Co-production of hydrogen and ethanol from glucose in Escherichia coli by activation of pentose-phosphate pathway through deletion of phosphoglucose isomerase (pgi) and overexpression of glucose-6-phosphate dehydrogenase (zwf) and 6-phosphogluconate dehydrogenase (gnd)

Background: Biologically, hydrogen (H-2) can be produced through dark fermentation and photofermentation. Dark fermentation is fast in rate and simple in reactor design, but H-2 production yield is unsatisfactorily low as < 4 mol H-2/ mol glucose. To address this challenge, simultaneous production of H-2 and ethanol has been suggested. Co-production of ethanol andH(2) requires enhanced formation of NAD(P) H during catabolism of glucose, which can be accomplished by diversion of glycolytic flux from the Embden-Meyerh-of-Parnas (EMP) pathway to the pentose-phosphate (PP) pathway in Escherichia coli. However, the disruption of pgi (phosphoglucose isomerase) for complete diversion of carbon flux to the PP pathway made E. coli unable to grow on glucose under anaerobic condition. Results: Here, we demonstrate that, when glucose-6-phosphate dehydrogenase (Zwf) and 6-phosphogluconate dehydrogenase (Gnd), two major enzymes of the PP pathway, are homologously overexpressed, E. coli.pgi can recover its anaerobic growth capability on glucose. Further, with additional deletions of Delta hycA,Delta hyaAB,Delta hybBC,Delta ldhA, and Delta frdAB, the recombinant.pgi mutant could produce 1.69 mol H-2 and 1.50 mol ethanol from 1 mol glucose. However, acetate was produced at 0.18 mol mol(-1) glucose, indicating that some carbon is metabolized through the Entner-Doudoroff (ED) pathway. To further improve the flux via the PP pathway, heterologous zwf and gnd from Leuconostoc mesenteroides and Gluconobacter oxydans, respectively, which are less inhibited by NADPH, were overexpressed. The new recombinant produced more ethanol at 1.62 mol mol(-1) glucose along with 1.74 mol H-2 mol(-1) glucose, which are close to the theoretically maximal yields, 1.67 mol mol(-1) each for ethanol andH(2). However, the attempt to delete the ED pathway in the.pgi mutant to operate the PP pathway as the sole glycolytic route, was unsuccessful. Conclusions: By deletion of pgi and overexpression of heterologous zwf and gnd in E. coli Delta hycA Delta hyaAB Delta hybBC Delta ldhA Delta frdAB, two important biofuels, ethanol andH(2), could be successfully co-produced at high yields close to their theoretical maximums. The strains developed in this study should be applicable for the production of other biofuels and biochemicals, which requires supply of excessive reducing power under anaerobic conditions

Co-production of hydrogen and ethanol from glucose by modification of glycolytic pathways in Escherichia coli - from Embden-Meyerhof-Parnas pathway to pentose phosphate pathway

Hydrogen (H-2) production from glucose by dark fermentation suffers from the low yield. As a solution to this problem, co-production of H-2 and ethanol, both of which are good biofuels, has been suggested. To this end, using Escherichia coli, activation of pentose phosphate (PP) pathway, which can generate more NADPH than the Embden-Meyhof-Parnas (EMP) pathway, was attempted. Overexpression of two key enzymes in the branch nodes of the glycolytic pathway, Zwf and Gnd, significantly improved the co-production of H-2 and ethanol with concomitant reduction of pyruvate secretion. Gene expression analysis and metabolic flux analysis (MFA) showed that, upon overexpression of Zwf and Gnd, glucose assimilation through the PP pathway, compared with that of the EMP or Entner-Doudoroff (ED) pathway, was greatly enhanced. The maximum co-production yields were 1.32 mol H-2 mol(-1) glucose and 1.38 mol ethanol mol(-1) glucose, respectively. It is noteworthy that the glycolysis and the amount of NAD(P)H formed under anaerobic conditions could be altered by modifying (the activity of) several key enzymes. Our strategy could be applied for the development of industrial strains for biological production of reduced chemicals and biofuels which suffers from lack of reduced co-factors.ope

Improvement of carbon monoxide-dependent hydrogen production activity in Citrobacter amalonaticus Y19 by over-expressing the CO-sensing transcriptional activator, CooA

Citrobacter amalonaticus Y19 (Y19) can produce hydrogen (H-2) from oxidation of carbon monoxide (CO) via the water-gas shift reaction. The reaction is catalyzed by two enzymes, carbon monoxide dehydrogenase (CODH) and carbon monoxide-dependent hydrogenase (CO-Hyd). The contig genome sequencing of Y19 exhibited the presence of unique CO oxidizing gene clusters encoding CODH (cooFS), CO-Hyd (cooMKLXUH) and a putative CO-responsive transcriptional activator (cooA). To improve CO-dependent H-2 production activity, we developed recombinant Y19 by homologously over-expressing cooA. The overexpression of cooA improved the whole-cell CO-dependent H-2 production activity (3.4-fold), and enzyme activities of CODH (5.3-fold) and CO-Hyd (1.2-fold). Furthermore, quantitative PCR analysis revealed a significant increase in the transcription of the genes located in CODH and CO-Hyd operons of recombinant Y19. The high CO-dependent H-2 production activity of the recombinant C. amalonaticus was stably maintained during repeated exposure to CO

Production of (R)-mandelic acid from styrene, L-phenylalanine, glycerol, or glucose via cascade biotransformations

10.1186/s40643-021-00374-6Bioresources and Bioprocessing812

Metabolic engineering of Klebsiella pneumoniae J2B for co-production of 3-hydroxypropionic acid and 1,3-propanediol from glycerol: Reduction of acetate and other by-products

Production of 3-hydroxypropionic acid (3-HP) or 1,3-propanediol (1,3-PDO) production from glycerol is challenging due to the problems associated with cofactor regeneration, coenzyme B12 synthesis, and the instability of pathway enzymes. To address these complications, simultaneous production of 3-HP and 1,3-PDO, instead of individual production of each compound, was attempted. With over-expression of an aldehyde dehydrogenase, recombinant Klebsiella pneumoniae could co-produce 3-HP and 1,3-PDO successfully. However, the production level was unsatisfactory due to excessive accumulation of many by-products, especially acetate. To reduce acetate production, we attempted; (i) reduction of glycerol assimilation through the glycolytic pathway, (ii) increase of glycerol flow towards co-production, and (iii) variation of aeration rate. These efforts were partially beneficial in reducing acetate and improving co-production: 21 g/L of 1,3-PDO and 43 g/L of 3-HP were obtained. Excessive acetate (&gt;150 mM) was still produced at the end of bioreactor runs, and limited co-production efficiency

Evaluation of Newly Isolated Klebsiella pneumoniae Strains for the Co-Production of 3-hydroxypropionic acid and 1,3-propanediol from Glycerol

Co-production of 3-hydroxypropionic acid (3-HP) and 1,3-propanediol (1,3-PDO) was suggested as an innovativestrategy to overcome several limitations occurring in the single production of 3-HP from glycerol. In this study, twonew isolates of Klebsiella pneumoniae, which produce less lipopolysaccharide (LPS) thus considered less pathogenicthan K. pneumoniae DSM 2026, were compared and evaluated for their potential for the co-production of 3-HP and 1,3-PDO. The newly isolated strains showed significantly faster sedimentation rate than DSM, which should be beneficial for downstream processing. Analysis of genome sequences of the isolates confirmed the presence of all genes necessary for glycerol assimilation, 1,3-PDO production and biosynthesis of coenzyme B12. Co-production yield was highest under anaerobic condition while cell growth was highest under aerobic condition. Both strains showed similarly good performance for the co-production although J2B gave the slightly higher co-production yield of 0.80 mol/mol than GSC021 (0.75 mol/mol). The evaluation of the newly developed strains presented here should be useful in designing similar evaluation experiments for other microorganisms

Legal Framework for Maritime Surveillance

Maritime border control is one of the European maritime domains to implement integrated maritime policy and European Union Maritime Security Strategy (MSS). According to the EU Regulation (EU) No 656/2014, maritime border control is not limited to the detection of attempts at unauthorized border crossings, but extends to the arrangements intended to address situations such as Search and Rescue (SAR). Hence, we present a legal framework for maritime surveillance concerning both border control and SAR operations in the context of operational cooperation coordinated by Frontex

Cloning and functional expression of Citrobacter amalonaticus Y19 carbon monoxide dehydrogenase in Escherichia coli

Carbon monoxide (CO) is highly toxic but is an abundant carbon source that can be utilized for the production of hydrogen (H-2). CO-dependent H-2 production is catalyzed by a unique enzyme complex composed of carbon monoxide dehydrogenase (CODH) and CO-dependent hydrogenase (CO-H(2)ase), both of which contain metal cluster(s). In this study, CODH and the required maturation proteins from the novel facultative anaerobic bacterium Citrobacter amalonaticus Y19 were cloned and heterologously expressed in Escherichia coli. For functional expression of CODH in E. coli, only CooF (ferredoxin-like protein) and CooS (CODH), not the maturation proteins, were needed. The recombinant E. coli BL21(DE3)-cooFS showed a 3.5-fold higher specific CODH activity (4.9 U mg protein(-1)) compared to C. amalonaticus Y19 (Y19) (1.4 U mg protein(-1)). Purified heterologous CODH from the soluble cell-free extract of the recombinant E. coli showed a specific activity of 170.6 U mg protein(-1). Recombinant E. coli harboring Y19 CODH and maturation proteins did not produce H-2 from CO, suggesting that the native hydrogenases present in E. coli could not substitute the Y19 CO-H(2)ase for CO-dependent H-2 production

Characterization of 1,3-propanediol oxidoreductase (DhaT) from Klebsiella pneumoniae J2B

1,3-propanediol oxidoreductase (DhaT) of Klebsiella pneumoniae converts 3-hydroxypropionaldehyde (3-HPA) to 1,3-propanediol (1,3-PD) during microbial production of 1,3-PD from glycerol. In this study, DhaT from newly isolated K. pneumoniae J2B was cloned, expressed, purified, and studied for its kinetic properties. It showed, on its physiological substrate 3-HPA, higher activity than similar aldehydes such as acetaldehyde, propionaldehyde and butyraldehyde. The turnover numbers (k (cat) , 1/s) were estimated as 59.4 for the forward reaction (3-HPA to 1,3-PD at pH 7.0) and 10.0 for the reverse reaction (1,3-PD to 3-HPA at pH 9.0). The Michaelis constants (K (m) , mM) were 0.77 (for 3-HPA) and 0.03 (for NADH) for the forward reaction (at pH 7.0), and 7.44 (for 1,3-PD) and 0.23 (for NAD(+)) for the reverse reaction (at pH 9.0). Between these forward and reverse reactions, the optimum temperature and pH were significantly different (37A degrees C and 7.0 vs. 55A degrees C and 9.0, respectively). These results indicate that, under physiological conditions, DhaT mostly catalyzes the forward reaction. The enzyme was seriously inhibited by heavy metal ions such as Ag+ and Hg2+. DhaT was highly unstable when incubated with its own substrate 3-HPA, indicating the necessity of enhancing its stability for improved 1,3-PD production from glycerol