Fermentative lactic acid production with a metabolically engineered yeast immobilized in photo-crosslinkable resins

In this study, the immobilization technique involving photo-crosslinkable resin gels was used for lactic acid production. Saccharomyces cerevisiae OC-2T T165R, a metabolically engineered yeast that produces optically pure l(+)-lactic acid, was immobilized in hydrophilic photo-crosslinked resin gels as a biocatalyst. Three resin gels, TEP 1, TEP 2 and TEP 3, were examined and all of them showed high performance as to lactic acid production. Resin gel TEP 1, which exhibited the highest productivity among the resin gels was used for 15 consecutive batch fermentations without decreases in productivity and mechanical deformation, indicating that it was a suitable carrier for long-term lactic acid fermentation. Moreover, the use of the immobilization technique can improve the productivity of the metabolically engineered yeast in the fermentation with or without extraction, showing promise for using the immobilized engineered yeast for lactic acid production.     

The effect of carbon dioxide on the growth kinetics of fructose-limited chemostat cultures of Acetobacterium woodii DSM 1030

The growth of the anaerobic acetogenic bacterium Acetobacterium woodii DSM 1030 was investigated in fructose-limited chemostat cultures. A defined medium was developed which contained fructose, mineral salts, cysteine · HCl and Ca pantothenate (1 mg · 1^–1) supplied in a vitamin supplement. Growth at high dilution rates was dependent on the presence of CO₂ in the gas phase. The ^max was found to be 0.16 h^–1 and the fructose maintenance requirement was 0.1 to 0.13 mmol fructose · (g dry wt)^–1 · h^–1. A growth yield of 61 g dry wt · (mol fructose)^–1, corrected for the cell maintenance requirement and for incorporation of fructose carbon into cell biomass, was determined from the fructose consumption. A corresponding growth yield of 69 g dry wt · (mol fructose)^–1 was calculated from the acetate production assuming that fructose fermentation was homoacetogenic. A Y_ATP of 12.2 to 13.8 g dry wt · (mol ATP)^–1 was calculated from these growth yields using a value of 5 mol ATP · (mol fructose)^–1 as an estimate of the amount of ATP synthesised from fructose fermentation. The addition of yeast extract (0.5 g · 1^–1) to the medium did not influence the ^max or cell yield. After prolonged growth under fructose-limited conditions the requirement of the culture for CO₂ in the gas phase was reduced.Abbreviations YE yeast extract - IC inorganic carbon - D fermenter dilution rate : h^–1 - M_X maintenance requirement for X: mmol X · (g dry wt)^–1 · h^–1 - X may be fructose (Fruct), fructose consumed in energy metabolism (Fruct [E]), acetate (Ac) - ATP CO₂, NH _inf4 ^sup+ or Pi - qX specific rate of utilisation or consumption of X: mmol X · (g dry wt)^–1 · h^–1 - V fermenter volume: litre - rC · Cell, fermenter cell carbon production: mmol C · h^–1 - Y_X yield of cells on X: g dry wt · (mol X)^–1 - Y _infx ^supmax the yield corrected for cell maintenance: g dry wt · (mol X)^–1 - S_ATP stoichiometry of ATP synthesis from fructose: mol ATP · (mol frucose)^–1 - x cell concentration: g dry wt · 1^–1 - specific growth rate : h^–1 - ^max maximum specific growth rate: h^–1     

High-level production of amorpha-4,11-diene in a two-phase partitioning bioreactor of metabolically engineered Escherichia coli

Reconstructing synthetic metabolic pathways in microbes holds great promise for the production of pharmaceuticals in large-scale fermentations. By recreating biosynthetic pathways in bacteria, complex molecules traditionally harvested from scarce natural resources can be produced in microbial cultures. Here we report on a strain of Escherichia coli containing a heterologous, nine-gene biosynthetic pathway for the production of the terpene amorpha-4,11-diene, a precursor to the anti-malarial drug artemisinin. Previous reports have underestimated the productivity of this strain due to the volatility of amorphadiene. Here we show that amorphadiene evaporates from a fermentor with a half-life of about 50 min. Using a condenser, we take advantage of this volatility by trapping the amorphadiene in the off-gas. Amorphadiene was positively identified using nuclear magnetic resonance spectroscopy and determined to be 89% pure as collected. We captured amorphadiene as it was produced in situ by employing a two-phase partitioning bioreactor with a dodecane organic phase. Using a previously characterized caryophyllene standard to calibrate amorphadiene production and capture, the concentration of amorphadiene produced was determined to be 0.5 g/L of culture medium. A standard of amorphadiene collected from the off-gas showed that the caryophyllene standard overestimated amorphadiene production by approximately 30%.     

Assessment of bacterial acyltransferases for an efficient lipid production in metabolically engineered strains of E. coli

Microbially produced lipids like triacylglycerols or fatty acid ethyl esters are currently of great interest as fuel replacements or other industrially relevant compounds. They can even be produced by non-oleaginous microbes, like Escherichia coli, upon metabolic engineering. However, there is still much room for improvement regarding the yield for a competitive microbial production of lipids or biofuels. We genetically engineered E. coli by expressing fadD, fadR, pgpB, plsB and ‘tesA in combination with atfA from Acinetobacter baylyi. A total fatty acid contents of up to 16% (w/w) was obtained on complex media, corresponding to approximately 9% (w/w) triacylglycerols and representing the highest titers of fatty acids and triacylglycerols obtained in E. coli under comparable cultivation conditions, so far. To evaluate further possibilities for an optimization of lipid production, ten promising bacterial wax ester synthase/acyl-Coenzyme A:diacylglycerol acyltransferases were tested and compared. While highest triacylglycerol storage was achieved with AtfA, the mutated variant AtfA-G355I turned out to be most suitable for fatty acid ethyl ester biosynthesis and enabled an accumulation of approx. 500 mg/L without external ethanol supplementation.     

Dry reforming of methane with CO2 on an electron-activated iron catalytic bed

A preliminary experimental investigation of dry reforming of methane with carbon dioxide, that has been performed on an iron bed activated with an electric current is reported. Operating conditions for the reaction included temperature ranging from 700 to 800° C and pressure close to 1 atm. The reaction, involving an excess of pure methane and carbon dioxide, was performed with and without addition of water vapour, provided by hot water saturation of the gaseous feed. According to syngas compositions, the electron flow has a dramatic effect on the conversion of both methane and carbon dioxide. It was shown also that hot water saturation of the CO(2) and CH(4) mixture allowed very good conversion, giving a syngas with a composition very close to what was expected from equilibrium calculations.     

In silico and in vivo stability analysis of a heterologous biosynthetic pathway for 1,4-butanediol production in metabolically engineered E. coli

Recently, several approaches have been published in order to develop a functional biosynthesis route for the non-natural compound 1,4-butanediol (BDO) in E. coli using glucose as a sole carbon source or starting from xylose. Among these studies, there was reported as high as 18 g/L product concentration achieved by industrial strains, however BDO production varies greatly in case of the reviewed studies. Our motivation was to build a simple heterologous pathway for this compound in E. coli and to design an appropriate cellular chassis based on a systemic biology approach, using constraint-based flux balance analysis and bi-level optimization for gene knock-out prediction. Thus, the present study reports, at the “proof-of concept” level, our findings related to model-driven development of a metabolically engineered E. coli strain lacking key genes for ethanol, lactate and formate production (ΔpflB, ΔldhA and ΔadhE), with a three-step biosynthetic pathway. We found this strain to produce a limited quantity of 1,4-BDO (.89 mg/L BDO under microaerobic conditions and .82 mg/L under anaerobic conditions). Using glycerol as carbon source, an approach, which to our knowledge has not been tackled before, our results suggest that further metabolic optimization is needed (gene-introductions or knock-outs, promoter fine-tuning) to address the redox potential imbalance problem and to achieve development of an industrially sustainable strain. Our experimental data on culture conditions, growth dynamics and fermentation parameters can consist a base for ongoing research on gene expression profiles and genetic stability of such metabolically engineered E. coli strains.     

Effects of carbon dioxide on metabolite production and bacterial communities during kimchi fermentation

Bacterial communities and metabolites in kimchi fermented under conventional conditions (CC) compared to CO₂-rich environments (CO₂) were analyzed. After a 20-day fermentation, lactic and acetic acid productions were 54 and 69 mM under CC, and 19 and 12 mM under CO₂, respectively. The final pH of kimchi fermented under CC (CC-fermenting) and CO₂ (CO₂-fermenting) were 4.1 and 4.7, respectively. For bacterial communities, OTU and Chao1 indices were both 35 in fresh kimchi, 10 and 15 in CC-fermenting kimchi, and 8 and 24 in CO₂-fermenting kimchi, respectively. Shannon and Simpson indices were 3.47 and 0.93 in fresh kimchi, 1.87–0.06 and 0.46–0.01 in CC-fermenting kimchi, and 1.65–0.44 and 0.63–0.12 in CO₂-fermenting kimchi, respectively. Non-lactic acid bacteria were eliminated in fermenting kimchi after 12 days under CC and 6 days under CO₂. I conclude that carbon dioxide can alter bacterial communities, reduce metabolite production, and improve fermented kimchi quality.     

Continuous production of enzymes under carbon-limited conditions by Trichoderma harzianum P49P11

Carbon-limited chemostat cultures were performed using different carbon sources (glucose, 10 and 20 g/L; sucrose, 10 g/L; fructose/glucose, 5.26/5.26 g/L; carboxymethyl cellulose, 10 g/L; and carboxymethyl cellulose/glucose, 5/5 g/L) to verify the capability of the wild type strain Trichoderma harzianum to produce extracellular enzymes. All chemostat cultures were carried out at a fixed dilution rate of 0.05 h^?1. Experiments using glucose, fructose/glucose and sucrose were performed in duplicate. Glucose condition was found to induce the production of enzymes that can catalyse the hydrolysis of p-nitrophenyl-β-d-glucopyranoside (PNPGase). A concentration of 20 g/L of glucose in the feed provided the highest productivity (1048 ± 16 U/mol h). Extracellular polysaccharides were considered the source of inducers. Based on the obtained results, a new PNPGase production process was developed using mainly glucose. This process raises interesting possibilities of synthesizing the inducer substrate and the induced enzymes in a single step using an easily assimilated carbon source under carbon-limited conditions.     

Continuous nisin production with bioengineered <Emphasis Type="Italic">Lactococcus lactis</Emphasis> strains

Nisin production in continuous cultures of bioengineered Lactococcus lactis strains that incorporate additional immunity and regulation genes was studied. Highest nisin activities were observed at 0.2 h^–1 dilution rate and 12.5 g l^–1 fructose concentration for all strains. Recombinant strains were able to produce greater amounts of nisin at dilution rates below 0.3 h⁻¹ compared to the control strain. However, this significant difference disappeared at dilution rates of 0.4 and 0.5 h^–1. For the strains LL27, LAC338, LAC339, and LAC340, optimum conditions for nisin production were determined to be at 0.29, 0.26, 0.27, and 0.27 h^–1 dilution rates and 11.95, 12.01, 11.63, and 12.50 g l^–1 fructose concentrations, respectively. The highest nisin productivity, 496 IU ml^–1 h^–1, was achieved with LAC339. The results of this study suggest that low dilution rates stabilize the high specific nisin productivity of the bioengineered strains in continuous fermentation. Moreover, response surface methodology analysis showed that regulation genes yielded high nisin productivity at wide ranges of dilution rates and fructose concentrations.     

Engineering Cupriavidus necator H16 for the autotrophic production of (R)-1,3-butanediol

Butanediols are widely used in the synthesis of polymers, specialty chemicals and important chemical intermediates. Optically pure R-form of 1,3-butanediol (1,3-BDO) is required for the synthesis of several industrial compounds and as a key intermediate of β-lactam antibiotic production. The (R)-1,3-BDO can only be produced by application of a biocatalytic process. Cupriavidus necator H16 is an established production host for biosynthesis of biodegradable polymer poly-3-hydroxybutryate (PHB) via acetyl-CoA intermediate. Therefore, the utilisation of acetyl-CoA or its upstream precursors offers a promising strategy for engineering biosynthesis of value-added products such as (R)-1,3-BDO in this bacterium. Notably, C. necator H16 is known for its natural capacity to fix carbon dioxide (CO₂) using hydrogen as an electron donor. Here, we report engineering of this facultative lithoautotrophic bacterium for heterotrophic and autotrophic production of (R)-1,3-BDO. Implementation of (R)-3-hydroxybutyraldehyde-CoA- and pyruvate-dependent biosynthetic pathways in combination with abolishing PHB biosynthesis and reducing flux through the tricarboxylic acid cycle enabled to engineer strain, which produced 2.97 g/L of (R)-1,3-BDO and achieved production rate of nearly 0.4 Cmol Cmol⁻¹ h⁻¹ autotrophically. This is first report of (R)-1,3-BDO production from CO₂.     

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.     

Fermentative production of L-glycerol 3-phosphate utilizing a Saccharomyces cerevisiae strain with an engineered glycerol biosynthetic pathway

Interest in L-glycerol 3-phosphate (L-G3P) production via microbial fermentation is due to the compound's potential to replace the unstable substrate dihydroxyacetone phosphate (DHAP) in one-pot enzymatic carbohydrate syntheses. A Saccharomyces cerevisiae strain with deletions in both genes encoding specific L-G3Pases (GPP1 and GPP2) and multicopy overexpression of L-glycerol 3-phosphate dehydrogenase (GPD1) was studied via small-scale (100 mL) batch fermentations under quasi-anaerobic conditions. Intracellular accumulation of L-G3P reached extremely high levels (roughly 200 mM) but thereafter declined. Extracellular L-G3P was also detected and its concentration continuously increased throughout the fermentation, such that most of the total L-G3P was found outside the cells as fermentation concluded. Moreover, in spite of the complete elimination of specific L-G3Pase activity, the strain showed considerable glycerol formation suggesting unspecific dephosphorylation as a mechanism to relieve cells of intracellular L-G3P accumulation. Up-scaling the process employed fed-batch fermentation with repeated glucose feeding, plus an aerobic growth phase followed by an anaerobic product accumulation phase. This produced a final product titer of about 325 mg total L-G3P per liter of fermentation broth.     

Simultaneous flue gas bioremediation and reduction of microalgal biomass production costs

A flue gas originating from a municipal waste incinerator was used as a source of CO₂ for the cultivation of the microalga Chlorella vulgaris, in order to decrease the biomass production costs and to bioremediate CO₂ simultaneously. The utilization of the flue gas containing 10–13% (v/v) CO₂ and 8–10% (v/v) O₂ for the photobioreactor agitation and CO₂ supply was proven to be convenient. The growth rate of algal cultures on the flue gas was even higher when compared with the control culture supplied by a mixture of pure CO₂ and air (11% (v/v) CO₂). Correspondingly, the CO₂ fixation rate was also higher when using the flue gas (4.4 g CO₂ l⁻¹ 24 h⁻¹) than using the control gas (3.0 g CO₂ l⁻¹ 24 h⁻¹). The toxicological analysis of the biomass produced using untreated flue gas showed only a slight excess of mercury while all the other compounds (other heavy metals, polycyclic aromatic hydrocarbons, polychlorinated dibenzodioxins and dibenzofurans, and polychlorinated biphenyls) were below the limits required by the European Union foodstuff legislation. Fortunately, extending the flue gas treatment prior to the cultivation unit by a simple granulated activated carbon column led to an efficient absorption of gaseous mercury and to the algal biomass composition compliant with all the foodstuff legislation requirements.     

Characterization of Xanthobacter strains H4-14 and 25a and enzyme profiles after growth under autotrophic and heterotrophic conditions

All Xanthobacter strains studied are versatile autotrophic bacteria, able to grow on methanol and other substrates. Strain 25a, a yellow-pigmented, pleomorphic, Gram-negative bacterium, capable of autotrophic growth on methanol, formate, thiosulfate, and molecular hydrogen, was isolated from an enrichment culture inoculated with soil from a subtropical greenhouse. Subsequent studies showed that the organism also grows on a wide range of multicarbon substrates. Ammonia, nitrate and molecular nitrogen were used as nitrogen sources. The taxonomic relationship of strains H4-14 and 25a with previously described Xanthobacter strains was studied by numerical classification. Strain H4-14 was identified as a X. flavus strain, but the precise position of strain 25a remained uncertain. It probably belongs to a new species of the genus Xanthobacter. The levels of various enzymes involved in autotrophic and heterotrophic metabolism were determined following growth of strains H4-14 and 25a in batch and continuous cultures. The mechanisms involved in controlling ribulose-1,5-bisphosphate carboxylase/oxygenase synthesis in Xanthobacter strains appear to be comparable to those observed for other autotrophic bacteria, namely repression by organic compounds and derepression by autotrophic energy sources, such as methanol and hydrogen.Abbreviations API appareils et procédés d'identification - CS citrate synthase - ED Entner-Doudoroff pathway - FBP fructose-1,6-bisphosphate - FDH formate dehydrogenase - HPS hexulose-6-phosphate synthase - ICDH isocitrate dehydrogenase - KDPG 2-keto-3-deoxy-6-phosphogluconate - MDH methanol dehydrogenase - PRK phosphoribulokinase - PQQ pyrrolo quinoline quinone - RuBisC/O ribulose-1,5-bisphosphate carboxylase/oxygenase - RuMP ribulose monophosphate     

Microalgal biomass production and on-site bioremediation of carbon dioxide, nitrogen oxide and sulfur dioxide from flue gas using Chlorella sp. cultures

The growth and on-site bioremediation potential of an isolated thermal- and CO?-tolerant mutant strain, Chlorella sp. MTF-7, were investigated. The Chlorella sp. MTF-7 cultures were directly aerated with the flue gas generated from coke oven of a steel plant. The biomass concentration, growth rate and lipid content of Chlorella sp. MTF-7 cultured in an outdoor 50-L photobioreactor for 6 days was 2.87 g L?1 (with an initial culture biomass concentration of 0.75 g L?1), 0.52 g L?1 d?1 and 25.2%, respectively. By the operation with intermittent flue gas aeration in a double-set photobioreactor system, average efficiency of CO? removal from the flue gas could reach to 60%, and NO and SO? removal efficiency was maintained at approximately 70% and 50%, respectively. Our results demonstrate that flue gas from coke oven could be directly introduced into Chlorella sp. MTF-7 cultures to potentially produce algal biomass and efficiently capture CO?, NO and SO? from flue gas.