivation and high solids cellulose fermentation. Three strain lineages comprising 16 strains total were examined. Two strain lineages in which genes involved in pathways leading to organic acids and/or sporulation had been knocked out resulted in four end-strains after adaptive laboratory evolution (ALE). A third strain lineage recapitulated mutations involving adhE that occurred spontaneously in some of the engineered strains. RESULTS: Contrary to lactate dehydrogenase, deleting phosphotransacetylase (pta, acetate) negatively affected steady-state biomass concentration and caused increased extracellular levels of free amino acids and pyruvate, while no increase in ethanol was detected. Adaptive laboratory evolution (ALE) improved growth and shifted elevated levels of amino acids and pyruvate towards ethanol, but not for all strain lineages. Three out of four end-strains produced ethanol at higher yield, and one did not. The occurrence of a mutation in the adhE gene, expanding its nicotinamide-cofactor compatibility, enabled two end-strains to produce more ethanol. A disruption in the hfsB hydrogenase is likely the reason why a third end-strain was able to make more ethanol. RNAseq analysis showed that the distribution of fermentation products was generally not regulated at the transcript level. At 120 g/L cellulose loadings, deletions of spo0A, ldh and pta and adaptive evolution did not negatively influence cellulose solubilization and utilization capabilities. Strains with a disruption in hfsB or a mutation in adhE produced more ethanol, isobutanol and 2,3-butanediol under these conditions and the highest isobutanol and ethanol titers reached were 5.1 and 29.9 g/L, respectively. CONCLUSIONS: Modifications in the organic acid fermentative pathways in Clostridium thermocellum caused an increase in extracellular pyruvate and free amino acids. Adaptive laboratory evolution led to improved growth, and an increase in ethanol yield and production due a mutation in adhE or a disruption in hfsB. Strains with deletions in ldh and pta pathways and subjected to ALE demonstrated undiminished cellulolytic capabilities when cultured on high cellulose loadings.

Development of both type I-B and type II CRISPR/Cas genome editing systems in the cellulolytic bacterium Clostridium thermocellum.

The robust lignocellulose-solubilizing activity of C. thermocellum makes it a top candidate for consolidated bioprocessing for biofuel production. Genetic techniques for C. thermocellum have lagged behind model organisms thus limiting attempts to improve biofuel production. To improve our ability to engineer C. thermocellum, we characterized a native Type I-B and heterologous Type II Clustered Regularly-Interspaced Short Palindromic Repeat (CRISPR)/cas (CRISPR associated) systems. We repurposed the native Type I-B system for genome editing. We tested three thermophilic Cas9 variants (Type II) and found that GeoCas9, isolated from Geobacillus stearothermophilus, is active in C. thermocellum. We employed CRISPR-mediated homology directed repair to introduce a nonsense mutation into pyrF. For both editing systems, homologous recombination between the repair template and the genome appeared to be the limiting step. To overcome this limitation, we tested three novel thermophilic recombinases and demonstrated that exo/beta homologs, isolated from Acidithiobacillus caldus, are functional in C. thermocellum. For the Type I-B system an engineered strain, termed LL1586, yielded 40% genome editing efficiency at the pyrF locus and when recombineering machinery was expressed this increased to 71%. For the Type II GeoCas9 system, 12.5% genome editing efficiency was observed and when recombineering machinery was expressed, this increased to 94%. By combining the thermophilic CRISPR system (either Type I-B or Type II) with the recombinases, we developed a new tool that allows for efficient CRISPR editing. We are now poised to enable CRISPR technologies to better engineer C. thermocellum for both increased lignocellulose degradation and biofuel production.

The redox-sensing protein Rex modulates ethanol production in Thermoanaerobacterium saccharolyticum.

Thermoanaerobacterium saccharolyticum is a thermophilic anaerobe that has been engineered to produce high amounts of ethanol, reaching ~90% theoretical yield at a titer of 70 g/L. Here we report the physiological changes that occur upon deleting the redox-sensing transcriptional regulator Rex in wild type T. saccharolyticum: a single deletion of rex resulted in a two-fold increase in ethanol yield (from 40% to 91% theoretical yield), but the resulting strains grew only about a third as fast as the wild type strain. Deletion of the rex gene also had the effect of increasing expression of alcohol dehydrogenase genes, adhE and adhA. After several serial transfers, the ethanol yield decreased from an average of 91% to 55%, and the growth rates had increased. We performed whole-genome resequencing to identify secondary mutations in the Δrex strains adapted for faster growth. In several cases, secondary mutations had appeared in the adhE gene. Furthermore, in these strains the NADH-linked alcohol dehydrogenase activity was greatly reduced. Complementation studies were done to reintroduce rex into the Δrex strains: reintroducing rex decreased ethanol yield to below wild type levels in the Δrex strain without adhE mutations, but did not change the ethanol yield in the Δrex strain where an adhE mutation occurred.

Transcriptional profiling in coronary artery disease: indications for novel markers of coronary collateralization.

BACKGROUND: The development of collateral circulation plays an important role in protecting tissues from ischemic damage, and its stimulation has emerged as one of principal approaches to therapeutic angiogenesis. Clinical observations have documented substantial differences in the extent of collateralization among patients with coronary artery disease (CAD), with some individuals demonstrating marked abundance and others showing nearly complete absence of these vessels. Recent studies have suggested that circulating monocytes play a major role in collateral growth. The present study was undertaken to determine transcriptional profiles of circulating monocytes in CAD patients with different extents of collateral growth. METHODS AND RESULTS: Monocyte transcriptomes from CAD patients with and without collateral vessels were obtained by use of high-throughput expression profiling. Using a newly developed redundancy-based data mining method, we have identified a set of molecular markers characteristic of a "noncollateralgenic" phenotype. Moreover, we show that these transcriptional abnormalities are independent of the severity of CAD or any other known clinical parameter thought to affect collateral development and correlated with protein expression levels in monocytes and plasma. CONCLUSIONS: Monocyte transcription profiling identifies sets of patients with extensive versus poorly developed collateral circulation. Thus, genetic factors may heavily influence coronary collateral vessel growth in CAD and affect prognosis and response to therapeutic interventions.

Metabolome analysis reveals a role for glyceraldehyde 3-phosphate dehydrogenase in the inhibition of C. thermocellum by ethanol.

BACKGROUND: Clostridium thermocellum is a promising microorganism for conversion of cellulosic biomass to biofuel, without added enzymes; however, the low ethanol titer produced by strains developed thus far is an obstacle to industrial application. RESULTS: Here, we analyzed changes in the relative concentration of intracellular metabolites in response to gradual addition of ethanol to growing cultures. For C. thermocellum, we observed that ethanol tolerance, in experiments with gradual ethanol addition, was twofold higher than previously observed in response to a stepwise increase in the ethanol concentration, and appears to be due to a mechanism other than mutation. As ethanol concentrations increased, we found accumulation of metabolites upstream of the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) reaction and depletion of metabolites downstream of that reaction. This pattern was not observed in the more ethanol-tolerant organism Thermoanaerobacterium saccharolyticum. We hypothesize that the Gapdh enzyme may have different properties in the two organisms. Our hypothesis is supported by enzyme assays showing greater sensitivity of the C. thermocellum enzyme to high levels of NADH, and by the increase in ethanol tolerance and production when the T. saccharolyticum gapdh was expressed in C. thermocellum. CONCLUSIONS: We have demonstrated that a metabolic bottleneck occurs at the GAPDH reaction when the growth of C. thermocellum is inhibited by high levels of ethanol. We then showed that this bottleneck could be relieved by expression of the gapdh gene from T. saccharolyticum. This enzyme is a promising target for future metabolic engineering work.

Synectin-dependent gene expression in endothelial cells.

Synectin (GIPC1), a receptor scaffold protein, has been isolated by our laboratory as a syndecan-4 cytoplasmic domain binding partner that regulates important aspects of cell motility (Gao Y, Li M, Chen W, Simons M. J Cell Physiol 184: 373-379, 2000; Tkachenko E, Elfenbein A, Tirziu D, Simons M. Circ Res 98: 1398-1404, 2006). Moreover, synectin plays a major role in arterial morphogenesis and in growth factor signaling in arterial endothelial cells by regulating Rac1 activity (Chittenden TW, Claes F, Lanahan AA, Autiero M, Palac RT, Tkachenko EV, Elfenbein A, Ruiz de Almodovar C, Dedkov E, Tomanek R, Li W, Westmore M, Singh J, Horowitz A, Mulligan-Kehoe MJ, Moodie KL, Zhuang ZW, Carmeliet P, Simons M. Dev Cell 10: 783-795, 2006). The present study was carried out to characterize changes in synectin-dependent gene expression induced by homozygous disruption of the gene in endothelial cells. Using a combination of suppression subtraction hybridization and high throughput microarray technology, we have identified aberrant biological processes of transcriptional regulation in synectin(-/-) primary endothelial cells including abnormal basal regulation of genes associated with development, cell organization and biogenesis, intracellular tracking, and cell adhesion. Analysis of gene expression following FGF2 treatment demonstrated significant abnormalities in transcription, cytoskeletal organization and biogenesis, and protein modification and transport in synectin(-/-) compared with synectin(+/+) endothelial cells. These results confirm synectin involvement in FGF2-dependent signal transduction and provide insights into synectin-dependent gene expression in the endothelium.

Development of a plasmid-based expression system in Clostridium thermocellum and its use to screen heterologous expression of bifunctional alcohol dehydrogenases (adhEs).

Clostridium thermocellum is a promising candidate for ethanol production from cellulosic biomass, but requires metabolic engineering to improve ethanol yield. A key gene in the ethanol production pathway is the bifunctional aldehyde and alcohol dehydrogenase, adhE. To explore the effects of overexpressing wild-type, mutant, and exogenous adhEs, we developed a new expression plasmid, pDGO144, that exhibited improved transformation efficiency and better gene expression than its predecessor, pDGO-66. This new expression plasmid will allow for many other metabolic engineering and basic research efforts in C. thermocellum. As proof of concept, we used this plasmid to express 12 different adhE genes (both wild type and mutant) from several organisms. Ethanol production varied between clones immediately after transformation, but tended to converge to a single value after several rounds of serial transfer. The previously described mutant C. thermocellum D494G adhE gave the best ethanol production, which is consistent with previously published results.

Verge: a novel vascular early response gene.

Vascular endothelium forms a continuous, semipermeable barrier that regulates the transvascular movement of hormones, macromolecules, and other solutes. Here, we describe a novel immediate early gene that is expressed selectively in vascular endothelial cells, verge (vascular early response gene). Verge protein includes an N-terminal region of approximately 70 amino acids with modest homology (approximately 30% identity) to Apolipoprotein L but is otherwise unique. Verge mRNA and protein are induced selectively in the endothelium of adult vasculature by electrical or chemical seizures. Verge expression appears to be responsive to local tissue conditions, because it is induced in the hemisphere ipsilateral to transient focal cerebral ischemia. In contrast to the transient expression in adult, Verge mRNA and protein are constitutively expressed at high levels in the endothelium of developing tissues (particularly heart) in association with angiogenesis. Verge mRNA is induced in cultured endothelial cells by defined growth factors and hypoxia. Verge protein is dramatically increased by cysteine proteinase inhibitors, suggesting rapid turnover, and is localized to focal regions near the periphery of the cells. Endothelial cell lines that stably express Verge form monolayers that show enhanced permeability in response to activation of protein kinase C by phorbol esters. This response is accompanied by reorganization of the actin cytoskeleton and the formation of paracellular gaps. These studies suggest that Verge functions as a dynamic regulator of endothelial cell signaling and vascular function.

Myosin VI and cardiomyopathy: Left ventricular hypertrophy, fibrosis, and both cardiac and pulmonary vascular endothelial cell defects in the Snell's waltzer mouse.

In mice and humans, loss of myosin VI (Myo6) function results in deafness, and certain Myo6 mutations also result in cardiomyopathies in humans. The current studies have utilized the Snell's waltzer (sv) mouse (a functional null mutation for Myo6) to determine if this mouse also exhibits cardiac defects and thus used to determine the cellular and molecular basis for Myo6-associated heart disease. Myo6 is expressed in mouse heart where it is predominantly expressed in vascular endothelial cells (VECs) based on co-localization with the VEC cell marker CD31. Sv/sv heart mass is significantly greater than that of sv/+ littermates, a result of left ventricle hypertrophy. The left ventricle of the sv/sv exhibits extensive fibrosis, both interstitial and perivascular, based on histologic staining, and immunolocalization of several markers for fibrosis including fibronectin, collagen IV, and the fibroblast marker vimentin. Myo6 is also expressed in lung VECs but not in VECs of intestine, kidney, or liver. Sv/sv lungs exhibit increased periaveolar fibrosis and enlarged air sacs. Electron microscopy of sv/sv cardiac and lung VECs revealed abnormal ultrastructure, including luminal protrusions and increased numbers of cytoplasmic vesicles. Previous studies have shown that loss of function of either Myo6 or its adaptor binding partner synectin/GIPC results in impaired arterial development due to defects in VEGF signaling. However, examination of synectin/GIPC-/- heart revealed no fibrosis or significantly altered VEC ultrastructure, suggesting that the cardiac and lung defects observed in the sv/sv mouse are not due to Myo6 function in arterial development.

Identifying promoters for gene expression in Clostridium thermocellum.

A key tool for metabolic engineering is the ability to express heterologous genes. One obstacle to gene expression in non-model organisms, and especially in relatively uncharacterized bacteria, is the lack of well-characterized promoters. Here we test 17 promoter regions for their ability to drive expression of the reporter genes ß-galactosidase (lacZ) and NADPH-alcohol dehydrogenase (adhB) in Clostridium thermocellum, an important bacterium for the production of cellulosic biofuels. Only three promoters have been commonly used for gene expression in C. thermocellum, gapDH, cbp and eno. Of the new promoters tested, 2638, 2926, 966 and 815 showed reliable expression. The 2638 promoter showed relatively higher activity when driving adhB (compared to lacZ), and the 815 promoter showed relatively higher activity when driving lacZ (compared to adhB).

Physiological roles of pyruvate ferredoxin oxidoreductase and pyruvate formate-lyase in Thermoanaerobacterium saccharolyticum JW/SL-YS485.

BACKGROUND: Thermoanaerobacter saccharolyticum is a thermophilic microorganism that has been engineered to produce ethanol at high titer (30-70 g/L) and greater than 90 % theoretical yield. However, few genes involved in pyruvate to ethanol production pathway have been unambiguously identified. In T. saccharolyticum, the products of six putative pfor gene clusters and one pfl gene may be responsible for the conversion of pyruvate to acetyl-CoA. To gain insights into the physiological roles of PFOR and PFL, we studied the effect of deletions of several genes thought to encode these activities. RESULTS: It was found that pyruvate ferredoxin oxidoreductase enzyme (PFOR) is encoded by the pforA gene and plays a key role in pyruvate dissimilation. We further demonstrated that pyruvate formate-lyase activity (PFL) is encoded by the pfl gene. Although the pfl gene is normally expressed at low levels, it is crucial for biosynthesis in T. saccharolyticum. In pforA deletion strains, pfl expression increased and was able to partially compensate for the loss of PFOR activity. Deletion of both pforA and pfl resulted in a strain that required acetate and formate for growth and produced lactate as the primary fermentation product, achieving 88 % theoretical lactate yield. CONCLUSION: PFOR encoded by Tsac_0046 and PFL encoded by Tsac_0628 are only two routes for converting pyruvate to acetyl-CoA in T. saccharolyticum. The physiological role of PFOR is pyruvate dissimilation, whereas that of PFL is supplying C1 units for biosynthesis.