Engineering a powerful green cell factory for robust photoautotrophic diterpenoid production

Diterpenoids display a large and structurally diverse class of natural compounds mainly found as specialized plant metabolites. Due to their diverse biological functions they represent an essential source for various industrially relevant applications as biopharmaceuticals, nutraceuticals, and fragrances. However, commercial production utilizing their native hosts is inhibited by low abundances, limited cultivability, and challenging extraction, while the precise stereochemistry displays a particular challenge for chemical synthesis. Due to a high carbon flux through their native 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway towards photosynthetically active pigments, green microalgae hold great potential as efficient and sustainable heterologous chassis for sustainable biosynthesis of plant-derived diterpenoids. In this study, innovative synthetic biology and efficient metabolic engineering strategies were systematically combined to re-direct the metabolic flux through the MEP pathway for efficient heterologous diterpenoid synthesis in C. reinhardtii. Engineering of the 1-Deoxy-D-xylulose 5-phosphate synthase (DXS) as the main rate-limiting enzyme of the MEP pathway and overexpression of diterpene synthase fusion proteins increased the production of high-value diterpenoids. Applying fully photoautotrophic high cell density cultivations demonstrate potent and sustainable production of the high-value diterpenoid sclareol up to 656 mg L⁻¹ with a maximal productivity of 78 mg L⁻¹ day⁻¹ in a 2.5 L scale photobioreactor, which is comparable to sclareol titers reached by highly engineered yeast. Consequently, this work represents a breakthrough in establishing a powerful phototrophic green cell factory for the competetive use in industrial biotechnology.     

Bacterium Hafnia alvei secretes l-methioninase enzyme: Optimization of the enzyme secretion conditions

I isolated bacteria from blue cheese in order to find bacterial strains secreting l-methioninase enzyme, and optimized the conditions for the most efficient enzyme secretion. The efficient isolate, identified according to the 16S rRNA gene sequence analysis, was Hafnia alvei belonging to Enterobacteriaceae. I confirmed that the H. alvei strain harbored the methionase gene, mdeA (1194 bp). The environmental (pH, temperature) and nutritional (carbon and nitrogen sources and Mg concentration) factors influencing the l-methioninase production of H. alvei were optimized. The highest yield of l-methioninase enzyme was reached after 48 h of incubation when the acidity of the growing medium was adjusted to pH 7.5 and the temperature was 35 °C. The following concentrations of the supplements increased the l-methioninase yield in the medium: galactose (2.0 g L⁻¹), MgSO₄ (0.25 g L⁻¹), l-methionine as an inducer (2.0 g L⁻¹), and l-asparagine as an additional N source (1.5 g L⁻¹). I introduce a bacterial strain of H. alvei that is previously unreported to secrete l-methioninase enzyme and show that a carbon source is a mandatory supplement whereas l-methionine is not a mandatory supplement for l-methioninase enzyme production of H. alvei.     

Effective biodegradation of 2,4,6-trinitrotoluene using a novel bacterial strain isolated from TNT-contaminated soil

In this environmental-sample based study, rapid microbial-mediated degradation of 2,4,6-trinitrotoluene (TNT) contaminated soils is demonstrated by a novel strain, Achromobacter spanius STE 11. Complete removal of 100 mg L⁻¹ TNT is achieved within only 20 h under aerobic conditions by the isolate. In this bio-conversion process, TNT is transformed to 2,4-dinitrotoluene (7 mg L⁻¹), 2,6-dinitrotoluene (3 mg L⁻¹), 4-aminodinitrotoluene (49 mg L⁻¹) and 2-aminodinitrotoluene (16 mg L⁻¹) as the key metabolites. A. spanius STE 11 has the ability to denitrate TNT in aerobic conditions as suggested by the dinitrotoluene and NO₃ productions during the growth period. Elemental analysis results indicate that 24.77 mg L⁻¹ nitrogen from TNT was accumulated in the cell biomass, showing that STE 11 can use TNT as its sole nitrogen source. TNT degradation was observed between pH 4.0–8.0 and 4–43 °C; however, the most efficient degradation was at pH 6.0–7.0 and 30 °C.     

Effect of oxygen supply on biomass and helvolic acid production in submerged fermentation of Cordyceps taii

The entomogenous fungus Cordyceps taii, a traditional Chinese medicinal mushroom, exhibits potent important pharmacological effects and it has great potential for health foods and medicine. In this work, the effects of oxygen supply on production of biomass and bioactive helvolic acid were studied in shake-flask fermentation of C. taii mycelia. The value of initial volumetric oxygen transfer coefficient (K_La) within 10.1–33.8 h⁻¹ affected the cell growth, helvolic acid production and expression levels of biosynthetic genes. The highest cell concentration of 17.2 g/L was obtained at 14.3 h⁻¹ of initial K_La. The highest helvolic acid production was 9.6 mg/L at 10.1 h⁻¹ of initial K_La. The expression levels of three genes encoding hydroxymethylglutaryl-CoA synthase, hydroxymethylglutaryl-CoA reductase and squalene synthase were down-regulated on day 2 and day 8 but up-regulated on day 14 at an initial K_La value of 10.1 h⁻¹ vs. 33.8 h⁻¹, which well corresponded to the helvolic acid biosynthesis in those conditions. The information obtained would be helpful for improving the biomass and helvolic acid production in large-scale fermentation of C. taii.     

High titer methyl ketone production with tailored Pseudomonas taiwanensis VLB120

Methyl ketones present a group of highly reduced platform chemicals industrially produced from petroleum-derived hydrocarbons. They find applications in the fragrance, flavor, pharmacological, and agrochemical industries, and are further discussed as biodiesel blends. In recent years, intense research has been carried out to achieve sustainable production of these molecules by re-arranging the fatty acid metabolism of various microbes. One challenge in the development of a highly productive microbe is the high demand for reducing power. Here, we engineered Pseudomonas taiwanensis VLB120 for methyl ketone production as this microbe has been shown to sustain exceptionally high NAD(P)H regeneration rates. The implementation of published strategies resulted in 2.1 g L_aq⁻¹ methyl ketones in fed-batch fermentation. We further increased the production by eliminating competing reactions suggested by metabolic analyses. These efforts resulted in the production of 9.8 g L_aq⁻¹ methyl ketones (corresponding to 69.3 g L_org⁻¹ in the in situ extraction phase) at 53% of the maximum theoretical yield. This represents a 4-fold improvement in product titer compared to the initial production strain and the highest titer of recombinantly produced methyl ketones reported to date. Accordingly, this study underlines the high potential of P. taiwanensis VLB120 to produce methyl ketones and emphasizes model-driven metabolic engineering to rationalize and accelerate strain optimization efforts.     

β-carotene production from soap stock by loofa-immobilized Rhodotorula rubra in an airlift photobioreactor

In this study, the soap stock as a sole carbon source was used for growing a carotenoid producing yeast (Rhodotorula rubra). The application of soap stock resulted in increase of carotenoids yield up to 5.36 folds when compared with the grown cultures on glucose. On the best Monod equation fitted on the specific growth rate (μ) data, the maximum specific growth rate (μ_m) and half-saturation concentration (K_S) were respectively determined at 0.064 h⁻¹ and 3.26 g L⁻¹ for total fatty acids presented in soap stock. Further tests on the carotenogenesis process were carried out in a cell-immobilized airlift photobioreactor where the natural loofa sponge was used for immobilization of the cells. The performance of the bioreactor was statistically studied by the response surface methodology (RSM) where aeration rate of 0.11 vvm and light irradiation intensity of 2517 Lx provided an optimum condition for producing β-carotene with a specific production rate of 22.65 mg g_cell⁻¹ day⁻¹.     

Cultivation of Chlorella vulgaris in tubular photobioreactors: A lipid source for biodiesel production

Chlorella vulgaris was cultivated in two different 2.0 L-helicoidal and horizontal photobioreactors at 5 klux using the bicarbonate contained in the medium and ambient air as the main CO₂ sources. The influence of bicarbonate concentration on biomass growth as well as lipid content and profile was first investigated in shake flasks, where the stationary phase was achieved in about one half the time required by the control. The best NaHCO₃ concentration (0.2 g L⁻¹) was then used in both photobioreactors. While the fed-batch run performed in the helicoidal photobioreactor provided the best result in terms of biomass productivity, which was (84.8 mg L⁻¹ d⁻¹) about 2.5-fold that of the batch run, the horizontal configuration ensured the highest lipid productivity (10.3 mg L⁻¹ d⁻¹) because of a higher lipid content of biomass (22.8%). These preliminary results suggest that the photobioreactor configuration is a key factor either for the growth or the composition of this microalga. The lipid quality of C. vulgaris biomass grown in both photobioreactors is expected to meet the standards for biodiesel, especially in the case of the helicoidal configuration, provided that further efforts will be made to optimize the conditions for its production as a biodiesel source.     

Modification of light intensity influence essential oils content,composition and antioxidant activity of thyme,marjoram and oregano

Thymus vulgaris L. (thyme), Origanum majorana L. (marjoram), and Origanum vulgare L. (oregano) were used to determine whether light modification (plants grown under nets with 40% shaded index or in un-shaded open field) could improve the quantity and quality of essential oils (EOs) and antioxidant activity. The yield of EOs of thyme, marjoram, and oregano obtained after 120 min of hydrodistillation was 2.32, 1.51, and 0.27 mL/100 g of plant material, respectively. At the same time under shading conditions plants synthetized more EOs (2.57, 1.68, and 0.32 mL/100 g of plant material). GC/MS and GC/FID analyses were applied for essential oils determinations. The main components of the thyme essential oil are thymol (8.05–9.35%); γ-terpinene (3.49–4.04%); p-cymene (2.80–3.60%) and caryophyllene oxide (1.54–2.15%). Marjoram main components were terpinene 4-ol (7.44–7.63%), γ-terpinene (2.82–2.86%) and linalool (2.04–2.65%) while oregano essential oil consisted of the following components: caryophyllene oxide (3.1–1.93%); germacrene D (1.17–2.0%) and (E)-caryophyllene (1.48–1.1%). The essential oil from thyme grown under shading (EC₅₀ value after 20 min of incubation) have shown the highest antioxidant activity – 0.85 mg mL⁻¹ in comparison to marjoram and oregano (shaded plants EC₅₀ 19.97 mg mL⁻¹ and 7.02 mg mL⁻¹ and unshaded, control plants EC₅₀ 54.01 mg mL⁻¹ and 7.45 mg mL⁻¹, respectively). The medicinal plants are a good source of natural antioxidants with potential application in the food and pharmaceutical industries. For production practice, it can be recommended to grow medicinal plants in shading conditions to achieve optimal quality parameters.     

Over expression of GroESL in Cupriavidus necator for heterotrophic and autotrophic isopropanol production

We previously reported a metabolic engineering strategy to develop an isopropanol producing strain of Cupriavidus necator leading to production of 3.4 g L⁻¹ isopropanol. In order to reach higher titers, isopropanol toxicity to the cells has to be considered. A toxic effect of isopropanol on the growth of C. necator has been indeed observed above a critical value of 15 g L⁻¹. GroESL chaperones were first searched and identified in the genome of C. necator. Native groEL and groES genes from C. necator were over-expressed in a strain deleted for PHA synthesis. We demonstrated that over-expressing groESL genes led to a better tolerance of the strain towards exogenous isopropanol. GroESL genes were then over-expressed within the best engineered isopropanol producing strain. A final isopropanol concentration of 9.8 g L⁻¹ was achieved in fed-batch culture on fructose as the sole carbon source (equivalent to 16 g L⁻¹ after taking into account evaporation). Cell viability was slightly improved by the chaperone over-expression, particularly at the end of the fermentation when the isopropanol concentration was the highest. Moreover, the strain over-expressing the chaperones showed higher enzyme activity levels of the 2 heterologous enzymes (acetoacetate carboxylase and alcohol dehydrogenase) of the isopropanol synthetic operon, translating to a higher specific production rate of isopropanol at the expense of the specific production rate of acetone. Over-expressing the native chaperones led to a 9–18% increase in the isopropanol yield on fructose.     

Production of ethanol and xylanolytic enzymes by yeasts inhabiting rotting wood isolated in sugarcane bagasse hydrolysate

Yeasts associated with rotting wood from four Atlantic Rain forest sites in Brazil were investigated using a culture medium based on sugarcane bagasse hydrolysate. A total of 330 yeast strains were isolated. Pichia manshurica, Candida pseudolambica, and Wickerhamomyces sp. 3 were the most frequently isolated species. Fourteen novel species were obtained in this study. All isolates were tested for their ability to ferment d-xylose and to produce xylanases. In the fermentation assays using d-xylose (30 g L⁻¹), the main ethanol producers were Scheffersomyces stipitis (14.08 g L⁻¹), Scheffersomyces sp. (7.94 g L⁻¹) and Spathaspora boniae (7.16 g L⁻¹). Sc. stipitis showed the highest ethanol yield (0.42 g g⁻¹) and the highest productivity (0.39 g L⁻¹h⁻¹). The fermentation results using hemicellulosic hydrolysate showed that Sc. stipitis was the best ethanol producer, achieving a yield of 0.32 g g⁻¹, while Sp. boniae and Scheffersomyces sp. were excellent xylitol producers. The best xylanase-producing yeasts at 50 °C belonged to the species Su. xylanicola (0.487 U mg⁻¹) and Saitozyma podzolica (0.384 U mg⁻¹). The results showed that rotting wood collected from the Atlantic Rainforest is a valuable source of yeasts able to grow in sugarcane bagasse hydrolysate, including species with promising biotechnological properties.     

d- and l-lactic acid production from fresh sweet potato through simultaneous saccharification and fermentation

The aim of this study was to develop a bioprocess for l- and d-lactic acid production from raw sweet potato through simultaneous saccharification and fermentation by Lactobacillus paracasei and Lactobacillus coryniformis, respectively. The effects of enzyme and nitrogen source concentrations as well as of the ratio of raw material to medium were investigated. At dried material concentrations of 136.36–219.51 g L⁻¹, yields of 90.13–91.17% (w/w) and productivities of 3.41–3.83 g L⁻¹ h⁻¹ were obtained with lactic acid concentrations as high as 198.32 g L⁻¹ for l-lactic acid production. In addition, d-lactic acid was produced with yields of 90.11–84.92% (w/w) and productivities of 2.55–3.11 g L⁻¹ h⁻¹ with a maximum concentration of 186.40 g L⁻¹ at the same concentrations of dried material. The simple and efficient process described in this study will benefit the tuber and root-based lactic acid industries without requiring alterations in plant equipment.     

Isolation and bioelectrochemical characterization of novel fungal sources with oxidasic activity applied in situ for the cathodic oxygen reduction in microbial fuel cells

Brazilian filamentous fungi Rhizopus sp. (SIS-31), Aspergillus sp. (SIS-18) and Penicillium sp. (SIS-21), sources of oxidases were isolated from Caatinga's soils and applied during the in situ cathodic oxygen reduction in fuel cells. All strains were cultivated in submerged cultures using an optimized saline medium enriched with 10 g L⁻¹ of glucose, 3.0 g L⁻¹ of peptone and 0.0005 g L⁻¹ of CuSO₄ as enzyme inducer. Parameters of oxidase activity, glucose consumption and microbial growth were evaluated. In-cell experiments evaluated by chronoamperometry were performed and two different electrode compositions were also compared. Maximum current densities of 125.7, 98.7 and 11.5 μA cm⁻² were observed before 24 h and coulombic efficiencies of 56.5, 46.5 and 23.8% were obtained for SIS-31, SIS-21 and SIS-18, respectively. Conversely, maximum power outputs of 328.73, 288.80 and 197.77 mW m⁻³ were observed for SIS-18, SIS-21 and SIS-31, respectively. This work provides the primary experimental evidences that fungi isolated from the Caatinga region in Brazil can serve as efficient biocatalysts during the oxygen reduction in air-cathodes to improve electricity generation in MFCs.     

Bench-scale fermentation of Trichoderma viride on wastewater sludge: Rheology,lytic enzymes and biocontrol activity

Conidiation and lytic enzyme production by Trichoderma viride at different solids concentration of pre-treated municipal wastewater sludge was examined in a 15-L fermenter. The maximum conidia concentration (5.94 × 10⁷ CFU mL⁻¹ at 96 h) was obtained at 30 g L⁻¹ suspended solids. The maximum lytic enzyme activities were achieved around 12–30 h of fermentation. Bioassay against a fungal phytopathogen, Fusarium sp. showed maximum activity in the sample drawn around 96 h of fermentation at 30 g L⁻¹ suspended solids concentration. Entomotoxicity against spruce budworm larvae showed maximum value ≈17290 SBU μL⁻¹ at 30 g L⁻¹ suspended solids concentration at the end of fermentation (96 h). Plant bioassay showed dual action of T. viride, i.e., disease prevention and growth promotion. The rheological analyses of fermentation sludges showed the pseudoplastic behaviour. In order to maintain required dissolved oxygen concentration ≥30%, the agitation and aeration requirements significantly increased at 35 g L⁻¹ compared to 30 and 25 g L⁻¹. The oxygen uptake rate and volumetric oxygen mass transfer coefficient, k_La at 35 g L⁻¹ did not increase in comparison to 30 g L⁻¹ due to rheological complexity of the broth during fermentation. Thus, the successful fermentation operation of the biocontrol fungus T. viride is a rational indication of its potential for mass-scale production for agriculture and forest sector as a biocontrol agent.     

Bioprocess development for kefiran production by Lactobacillus kefiranofaciens in semi industrial scale bioreactor

Lactobacillus kefiranofaciens is non-pathogenic gram positive bacteria isolated from kefir grains and able to produce extracellular exopolysaccharides named kefiran. This polysaccharide contains approximately equal amounts of glucose and galactose. Kefiran has wide applications in pharmaceutical industries. Therefore, an approach has been extensively studied to increase kefiran production for pharmaceutical application in industrial scale. The present work aims to maximize kefiran production through the optimization of medium composition and production in semi industrial scale bioreactor. The composition of the optimal medium for kefiran production contained sucrose, yeast extract and K₂HPO₄ at 20.0, 6.0, 0.25 g L⁻¹, respectively. The optimized medium significantly increased both cell growth and kefiran production by about 170.56% and 58.02%, respectively, in comparison with the unoptimized medium. Furthermore, the kinetics of cell growth and kefiran production in batch culture of L. kefiranofaciens was investigated under un-controlled pH conditions in 16-L scale bioreactor. The maximal cell mass in bioreactor culture reached 2.76 g L⁻¹ concomitant with kefiran production of 1.91 g L⁻¹.     

Effects of terrigenous organic substrates and additional phosphorus on bacterioplankton metabolism and exoenzyme stoichiometry

1. Bamboo, as a pioneer vegetation, often forms forests on bare lands after catastrophic landslides. Compared to evergreen forest soil, bamboo forest soil is much more labile, with a higher percentage of microbially derived organic carbon (OC), lower molecular weight, and lower humic acid content. We hypothesised that different terrigenous organic matter (tOM) sources with varying lability and phosphorus (P) availability select for bacterioplankton with distinct metabolic pathways.
2. We incubated natural bacterioplankton assemblages with tOM leached from bamboo forest soil (BOM) and evergreen forest soil (EOM) and compared these to a lake water control. To test if microbial metabolism would be limited by OC or P availability of each tOM treatment, we used acetate as an extra labile OC source and phosphate as an inorganic P source. Bacterial metabolism was measured by analysing respiration via O₂ consumption and production via tritiated thymidine (TdR) assimilation.
3. Bacterioplankton metabolism is limited by the availability of P in BOM substrates. When using BOM, bacteria had higher enzymatic activities for phosphatase. The nutrients required for bacterial biomass seemed to be derived from organic matter. Under BOM treatment, bacterial production (BP) (0.92 ± 0.13 μg C L⁻¹ hr⁻¹) and cell specific TdR assimilation rates (0.015 ± 0.002 10^–18 M TdR cell⁻¹ hr⁻¹) were low. Adding P enhanced BP (BOM_+P 1.52 ± 0.31 and BOM_+C+P 2.25 ± 0.37 μg C L⁻¹ hr⁻¹) while acetate addition had no significant effect on BOM treatment.
4. This indicated that the bacteria switched to using added inorganic P to respire a P-limited BOM substrate, which increased total BP and abundance, resulting in even more active respiration and lower growth efficiency. We also found higher activities for chitin-degrading enzyme β-N-acetylglucosaminidase, which is associated with N mining from aminosaccharides.
5. Microbes using EOM, however, did not change metabolic strategies with additional acetate or/and inorganic P. This is due to higher concentrations of organic P in EOM substrates and the presence of inorganic N in the EOM leachates an alternative nutrient source. Bacteria produced β-glucosidase and leucyl-aminopeptidase in order to utilise the humic substances, which sustained greater bacterial abundance, higher BP (2.64 ± 0.39 μg C L⁻¹ hr⁻¹), and lower cell-specific respiration. This yielded a much higher bacterial growth efficiency (15 ± 9.2%) than the lake water control.
6. Our study demonstrated the aquatic metabolic discrepancy between tOM of different forest types. Bacterioplankton in BOM and EOM exhibit distinct metabolic responses. Bacterial metabolic strategy when using BOM implied that the supposedly stabilised biomass OM might be efficiently used by aquatic bacterioplankton. As the labile and nutrient-deficient BOM is more susceptible to the influence of additional nutrients, fertiliser residues in bamboo forest catchments might have a stronger effect on aquatic bacterial metabolic pathways. Thus, it is important to take tOM differences into consideration when building models to estimate soil carbon turnover rates along a terrestrial–aquatic continuum.

     

Seasonal variations in photosynthetic irradiance response curves of macrophytes from a Mediterranean coastal lagoon

The main photosynthesis and respiration parameters (dark respiration rate, light saturated production rate, saturation irradiance, photosynthetic efficiency) were measured on a total of 23 macrophytes of the Thau lagoon (2 Phanerogams, 5 Chlorophyceae, 10 Rhodophyceae and 6 Phaeophyceae). Those measurements were performed in vitro under controlled conditions, close to the natural ones, and at several seasons. Concomitantly, measurements of pigment concentrations, carbon, phosphorous and nitrogen contents in tissues were performed. Seasonal intra-specific variability of photosynthetic parameters was found very high, enlightening an important acclimatation capacity. The highest photosynthetic capacities were found for Chlorophyceae (e.g. Monostroma obscurum thalli at 17 °C, 982 μmol O₂ g⁻¹ dw h⁻¹ and 9.1 μmol O₂ g⁻¹ dw h⁻¹/μmol photons m⁻² s⁻¹, respectively for light saturated net production rate and photosynthetic efficiency) and Phanerogams (e.g. Nanozostera noltii leaves at 25 °C, 583 μmol O₂ g⁻¹ dw h⁻¹ and 2.6 μmol O₂ g⁻¹ dw h⁻¹/μmol photons m⁻² s⁻¹ respectively for light saturated net production rate and photosynthetic efficiency). As expected, species with a high surface/volume ratio were found to be more productive than coarsely branched thalli and thick blades shaped species. Contrary to R_d (ranging 6.7–794 μmol O₂ g⁻¹ dw h⁻¹, respectively for Rytiphlaea tinctoria at 7 °C and for Dasya sessilis at 25 °C) for which a positive relationship with water temperature was found whatever the species studied, the evolution of P/I curves with temperature exhibited different responses amongst the species. The results allowed to show summer nitrogen limitation for some species (Gracilaria bursa-pastoris and Ulva spp.) and to propose temperature preferences based on the photosynthetic parameters for some others (N. noltii, Zostera marina, Chaetomorpha linum).     

Continuous succinic acid production by Actinobacillus succinogenes in a biofilm reactor: Steady-state metabolic flux variation

Continuous anaerobic fermentations were performed in a novel external-recycle, biofilm reactor using d-glucose and CO₂ as carbon substrates. Succinic acid (SA) yields were found to be an increasing function of glucose consumption with the succinic acid to acetic acid ratio increasing from 2.4 g g⁻¹ at a glucose consumption of 10 g L⁻¹, to 5.7 g g⁻¹ at a glucose consumption of 50 g L⁻¹. The formic acid to acetic acid ratio decreased from an equimolar value (0.77 g g⁻¹) at a glucose consumption of 10 g L⁻¹ to a value close to zero at 50 g L⁻¹. The highest SA yield on glucose and highest SA titre obtained were 0.91 g g⁻¹ and 48.5 g L⁻¹ respectively. Metabolic flux analysis based on the established C₃ and C₄ metabolic pathways of Actinobacillus succinogenes revealed that the increase in the succinate to acetate ratio could not be attributed to the decrease in formic acid and that an additional source of NADH was present. The fraction of unaccounted NADH increased with glucose consumption, suggesting that additional reducing power is present in the medium or is provided by the activation of an alternative metabolic pathway.     

Acetone production with metabolically engineered strains of Acetobacterium woodii

Expected depletion of oil and fossil resources urges the development of new alternative routes for the production of bulk chemicals and fuels beyond petroleum resources. In this study, the clostridial acetone pathway was used for the formation of acetone in the acetogenic bacterium Acetobacterium woodii. The acetone production operon (APO) containing the genes thlA (encoding thiolase A), ctfA/ctfB (encoding CoA transferase), and adc (encoding acetoacetate decarboxylase) from Clostridium acetobutylicum were cloned under the control of the thlA promoter into four vectors having different replicons for Gram-positives (pIP404, pBP1, pCB102, and pCD6). Stable replication was observed for all constructs. A. woodii [pJIR_act_thlA] achieved the maximal acetone concentration under autotrophic conditions (15.2±3.4 mM). Promoter sequences of the genes ackA from A. woodii and pta-ack from C. ljungdahlii were determined by primer extension (PEX) and cloned upstream of the APO. The highest acetone production in recombinant A. woodii cells was achieved using the promoters P_thlA and P_pta-ack. Batch fermentations using A. woodii [pMTL84151_act_thlA] in a bioreactor revealed that acetate concentration had an effect on the acetone production, due to the high K_m value of the CoA transferase. In order to establish consistent acetate concentration within the bioreactor and to increase biomass, a continuous fermentation process for A. woodii was developed. Thus, acetone productivity of the strain A. woodii [pMTL84151_act_thlA] was increased from 1.2 mg L⁻¹ h⁻¹ in bottle fermentation to 26.4 mg L⁻¹ h⁻¹ in continuous gas fermentation.     

Implication of storage conditions on luminescence response from Photobacterium phosphoreum in free and immobilized form

Bioluminescent bacteria in the form of a cell suspension for on-site hazard analysis are not suitable as in vivo luminescence in free cells fluctuates and may lead to erroneous results. Furthermore, the culture broth cannot be stored for long durations to continue sensing analytes as the luminescence ceases over time. Factors that affect luminescence response include growth dynamism, and ambient environmental conditions. The present study investigated the effect of storage conditions such as temperature (25 ± 2°C, room temperature; 4°C; and −20°C) and ambient aqueous environment (M1: sucrose, 1.02 M; M2, bioluminescent media [tryptone, 10 g L⁻¹; NaCl, 28.5 g L⁻¹; MgCl₂.7H₂O, 4.5 g L⁻¹; CaCl₂, 0.5 g L⁻¹; KCl 0.5 g L⁻¹; yeast extract, 1 g L⁻¹; H₂O, 1 L]; M3, bioluminescent media and 95% glycerol, 1:1 ratio) on the luminescence emission from the calcium alginate-immobilized Photobacterium phosphoreum (S_b) against the cells in free suspension for an extended period. The results indicated that both the parameters that were undertaken markedly affected the luminescence. In the study, S_b showed an enhanced luminescence emission than the control up to 18.5-fold and for a prolonged period which can be efficiently utilized for rapid biosensing of hazardous materials.     

Unusual ent-atisane type diterpenoids with 2-oxopropyl skeleton from the roots of Euphorbia ebracteolata and their antiviral activity against human rhinovirus 3 and enterovirus 71

Two novel ent-atisane type diterpenoids possessing the extra unusal 2-oxopropyl moiety (1 and 2) and four known analogues have been isolated from the roots of Euphorbia ebracteolata. The structures and absolute configurations of these compounds were determined by extensive spectroscopic data analysis, including 2D NMR, single-crystal X-ray crystallography, ¹³C NMR calculation, and electronic circular dichroism spectra calculation. Compounds 1 and 2 are the first examples of natural products with ent-atisane type diterpenoids possessing 2-oxopropyl skeleton. Compounds 2, 3, 5, and 6 show antiviral activities against human rhinovirus 3, with IC₅₀ values of 25.27–90.35 μM. Compounds 5 and 6 showed moderate antiviral activities against EV71 at a concentration of 100 μM.