Engineered Respiro-Fermentative Metabolism for the Production of Biofuels and Biochemicals from Fatty Acid-Rich Feedstocks

Although lignocellulosic sugars have been proposed as the primary feedstock for the biological production of renewable fuels and chemicals, the availability of fatty acid (FA)-rich feedstocks and recent progress in the development of oil-accumulating organisms make FAs an attractive alternative. In addition to their abundance, the metabolism of FAs is very efficient and could support product yields significantly higher than those obtained from lignocellulosic sugars. However, FAs are metabolized only under respiratory conditions, a metabolic mode that does not support the synthesis of fermentation products. In the work reported here we engineered several native and heterologous fermentative pathways to function in Escherichia coli under aerobic conditions, thus creating a respiro-fermentative metabolic mode that enables the efficient synthesis of fuels and chemicals from FAs. Representative biofuels (ethanol and butanol) and biochemicals (acetate, acetone, isopropanol, succinate, and propionate) were chosen as target products to illustrate the feasibility of the proposed platform. The yields of ethanol, acetate, and acetone in the engineered strains exceeded those reported in the literature for their production from sugars, and in the cases of ethanol and acetate they also surpassed the maximum theoretical values that can be achieved from lignocellulosic sugars. Butanol was produced at yields and titers that were between 2- and 3-fold higher than those reported for its production from sugars in previously engineered microorganisms. Moreover, our work demonstrates production of propionate, a compound previously thought to be synthesized only by propionibacteria, in E. coli. Finally, the synthesis of isopropanol and succinate was also demonstrated. The work reported here represents the first effort toward engineering microorganisms for the conversion of FAs to the aforementioned products.Concerns about climate change and the depletion and cost of petroleum resources have ignited interest in the establishment of a bio-based industry (5, 49, 61), and the conceptual model of a biorefinery has emerged (27, 28, 45). Given its abundance in nature, the carbohydrate portion of edible crops such as sugarcane, sugar beet, maize (corn), and sorghum is currently used as the primary feedstock in the biological production of fuels and chemicals (12, 49, 52). Although the use of nonedible lignocellulosic sugars has been proposed as an efficient and sustainable avenue to the aforementioned processes, the availability of fatty acid (FA)-rich feedstocks and recent progress in the development of oil-accumulating organisms make FAs an attractive alternative. Edible oil-rich crops such as rapeseed, sunflower, soybean, and palm are currently available and widely used as feedstocks for chemical conversion to biodiesel (6), while oleaginous algae and nonedible FA-rich crops along with industrial by-products are receiving greater attention as longer-term alternatives. These nonedible FA-rich feedstocks are presently generated in large amounts and can be exploited for the biological production of fuels and chemicals (14, 22, 51, 56, 57). Unfortunately, microbial platforms to enable this are at present almost absent.FAs not only are abundant but also offer several advantages when used for fuel and chemical production. For example, their metabolism to the key intermediate metabolite acetyl coenzyme A (acetyl-CoA) is very efficient, as it results in 100% carbon recovery (Fig. ​(Fig.1).1). Since many fuels and chemicals can be derived from acetyl-CoA, high yields can be realized if FAs are used as the carbon source. In contrast, sugar metabolism generates one molecule of carbon dioxide (or formate) per molecule of acetyl-CoA, limiting the yield of products derived from acetyl-CoA (Fig. ​(Fig.1).1). The product yield advantage of FAs over sugars is also supported by the more highly reduced nature of their carbon atoms. Table ​Table11 provides a comparison of maximum theoretical yields, on both weight and carbon bases, for the production of biofuels and biochemicals from FAs and lignocellulosic sugars. Maximum theoretical yields have been calculated from stoichiometry based on the pathways shown in Fig. ​Fig.11 for the utilization of FAs and glucose, the synthesis of products, the tricarboxylic acid (TCA) cycle, and oxidative phosphorylation. The stoichiometric coefficients were obtained by conducting elemental balances on carbon, hydrogen, and oxygen, and an ATP balance was also included in the analysis. As an example, when production of biofuels (e.g., ethanol and butanol) is considered, utilization of FAs (e.g., palmitic acid [C_16:0]) as a substrate returns product yields 2.7-fold (wt/wt) or 1.4-fold (C/C) higher than those for sugars (calculations are provided for glucose but are equally valid for other lignocellulosic sugars). Although the current prices of feedstocks on a weight basis are comparable (lower than 20¢/pound), the data reported in Fig. S1a in the supplemental material show that the price per carbon for glucose derived from corn is remarkably higher. Regardless of the basis used for calculations (i.e., weight or carbon basis), when maximum theoretical yields and costs of FA and sugar feedstocks are accounted for, the advantages of using FAs are remarkable (see Fig. S1b in the supplemental material).Open in a separate windowFIG. 1.Pathways engineered in E. coli for the conversion of fatty acids to fuels (red) and chemicals (green). Also shown is the catabolism of fatty acids via the β-oxidation pathway (orange) and of glucose through the Embden-Meyerhof-Parnas pathway (blue). Relevant reactions are represented by the names of the genes coding for the enzymes (E. coli genes unless otherwise specified in parentheses as follows: C. acetobutylicum, ca; C. beijerinckii, cb): aceA, isocitrate lyase; aceB, malate synthase A; adc, acetoacetate decarboxylase (ca); ackA, acetate kinase; adh, secondary alcohol dehydrogenase (cb); adhE, acetaldehyde/alcohol dehydrogenase; adhE2, secondary alcohol dehydrogenase (ca); atoA and atoD, acetyl-CoA:acetoacetyl-CoA transferase; atoB, acetyl-CoA acetyltransferase; bcd, butyryl-CoA dehydrogenase (ca); crt, crotonase (ca); etfAB, electron transfer flavoprotein (ca); fadA, 3-ketoacyl-CoA thiolase; fadB, enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase; fadD, acyl-CoA synthetase; fadE, acyl-CoA dehydrogenase; hbd, β-hydroxybutyryl-CoA dehydrogenase (ca); icd, isocitrate dehydrogenase; pta, phosphate acetyltransferase; sdhABCD, succinate dehydrogenase; scpA, methylmalonyl-CoA mutase; scpB, methylmalonyl-CoA decarboxylase; scpC, propionyl-CoA:succinate CoA transferase; sucA, 2-oxoglutarate dehydrogenase; sucB, dihydrolipoyltranssuccinylase; and sucCD, succinyl-CoA synthetase. Abbreviations: 2[H] = NADH = FADH₂ = QH₂ = H₂; P/O, amount of ATP produced per oxygen consumed in the oxidative phosphorylation.

TABLE 1.

Comparison of maximum theoretical yields for the production of biofuels and biochemicals from fatty acids (palmitic acid) and lignocellulosic sugars (glucose)

Structure-activity relationships of chromogranin A in cell adhesion. Identification of an adhesion site for fibroblasts and smooth muscle cells

Previous studies showed that chromogranin A (CgA), a glycoprotein stored and co-released with various hormones by neuroendocrine cells and neurons, can modulate cell adhesion. We have investigated the structure-activity relationships of CgA using fibroblasts and coronary artery smooth muscle cells in adhesion assays. A recombinant CgA fragment 1-78 and a peptide 7-57 containing reduced and alkylated cysteines (Cys(17) and Cys(38)) induced cell adhesion after adsorption onto solid phases at 50-100 nm. Peptides lacking the disulfide loop region, including residues 47-68, 39-59, and 39-68, induced cell adhesion, either bound to solid phases at 200-400 nm or added to the liquid phase at 5-10 microm, whereas peptide 60-68 was inactive, suggesting that residues 47-57 are important for activity. The effect of CgA-(1-78) was blocked by anti-CgA antibodies against epitopes including residues Arg(53), His(54), and Leu(57). Substitutions of residues His(54), Gln(55), and Asn(56) with alanine decreased the cell adhesion activity of peptide 47-68. These results suggest that the region 47-57 (RILSILRHQNL) contains a cell adhesion site and that the disulfide bridge is not necessary for the proadhesive activity. The ability of soluble peptides to elicit proadhesive effects suggests an indirect mechanism. The high sequence conservation and accessibility to antibodies suggest that this region is important for the physiological role of CgA.     

Plasticity of glutamatergic control of striatal acetylcholine release in experimental parkinsonism: opposite changes at group-II metabotropic and NMDA receptors

To investigate whether adaptive changes of glutamatergic transmission underlie dysfunction of the cholinergic system in experimental parkinsonism, the effects of group-II metabotropic glutamate and NMDA receptor ligands on acetylcholine release was studied in striatal slices and synaptosomes obtained from naive rats, 6-hydroxydopamine hemi-lesioned rats and 6-hydroxydopamine hemi-lesioned rats chronically treated with levodopa (L-DOPA) plus benserazide (non-dyskinetic). Group-II metabotropic glutamate receptor agonists LY354740, DCG-IV and L-CCG-I inhibited the electrically-evoked endogenous acetylcholine release from slices, while NMDA facilitated it. LY354740 also inhibited K+-evoked acetylcholine release from synaptosomes. LY354740-induced inhibition was prevented by the group-II metabotropic glutamate receptor antagonist LY341495. In hemi-parkinsonian rats, sensitivity towards LY354740 was reduced while that to NMDA was enhanced in the lesioned (denervated) compared with unlesioned striatum. Moreover, dizocilpine inhibited acetylcholine release in the lesioned compared with unlesioned striatum. Chronic treatment with L-DOPA normalized sensitivity towards glutamatergic agonists. We conclude that striatal dopamine denervation results in plastic changes at group-II metabotropic glutamate and NMDA receptors that may shift glutamatergic control of acetylcholine release towards facilitation. From a clinical perspective, L-DOPA and NMDA antagonists appear effective in counteracting overactivity of striatal cholinergic interneurones associated with Parkinson's disease.     

Synthesis and beta-blocking activity of (R,S)-(E)-oximeethers of 2,3-dihydro-1,8-naphthyridine and 2,3-dihydrothiopyrano[2,3-b]pyridine: identification of beta 3-antagonists

Drugs acting on beta(1)- and beta(2)-adrenergic receptors are widely used for the clinical management of a large number of cardiovascular and respiratory pathologies. In the last decade, the discovery of the third subtype of beta receptors, the beta(3)-adrenoceptor, gave a further pharmacological target for the development of new selective drugs. Initially, a potential therapeutic use of beta(3)-selective agents seemed to be restricted to agonists, for the treatment of metabolic diseases, such as obesity, non-insulin-dependent diabetes, urinary frequency and incontinence. More recently, some interesting theories about a negative role played by the cardio-depressant activity of myocardial beta(3)-adrenoceptors in heart failure, seemed to justify a clinical use of beta(3)-antagonists in the last phases of this cardiac disease. Following the indications deriving from previous experimental work, the beta-antagonist properties of newly-synthesised (R,S)-(E)-oximeethers of 2,3-dihydro-1,8-naphthyridine and of 2,3-dihydrothiopyrano[2,3-b]pyridine were evaluated, in order to identify some useful structure-activity relationships, which might account for selectivity towards the three beta-subtypes and, in particular, the beta(3)-adrenoceptor. Among the various observations regarding possible structure-activity relationships, able to explain the pharmacodynamic patterns of the synthesised compounds on the three subtypes of beta-adrenoceptors, the most significant data derived from the evaluation of the beta(3)-blocking properties of some oximeethers of 1,8-naphthyridine derivatives. In these molecules, although the presence of the large substituents in position 7, such as 4-chloro-phenoxy- or 4-t-butyl-phenoxy groups determined a dramatic decline in both the beta(1)- and beta(2)-activities, this structural characteristic had a modest influence on the beta(3)-affinity, which was only slightly lower. Hence, this last structural requirement of oximeethers of 1,8-naphthyridine derivatives seems to represent a useful expedient to induce an appreciable selectivity towards the beta(3)-receptor, through a markedly negative effect on the beta(1)- and beta(2)-activities rather than an increase in the beta(3)-affinity.     

Surface plamon resonance imaging of DNA based biosensors for potential applications in food analysis

The adsorption processes of oligonucleotides immobilised onto suitable photolithographic patterned gold substrates have been investigated in aqueous buffer solution by using a home made surface plasmon resonance (SPR) imaging equipment. A rapid self-assembled method for the construction of DNA chips to be used in SPR imaging experiments have been followed. The immobilised DNA molecules (probes) adopted in our SPR experiments anchored to a gold surface via thiol group were 5'thiol-modified containing a (CH(2))(15) tail. The hybridisation processes taking place with its complementary sequence have been observed and characterized by monitoring phenomena by a SPR imaging system. The two analysed oligonucleotides (probes and target) are of interest in plant gene biotechnological application and differing for the presence at the 5'-end of a poly T16 spacer. Dynamic investigation of smallest changes in SPR imaging pictures performed in liquid phase in the presence of DNA complementary probes have been performed. Quantitative information in terms of threshold of sensitivity has been extracted by using a specific images treatment.     

Striatal dopamine-NMDA receptor interactions in the modulation of glutamate release in the substantia nigra pars reticulata in vivo: opposite role for D1 and D2 receptors

To investigate the antioxidative capacities of oligodendrocytes, rat brain cultures enriched for oligodendroglial cells were prepared and characterized. These cultures contained predominantly oligodendroglial cells as determined by immunocytochemical staining for the markers galactocerebroside and myelin basic protein. If oligodendroglial cultures were exposed to exogenous hydrogen peroxide (100 micro m), the peroxide disappeared from the incubation medium following first order kinetics with a half-time of approximately 18 min. Normalization of the disposal rate to the protein content of the cultures by calculation of the specific hydrogen peroxide detoxification rate constant revealed that the cells in oligodendroglial cultures have a 60% to 120% higher specific capacity to dispose of hydrogen peroxide than cultures enriched for astroglial cells, microglial cells or neurones. Oligodendroglial cultures contained specific activities of 133.5 +/- 30.4 nmol x min(-1) x mg protein(-1) and 27.5 +/- 5.4 nmol x min(-1) x mg protein(-1) of glutathione peroxidase and glutathione reductase, respectively. The specific rate constant of catalase in these cultures was 1.61 +/- 0.54 min(-1) x mg protein(-1). Comparison with data obtained by identical methods for cultures of astroglial cells, microglial cells and neurones revealed that all three of the enzymes which are involved in hydrogen peroxide disposal were present in oligodendroglial cultures in the highest specific activities. These results demonstrate that oligodendroglial cells in culture have a prominent machinery for the disposal of hydrogen peroxide, which is likely to support the protection of these cells in brain against peroxides when produced by these or by surrounding brain cells.     

Histopathology, ultrastructure and immunohistochemistry of Coregonus lavaretus hearts naturally infected with Ichthyocotylurus erraticus (Trematoda)

Histopathological, ultrastructural and immunohistochemical investigations were conducted on 26 specimens of powan Coregonus lavaretus (L.) from Loch Lomond (Scotland). The hearts of all 26 powan (15 females and 11 males) investigated harboured metacercariae of the digenean trematode Ichthyocotylurus erraticus (Rudolphi, 1809). The vast majority of metacercariae were located either singly or as an aggregation of white cysts on the surface of the bulbus arteriosus. The intensity of infection ranged from 2 to 200 larvae heart(-1), although the number of metacercariae found on male powan did not exceed 13. Histochemically, the parasite cyst wall gave a strong positive reaction with periodic acid schiff (PAS) and a faint positive signal with Azan-Mallory stain. All the metacercariae cysts were embedded in a granulomatous proliferation of heart epicardium tissue, forming a reactive fibroconnective capsule around the parasite. The capsule enclosing the parasite (produced by the host's reaction to the parasite) measured 13.57 to 90.20 microm (37.43 +/- 3.56) in thickness. Within the capsule wall, eosinophilic granular cells (EGCs), granulocytes, melanocytes and, in some instances, partially degenerated or vacuolated epithelioid cells were observed in close proximity to the cyst wall. Pigment-bearing macrophages were scattered throughout the granulomatous host-tissue reaction and as macrophage aggregates (MAs) within the capsules surrounding parasites. Immunohistochemical tests were applied to infected heart sections using 12 different antisera. Nerve fibres immunoreactive to bombesin, substance P (SP), and atrial natriuretic peptide (ANP) antisera were observed in close proximity to the parasite larvae. The presence of a serotonin-like substance was also observed within host immune-cells surrounding trematode cysts. Large cells of the epicardium were found to be immunoreactive to met-enkephalin and vasoactive intestinal peptide (VIP) antisera but not immunoreactive to anti-protein gene-product 9.5 (PGP9.5) sera.     

Sulphated glycoconjugates are powerful inhibitors of spermatozoa binding to the vitelline envelope in amphibian eggs

BACKGROUND INFORMATION: In amphibians, the role of sulphated glycans has not been determined in spermatozoa-egg interaction, although they are known to be involved in other systems. In previous studies, it was found that, in Discoglossus pictus, a VE (vitelline envelope) glycoprotein of 63 kDa exhibits high homology to Xenopus laevis gp69/gp64 and to ZP2 of mammals. gp63 and a glycoprotein of 75 kDa are both capable of binding the spermatozoa in in vitro assays and, having similar peptide maps and different glycosylation, are probably two glycoforms of the same protein. RESULTS: In the present study, binding assays performed by treating dejellied eggs with metaperiodate suggest that hydroxy groups of sugars are not directly involved in spermatozoa-vitelline envelope binding. Competition assays between dejellied eggs and spermatozoa preincubated with dextran, dextran sulphate or fucoidan indicated that sulphated oligosaccharides have an inhibitory effect on spermatozoa binding. In similar competition assays, Le(x) (Lewis(x)) trisaccharide 3'-sulphate inhibited spermatozoa binding to VE in contrast with 3'-sialyl-Le(x) tetrasaccharide. Assays performed with gp75- or gp63-coated beads and spermatozoa treated with fucoidan or dextran sulphate indicated that sulphated oligosaccharides competitively inhibit spermatozoa binding to gp75-coated beads, yet not to gp63-coated beads. Finally, solubilized VE digested with N-glycosidase F retains the inhibitory activity in spermatozoa-VE binding assays in contrast with VE treated with alpha-N-acetylgalactosaminidase. CONCLUSION: It was concluded that VE sulphate groups are involved in spermatozoa binding. These groups are present in gp75 glycoconjugates and are probably located in O-linked glycoconjugates.     

Post-ischemic recovery of acetylcholine release in vitro: influence of different excitatory amino acid receptor subtype antagonists

The release of endogenous acetylcholine was measured in electrically (5–20 Hz) stimulated guinea pig cerebral cortex and caudate nucleus slices under ischemic (hypoxic and glucose-free) conditions. Ischemia reduced acetylcholine release by 40–90%; the inhibition depended on the duration of ischemia (10–30 min) while the extent of post-ischemic recovery was inversely related to it. Caudate nucleus slices displayed a higher sensitivity to ischemia than did cortical slices. To test the effects of excitatory amino acid receptor antagonists on the ischemia-induced reduction of acetylcholine release and on its post-ischemic recovery, the following drugs were used: 5-methyl-10,11-dihydro-5-H-dibenzo-[a,b]-cyclohepten-5,10-imine (MK-801, a blocker of the N-methyl-D-aspartate [NMDA] receptor-linked channel), 7-chloro-kynurenic acid (7-Cl-KYN) and (E)-3-[2(phenylcarbamoyl)ethenyl]-4,6-dichloroindole-2-carboxylic acid sodium salt (GV150526A, blockers of the glycine site of the NMDA receptor), eliprodil, (an antagonist at the polyamine site of the NMDA receptor), and 6-cyano- 7-nitro-quinoxalin-2,3-dione (CNQX, a D,L--amino-3-hydroxy-5-methyl-4-isoxalone propionic acid [AMPA] receptor antagonist). These did not modify the time-course and the extent of ischemia-induced inhibition but improved post-ischemic recovery in a concentration dependent manner. GV 150526A and CNQX appeared to be more effective in the cerebral cortex. Only eliprodil was devoid of any effect in both areas.

The evaluation of acetylcholine release from brain slices represents a suitable in vitro model to quantify the effectiveness of drugs in favouring recovery from the cholinergic presynaptic failure induced by ischemic conditions. The different effects of the excitatory amino acid receptor antagonists cited above, depending on the brain areas considered and the receptor subtypes involved, may be of interest in view of their therapeutic potential.     

A Feasibility Study with Image-Based Rendered Virtual Reality in Patients with Mild Cognitive Impairment and Dementia

Virtual Reality (VR) has emerged as a promising tool in many domains of therapy and rehabilitation, and has recently attracted the attention of researchers and clinicians working with elderly people with MCI, Alzheimer’s disease and related disorders. Here we present a study testing the feasibility of using highly realistic image-based rendered VR with patients with MCI and dementia. We designed an attentional task to train selective and sustained attention, and we tested a VR and a paper version of this task in a single-session within-subjects design. Results showed that participants with MCI and dementia reported to be highly satisfied and interested in the task, and they reported high feelings of security, low discomfort, anxiety and fatigue. In addition, participants reported a preference for the VR condition compared to the paper condition, even if the task was more difficult. Interestingly, apathetic participants showed a preference for the VR condition stronger than that of non-apathetic participants. These findings suggest that VR-based training can be considered as an interesting tool to improve adherence to cognitive training in elderly people with cognitive impairment.