Accumulation of phycocyanin in heterotrophic and mixotrophic cultures of the acidophilic red alga Galdieria sulphuraria

The relationship between light intensity, nitrogen availability and pigmentation was investigated in mixotrophic and heterotrophic cultures of the unicellular red alga Galdieria sulphuraria 074G, a potential host for production of the blue pigment, phycocyanin (PC). During the exponential growth phase of batch cultures, G. sulphuraria 074G contained 2–4 mg phycocyanin per g dry weight. In carbon-limited and nitrogen-sufficient batch cultures grown in darkness, this value increased to 8–12 mg g⁻¹ dry weight during the stationary phase, whereas the phycocyanin content in nitrogen-deficient cells decreased to values below 1 mg g⁻¹ dry weight during stationary phase. Light intensities between 0 and 100 μmol photons m⁻² s⁻¹ had no influence on phycocyanin accumulation in mixotrophic cultures grown on glucose or fructose, while light stimulated phycocyanin synthesis in cultures grown on glycerol, in which the phycocyanin content in stationary phase was increased from 10 mg g⁻¹ dry weight in darkness to 20 mg g⁻¹ dry weight at a light intensity of 80 μmol photons m⁻² s⁻¹. At higher light intensities, less phycocyanin accumulated than at lower intensities, irrespective of the carbon substrate used. In carbon-limited continuous flow cultures grown on glucose or glycerol at a dilution rate of 0.63 day⁻¹, corresponding to 50% of the maximum specific growth rate, the highest steady-state phycocyanin content of 15–28 mg g⁻¹ dry weight was found at 65 μmol photons m⁻² s⁻¹. In contrast to the apparent glucose repression of light-induced PC synthesis observed in batch cultures, no glucose repression of the light stimulation was observed in continuous flow cultures because the glucose concentration in the culture supernatant always remained at limiting levels. Despite the fact that G. sulphuraria 074G contains less phycocyanin than some other microalgae and cyanobacteria, the ability of G. sulphuraria 074G to grow and synthesize phycocyanin in heterotrophic or mixotrophic cultures makes it an interesting alternative to the cyanobacterium, Spirulina platensis presently used for synthesis of phycocyanin.     

Influence of specific growth rate over the secretory expression of recombinant potato carboxypeptidase inhibitor in fed-batch cultures of Escherichia coli

A high cell density cultivation protocol was developed for the secretory production of potato carboxypeptidase inhibitor (PCI) in Escherichia coli. The strain BW25113 (pIMAM3) was cultured in fed-batch mode employing minimal media and an exponential feed profile where the specific growth rate was fixed by limitation of the fed carbon source (glycerol). Plasmid loss rates were found to be proportional to the specific growth rate. Distribution of PCI along the cell compartments and the culture media was also dependent on the fixed growth rate. When specific growth rate was kept at μ = 0.10 h⁻¹, 1.4 g PCI L⁻¹ were obtained when adding the product present in periplasmic extracts and supernatant fractions, with a 50% of the total expressed protein recovered from the extracellular medium. This constituted a 1.2-fold increase compared to growth at μ = 0.15 h⁻¹, and 2.0-fold compared to μ = 0.25 h⁻¹. Last, a cell permeabilization treatment with Triton X-100 and glycine was employed to direct most of the product to the culture media, achieving over 81% of extracellular PCI. Overall, our results point out that production yields of secretory proteins in fed-batch cultures of E. coli can be improved by means of process variables, with applications to the production of small disulfide-bridged proteins. Overall, our results point out that control of the specific growth rate is a successful strategy to improve the production yields of secretory expression in fed-batch cultures of E. coli, with applications to the production of small disulfide-bridged proteins.     

Modulation of guanosine 5′-diphosphate-d-mannose metabolism in recombinant Escherichia coli for production of guanosine 5′-diphosphate-l-fucose

Guanosine 5’-diphosphate (GDP)-l-fucose, an activated form of a nucleotide sugar, plays an important role in a wide range of biological functions. In this study, the enhancement of GDP-l-fucose production was attempted by supplementation of mannose, which is a potentially better carbon source to be converted into GDP-l-fucose than glucose, and combinatorial overexpression of the genes involved in the biosynthesis of GDP-d-mannose, a precursor of GDP-l-fucose. Supply of a mannose and glucose led to a 1.3-fold-increase in GDP-l-fucose concentration (52.5 ± 0.8 mg l^?1) in a fed-batch fermentation of recombinant E. coli BL21star(DE3) overexpressing the gmd and wcaG genes, compared with the case using glucose as a sole carbon source. A maximum GDP-l-fucose concentration of 170.3 ± 2.3 mg l^?1, corresponding to a 4.4-fold enhancement compared with the control strain overexpressing gmd and wcaG genes only, was achieved in a glucose-limited fed-batch fermentation of a recombinant E. coli BL21star(DE3) strain overexpressing manB, manC, gmd and wcaG genes. Further improvement of GDP-l-fucose production was not obtained by additional overexpression of the manA gene.     

Enhanced anaerobic succinic acid production by Escherichia coli NZN111 aerobically grown on gluconeogenic carbon sources

Escherichia coli strain NZN111, a pflB and ldhA double mutant of E. coli W1485, is considered a candidate of succinic acid producer. However, it is reported that this strain fails to ferment glucose anaerobically. In this study, it was demonstrated that when a gluconeogenic carbon source was used to replace glucose in aerobic culture, the NZN111 cells restored the ability to ferment glucose in the subsequent anaerobic culture with succinic acid as the major product even though no further genetic manipulation had been carried out. Activities of enzymes including phosphoenolpyruvate (PEP) carboxykinase, PEP carboxylase, isocitrate lyase, malate dehydrogenase, malic enzyme, and pyruvate kinase in the NZN111 cells aerobically grown on different carbon sources were measured, and enhanced anaplerotic and oxaloacetate-reducing activities were revealed. Furthermore, supply of MgCO₃ or NaHCO₃ greatly improved succinate production by the malate-grown NZN111 cells. At the same time, pyruvic acid production was significantly reduced. When the malate-grown cells were anaerobically cultured in a salt medium with high pH buffering capacity, succinic acid was produced at a specific productivity of 308 mg/(g DCW h) with a molar yield of 1.31 mol succinic acid/mol glucose.     

Production of glucaric acid from myo-inositol in engineered Pichia pastoris

A potential myo-inositol oxygenase (ppMIOX) was identified as a functional enzyme and a glucaric acid synthetic pathway was firstly constructed in Pichia pastoris. Coexpression of the native ppMIOX and the urinate dehydrogenase (Udh) from Pseudomonas putida KT2440 led to obvious accumulation of glucaric acid (90.46 ± 0.04 mg/L) from myo-inositol whereas no glucaric acid was detected from glucose. In comparison, coexpression of the heterologous mouse MIOX (mMIOX) and Udh resulted in higher titers of glucaric acid from glucose and myo-inositol, 107.19 ± 11.91 mg/L and 785.4 ± 1.41 mg/L, respectively. By applying a fusion expression strategy with flexible peptides, the mMIOX specific activity and the glucaric acid concentration were significantly increased. Using glucose and myo-inositol as carbon substrates, the production of glucaric acid was substantially enhanced to 6.61 ± 0.30 g/L in fed-batch cultures. To the best of our knowledge, this is the highest reported value to date.     

Expression,characterization and mutagenesis of an FAD-dependent glucose dehydrogenase from Aspergillus terreus

An FAD-dependent glucose dehydrogenase (FAD-GDH) from Aspergillus terreus NIH2624 was expressed in Escherichia coli with a yield of 228 ± 16 U/L of culture. Co-expression with chaperones DnaK/DnaJ/GrpE and osmotic stress induced by simple carbon sources enhanced productivity significantly, improving the yield to 23883 ± 563 U/L after optimization. FAD-GDH was purified in two steps with the specific activity of 604 U/mg. Using d-glucose as substrate, the optimal pH and temperature for FAD-GDH were determined to be 7.5 and 50 °C, respectively. Activity was stable across the pH range 3.5–9.0, and the half-life was 52 min at 42 °C. K_m and V_max were calculated as 86.7 ± 5.3 mM and 928 ± 35 U/mg, and the molecular weight was approximately 65.6 kDa based on size exclusion chromatography, indicating a monomeric structure. The 3D structure of FAD-GDH was simulated by homology modelling using the structure of A. niger glucose oxidase (GOD) as template. From the model, His551, His508, Asn506 and Arg504 were identified as key residues, and their importance was verified by site-directed mutagenesis. Furthermore, three additional mutants (Arg84Ala, Tyr340Phe and Tyr406Phe) were generated and all exhibited a higher degree of substrate specificity than the native enzyme. These results extend our understanding of the structure and function of FAD-GDH, and could assist potential commercial applications.     

Roles of extracellular lactose hydrolysis in cellulase production by Trichoderma reesei Rut C30 using lactose as inducing substrate

Lactose, an inexpensive, soluble substrate, offers reasonably good induction for cellulase production by Trichoderma reesei. The fungus does not uptake lactose directly. Lactose is hydrolyzed to extracellular glucose and galactose for subsequent ingestion. The roles of this extracellular hydrolysis step were investigated in this study. Batch and continuous cultures were grown on the following substrates: lactose, lactose–glycerol mixtures, glucose, galactose, and glucose–galactose mixtures. Cell growth, substrate consumption, lactose hydrolysis, and lactase and cellulase production were followed and modeled. Cells grew much faster on glucose than on galactose, but with comparable cell yields. Glucose (at >0.3 g/L) repressed the galactose consumption. Cellulase synthesis was growth-independent while lactase synthesis was growth-dependent, except at D < ∼0.065 h⁻¹ where a basal level lactase production was observed. For cellulase production the optimal D was 0.055–0.065 h⁻¹ where the enzyme activity and productivity were both near maxima. The model suggested that lactase synthesis was subject to weak galactose repression. As the galactose concentration increased at high D (>0.1 h⁻¹), lactase synthesis became repressed. The insufficient lactase synthesis limited the lactose hydrolysis rate. Extracellular lactose hydrolysis was concluded to be the rate-limiting step for growth of T. reesei Rut C30 on lactose.     

Quantitative analysis on inactivation and reactivation of recombinant glycerol dehydratase from Klebsiella pneumoniae XJPD-Li

The genes encoding glycerol dehydratase were cloned and characterized by genomic DNA from Klebsiella pneumoniae XJPD-Li, and the assigned accession number EF634063 was available from the GenBank database. The DNA sequence analysis showed that the clone included three ORFs (dhaB, dhaC and dhaE, encoding α, β and γ subunit of glycerol dehydratase, respectively). Among three subunits of glycerol dehydratase, amino acid residues H₁₃, S₁₉₃, N₃₅₉, E₄₀₇, and M₅₁₅ of α subunit, N₄₇, L₁₅₀, V₁₈₉ of β subunit are different with what had been reported. Subsequently, the expression vector was constructed and transformed into E. coli BL21, and the colony carried genes of glycerol dehydratase were selected. SDS-PAGE examination showed that the three subunits were well expressed. The specific activity of recombined glycerol dehydratase reached to 0.299 U mg^?1, which was about 3 times comparing with that of the wild strain. The research also displayed that both glycerol and O₂ could inactive the glycerol dehydratase expressed in E. coli quickly in 10 min. The inactivated glycerol dehydratase could be effectively reactivated under the system as follows: the concentration of ATP, Mg²⁺ and coenzyme B₁₂ were 50 mM, 10 mM and 3 μM, respectively, when the ratio (W/W) of glycerol dehydratase to reactivation factor was 4:1. The O₂-inactivated and glycerol-inactivated dehydratase could be reactivated to 97.3% and 98.9% of initial activity in 10 min in above-mentioned conditions, respectively. The reactivation factor together with ATP was considered as the “ON/OFF” reactivating condition.     

Cloning and characterization of a recombinant family 5 endoglucanase from Bacillus licheniformis strain B-41361

The gene encoding a family 5 endoglucanase, cel5A, was cloned from the moderate thermophile Bacillus licheniformis strain B-41361. The primary structure of the translated cel5A gene predicts a 49 amino acid putative secretion signal and a 485 residue endoglucanase consisting of an N-terminal family 5 catalytic domain and C-terminal family 3 cellulose binding domain. The endoglucanase portion of the gene was expressed in Escherichia coli, but soluble activity in cell lysates was due to a truncated enzyme with an apparent mass of 42 kDa, the equivalent of the predicted catalytic domain. Insoluble protein renatured from inclusion bodies was protected against truncation, yielding an active holoenzyme (rCel5A) with apparent mass of 62 kDa. The recombinant rCel5A was optimally active at 65 °C and pH 6.0, but retained only 10% activity after 1 h incubation at this temperature. At 55 °C, rCel5A had a broad pH range for activity and stability, with greater than 75% relative activity from pH 4.5–7.0, and retaining greater than 80% relativity activity across the range pH 4.5–8.0 following 1 h incubation at 55 °C. It readily hydrolyzed pNPC, carboxymethylcellulose, barley β-glucan, and lichenan, but despite binding to cellulose, had only weak activity against avicel. Hydrolysis products from soluble polysaccharides included glucose, cellobiose, cellotriose, and cellotetraose. The catalytic properties, broad pH range and thermostability of the recombinant B. licheniformis endoglucanase may prove suitable for industrial applications.     

Osmoregulation in Dunaliella,Part II: Photosynthesis and starch contribute carbon for glycerol synthesis during a salt stress in Dunaliella tertiolecta

In response to an osmotic stress, Dunaliella tertiolecta osmoregulates by metabolizing intracellular glycerol as compatible solute. Upon the application of a salt stress to 0.17 M or 0.7 M NaCl grown D. tertiolecta cells, rates of total glycerol synthesis were substantially higher than that arising from photosynthetic ¹⁴CO₂ fixation into glycerol. The source of this extra carbon is the reserve starch pool. The contribution of carbon from the starch breakdown to glycerol synthesis was estimated from the difference between the total glycerol synthesized and that arising from ¹⁴CO₂ fixation. The maximum observed flux of carbon from ¹⁴CO₂ to glycerol from photosynthesis was of the order of 15–20 μmol ¹⁴C-glycerol mg⁻¹ Chl h⁻¹, whereas the total glycerol synthesis reached about 70 μmol glycerol mg⁻¹ Chl h⁻¹. The contribution of products of starch breakdown to glycerol synthesis increased progressively with increasing salt stress. In light, contrary to prevailing assumptions, both the photosynthesis and the starch breakdown contribute carbon to glycerol biosynthesis. The relative contributions of these two processes in the light, while cells were actively photosynthesizing, depended on the magnitude of the salt stress. On application of dilution stress, the flux of carbon from newly photosynthetically fixed ¹⁴CO₂ into glycerol was reduced progressively with increasing dilution stress that was also accompanied by a decline in total glycerol contents of the cell. The maximum observed rate of glycerol dissimilation was about 135 μmol glycerol mg⁻¹ Chl h⁻¹.     

Dihydroxyacetone production in an engineered Escherichia coli through expression of Corynebacterium glutamicum dihydroxyacetone phosphate dephosphorylase

Dihydroxyacetone (DHA) has several industrial applications such as a tanning agent in tanning lotions in the cosmetic industry; its production via microbial fermentation would present a more sustainable option for the future. Here we genetically engineered Escherichia coli (E. coli) for DHA production from glucose. Deletion of E. coli triose phosphate isomerase (tpiA) gene was carried out to accumulate dihydroxyacetone phosphate (DHAP), for use as the main intermediate or precursor for DHA production. The accumulated DHAP was then converted to DHA through the heterologous expression of Corynebacterium glutamicum DHAP dephosphorylase (cghdpA) gene. To conserve DHAP exclusively for DHA production we removed methylglyoxal synthase (mgsA) gene in the ΔtpiA strain. This drastically improved DHA production from 0.83 g/l (0.06 g DHA/g glucose) in the ΔtpiA strain bearing cghdpA to 5.84 g/l (0.41 g DHA/g glucose) in the ΔtpiAΔmgsA double mutant containing the same gene. To limit the conversion of intracellular DHA to glycerol, glycerol dehydrogenase (gldA) gene was further knocked out resulting in a ΔtpiAΔmgsAΔgldA triple mutant. This triple mutant expressing the cghdpA gene produced 6.60 g/l of DHA at 87% of the maximum theoretical yield. In summary, we demonstrated an efficient system for DHA production in genetically engineered E. coli strain.     

Horseradish peroxidase production from Spodoptera frugiperda larvae: A simple and inexpensive method

Horseradish peroxidase is used in many biotechnological fields including diagnostics, biocatalysts and biosensors. Horseradish peroxidase isozyme C (HRPC) was extracellularly expressed in Spodoptera frugiperda Sf9 cell culture and in intact larvae. At day 6 post-infection, the concentration of active HRPC in suspension cultures was 3.0 ± 0.1 μg per 1 × 10⁶ cells or 3.0 ± 0.1 mg l⁻¹ with a multiplicity of infection of 1 in the presence of 7.2 μM hemin. Similar yields were obtained in monolayer cultures. In larvae, the HRPC expression level was 137 ± 17 mg HRPC kg⁻¹ larvae at day 6 post-infection with a single larvae thus producing approximately 41 μg HRPC. The whole larval extract was separated by ion exchange chromatography and HRPC was purified in a single step with a yield of 75% and a purification factor of 117. The molecular weight of recombinant HRPC was 44,016 Da, and its glycosylation pattern agreed with that expected for invertebrates. The K_m and V_max were 12.1 ± 1.7 mM and 2673 ± 113 U mg⁻¹, respectively, similar to those of HRP purified from Armoracia rusticana roots. The method described in this study, based on overexpression of HRPC in S. frugiperda larvae, is a simple and inexpensive way to obtain high levels of active enzyme for research and other biotechnological applications.     

High-level production of biologically active chemokines in Escherichia coli

The chemokines eotaxin-1 (CCL11) and eotaxin-2 (CCL24), belonging to the CC chemokines family, play key roles in the inflammatory response, allergic asthma and other diseases. When expressed in Escherichia coli, chemokines are prone to form inclusion bodies devoid of biological activity, and it is hard to refold them properly. Here an expression and purification protocol for high-level production of soluble and biologically active CCL11 and CCL24 in E. coli has been established. A final yield of 8.7 mg/l for CCL11 and 3.9 mg/l for CCL24 has been obtained and the purified proteins were characterized with SDS-PAGE, mass spectrometry and circular dichroism. High binding affinity of purified chemokines with CC chemokine receptor type 3 (CCR3) has been confirmed with surface plasmon resonance (SPR) and the K_D values are 3.7 × 10⁻⁷ M and 3.0 × 10⁻⁷ M, respectively, for CCL11 and CCL24. This report provides a straightforward strategy for the efficient production of soluble and biologically active chemokines in E. coli.     

Production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) from unrelated carbon sources by metabolically engineered Escherichia coli

A metabolically engineered Escherichia coli has been constructed for the production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] from unrelated carbon sources. Genes involved in succinate degradation in Clostridium kluyveri and P(3HB) accumulation pathway of Ralstonia eutropha were co-expressed for the synthesis of the above copolyester. E. coli native succinate semialdehyde dehydrogenase genes sad and gabD were both deleted for eliminating succinate formation from succinate semialdehyde, which functioned to enhance the carbon flux to 4HB biosynthesis. The metabolically engineered E. coli produced 9.4 g l^?1 cell dry weight containing 65.5% P(3HB-co-11.1 mol% 4HB) using glucose as carbon source in a 48 h shake flask growth. The presence of 1.5–2 g l^?1 α-ketoglutarate or 1.0 g l^?1 citrate enhanced the 4HB monomer content from 11.1% to more than 20%. In a 6 l fermentor study, a 23.5 g l^?1 cell dry weight containing 62.7% P(3HB-co-12.5 mol% 4HB) was obtained after 29 h of cultivation. To the best of our knowledge, this study reports the highest 4HB monomer content in P(3HB-co-4HB) produced from unrelated carbon sources.     

Metabolic engineering of Escherichia coli for the production of 5-aminovalerate and glutarate as C5 platform chemicals

5-Aminovalerate (5AVA) is the precursor of valerolactam, a potential building block for producing nylon 5, and is a C5 platform chemical for synthesizing 5-hydroxyvalerate, glutarate, and 1,5-pentanediol. Escherichia coli was metabolically engineered for the production of 5-aminovalerate (5AVA) and glutarate. When the recombinant E. coli WL3110 strain expressing the Pseudomonas putida davAB genes encoding delta-aminovaleramidase and lysine 2-monooxygenase, respectively, were cultured in a medium containing 20 g/L of glucose and 10 g/L of l-lysine, 3.6 g/L of 5AVA was produced by converting 7 g/L of l-lysine. When the davAB genes were introduced into recombinant E. coli strainXQ56allowing enhanced l-lysine synthesis, 0.27 and 0.5 g/L of 5AVA were produced directly from glucose by batch and fed-batch cultures, respectively. Further conversion of 5AVA into glutarate could be demonstrated by expression of the P. putida gabTD genes encoding 5AVA aminotransferase and glutarate semialdehyde dehydrogenase. When recombinant E. coli WL3110 strain expressing the davAB and gabTD genes was cultured in a medium containing 20 g/L glucose, 10 g/L l-lysine and 10 g/L α-ketoglutarate, 1.7 g/L of glutarate was produced.     

Development of a recombinant Escherichia coli-based biocatalyst to enable high styrene epoxidation activity with high product yield on energy source

A highly active recombinant whole-cell biocatalyst, Escherichia coli pETAB2/pG-KJE1, was developed for the efficient production of (S)-styrene oxide from styrene. The recombinant E. coli overexpressed styAB the genes of styrene monooxygenase of Pseudomonas putida SN1 and coexpressed the genes encoding chaperones (i.e., GroEL–GroES and DnaK–DnaJ–GrpE). The styrene monooxygenases were produced to ca. 40% of the total soluble proteins, enabling the whole-cell activity of the recombinant of 180 U/g CDW. The high StyAB activity in turn appeared to direct cofactors and molecular oxygen to styrene epoxidation. The product yield on energy source (i.e., glucose) reached ca. 40%. In addition, biotransformation in an organic/aqueous two-liquid phase system allowed the product to accumulate to 400 mM in the organic phase within 6 h, resulting in an average specific and volumetric productivity of 6.4 mmol/g dry cells/h (106 U/g dry cells) and 67 mM/h (1110 U/L_aq), respectively, under mild reaction conditions. These results indicated that the high productivity and the high product yield on energy source were driven by the high enzyme activity. Therefore, it was concluded that oxygenase activity of whole-cell biocatalysts is one of the critical factors to determine their catalytic performance.     

Engineering of Saccharomyces cerevisiae for the production of dihydroxyacetone (DHA) from sugars: A proof of concept

Dihydroxyacetone (DHA) has numerous industrial applications. In this work, we pursue the idea to produce DHA from sugars in the yeast Saccharomyces cerevisiae, via glycerol as an intermediate. Firstly, three glycerol dehydrogenase (GDH) genes from different microbial sources were expressed in yeast. Among them, the NAD⁺-dependent GDH of Hansenula polymorpha showed the highest glycerol-oxidizing activity. DHA concentration in shake-flask experiments was roughly 100 mg/l DHA from 20 g/l glucose, i.e. five times the wild-type level. This level was achieved only when cultures were subjected to osmotic stress, known to enhance glycerol production and accumulation in S. cerevisiae. Secondly, DHA kinase activity was abolished to prevent conversion of DHA to dihydroxyacetone phosphate (DHAP). The dak1Δdak2Δ double-deletion mutant overexpressing H. polymorpha gdh produced 700 mg/l DHA under the same conditions. Although current DHA yield and titer still need to be optimized, our approach provides the proof of concept for producing DHA from sugars in yeast.     

Recombinant human sperm-specific glyceraldehyde-3-phosphate dehydrogenase: Structural basis for enhanced stability

Sperm-specific glyceraldehyde-3-phosphate dehydrogenase (GAPDS) is bound to the fibrous sheath of the sperm flagellum through the hydrophobic N-terminal domain of the enzyme molecule. Expression of human GAPDS in E.coli cells yields inactive and insoluble protein. Presumably, the N-terminal domain prevents correct folding of the full-length recombinant enzyme. To obtain GAPDS in a soluble and active form, a recombinant enzyme lacking in 68 amino acids of the N-terminal domain (dN-GAPDS) was expressed in E.coli cells. Purified dN-GAPDS was shown to be a protein of 9.3 nm in diameter (by dynamic light scattering), which is close to the size of the muscle tetrameric glyceraldehyde-3-phosphate dehydrogenase (8.6 nm). The catalytic properties of the protein differed a little from those of the muscle glyceraldehyde-3-phoshate dehydrogenase. However, compared to muscle glyceraldehyde-3-phoshate dehydrogenase, dN-GAPDS exhibited enhanced thermostability (the transition midpoints values are 60.8 and 67.4 °C, respectively) and was much more resistant towards action of guanidine hydrochloride (inactivation constants are 2.45 ± 0.018 and 0.118 ± 0.008 min^? 1, respectively). The enhanced stability of dN-GAPDS is likely to be related to some specific features of the GAPDS structure compared to that of the muscle enzyme: 1) reduced number of solvent-exposed salt bridges; 2) 2 additional buried salt bridges; and 3) 6 additional proline residues in GAPDS meeting the “proline rule”. It is assumed that high stability of the sperm-specific GAPDS is of importance for the efficiency of fertilization.     

Cyanobacterial production of 1,3-propanediol directly from carbon dioxide using a synthetic metabolic pathway

Production of chemicals directly from carbon dioxide using light energy is an attractive option for a sustainable future. The 1,3-propanediol (1,3-PDO) production directly from carbon dioxide was achieved by engineered Synechococcus elongatus PCC 7942 with a synthetic metabolic pathway. Glycerol dehydratase catalyzing the conversion of glycerol to 3-hydroxypropionaldehyde in a coenzyme B₁₂-dependent manner worked in S. elongatus PCC 7942 without addition of vitamin B₁₂, suggesting that the intrinsic pseudovitamin B₁₂ served as a substitute of coenzyme B₁₂. The highest titers of 1,3-PDO (3.79±0.23 mM; 288±17.7 mg/L) and glycerol (12.62±1.55 mM; 1.16±0.14 g/L), precursor of 1,3-PDO, were reached after 14 days of culture under optimized conditions in this study.