Characterization of rhamnolipid biosurfactants produced by recombinant <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> strain DAB with removal of crude oil

Objectives

To improve rhamnolipid production and its potential application in removal of crude oil, the recombinant Pseudomonas aeruginosa strain DAB was constructed to enhance yield of rhamnolipids.

Results

Strain DAB had a higher yield of 17.3 g rhamnolipids l^?1 in the removal process with crude oil as the sole carbon source than 10 g rhamnolipids l^?1 of wild-type strain DN1, where 1% crude oil was degraded more than 95% after 14 days cultivation. These rhamnolipids reduced the surface tension of water from 72.92 to 26.15 mN m^?1 with CMC of 90 mg l^?1. The predominant rhamnolipid congeners were Rha–C₁₀–C₁₀ and Rha–Rha–C₁₀–C₁₀ detected by MALDI-TOF MS analysis with approx. 70% relative abundance, although a total of 21 rhamnolipid congeners were accumulated.

Conclusion

Increasing the copy number of rhlAB genes efficiently enhanced the production of rhamnolipids by the recombinant P. aeruginosa DAB and thus presents a promising application for the bioremediation process.

     

Assessing biodegradation of furfuryl alcohol using <Emphasis Type="Italic">Pseudomonas putida</Emphasis> MTCC 1194 and <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> MTCC 1034 and its kinetics

The biodegradation of furfuryl alcohol (FA) in shake flask experiments using a pure culture of Pseudomonas putida (MTCC 1194) and Pseudomonas aeruginosa (MTCC 1034) was studied at 30 °C and pH 7.0. Experiments were performed at different FA concentrations ranging from 50 to 500 mg/l. Before carrying out the biodegradation studies, the bacterial strains were acclimatized to the concentration of 500 mg/l of FA by gradually raising 100 mg/l of FA in each step. The well acclimatized culture of P. putida and P. aeruginosa degraded about 80 and 66% of 50 mg/l FA, respectively. At higher concentration of FA, the percentage of FA degradation decreased. The purpose of this study was to determine the kinetics of biodegradation of FA by measuring biomass growth rates and concentration of FA as a function of time. Substrate inhibition was calculated from experimental growth parameters using the Haldane equation. Data for P. putida were determined as µ _max ?=?0.23 h^?1, K _s ?=?23.93 mg/l and K _i ?=?217.1 mg/l and for P. aeruginosa were determined as µ _max ?=?0.13 h^?1, K _s ?=?21.3 mg/l and K _i ?=?284.9 mg/l. The experimental data were fitted in Haldane, Aiba and Edwards inhibition models.     

Expression of extracellular polysaccharides and proteins by clinical isolates of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> in response to environmental conditions

The opportunistic pathogen Pseudomonas aeruginosa causes chronic respiratory infections in patients with cystic fibrosis (CF). Persistence of this bacterium is attributed to its ability to form biofilms which rely on an extracellular polymeric substance matrix. Extracellular polysaccharides (EPS) and secreted proteins are key matrix components of P. aeruginosa biofilms. Recently, nebulized magnesium sulfate has been reported as a significant bronchodilator for asthmatic patients including CF. However, the impact of magnesium sulfate on the virulence effect of P. aeruginosa is lacking. In this report, we investigated the influence of magnesium sulfate and other environmental factors on the synthesis of alginate and secretion of proteins by a mucoid and a non-mucoid strain of P. aeruginosa, respectively. By applying the Plackett-Burman and Box-Behnken experimental designs, we found that phosphates (6.0 g/l), ammonium sulfate (4.0 g/l), and trace elements (0.6 mg/l) markedly supported alginate production by the mucoid strain. However, ferrous sulfate (0.3 mg/l), magnesium sulfate (0.02 g/l), and phosphates (6.0 g/l) reinforced the secretion of proteins by the non-mucoid strain.     

Terpenoid bioactive compound from <Emphasis Type="Italic">Streptomyces rochei</Emphasis> (M32): taxonomy,fermentation and biological activities

The present study emphasized the production of biologically active terpenoid compound from Streptomyces rochei M32, which was isolated from Western Ghats ecosystem, South India. The presence of resistant genes like mecA, vanA of Staphylococcus aureus and bla _SHV, bla _TEM of Pseudomonas aeruginosa was confirmed by molecular studies. The isolated compound from Streptomyces rochei M32 inhibited wide range of standard and clinical drug resistant pathogens and enteric pathogens. The rice bran supplemented basal medium influenced the active compound production on 8th day of fermentation and yielded 1875 mg of crude extract from 10 g of rice bran substrate. Purification and characterization of crude ethyl acetate extract was achieved by preparative thin layer chromatography. The active fraction was identified as terpenoid class compound by chemical screening. Based on the results of spectral studies (NMR, LC–MS, FTIR, etc.), the active compound was tentatively identified as 1, 19-bis (3-hydroxyazetidin-1-yl) nonadeca-5, 14-diene-1, 8, 12, 19-tetraone with molecular weight 462.41 g/mol. Minimum inhibitory concentration value ranges between 7.6 and 31.2 µg/mL against test organisms was observed. The cytotoxicity results on cervical cancer (HeLa) cell line showed IC₅₀ value of 2.034 µg/mL. The corresponding compound is not previously reported from any microbial resources.     

Efficient production of indigoidine in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Indigoidine is a bacterial natural product with antioxidant and antimicrobial activities. Its bright blue color resembles the industrial dye indigo, thus representing a new natural blue dye that may find uses in industry. In our previous study, an indigoidine synthetase Sc-IndC and an associated helper protein Sc-IndB were identified from Streptomyces chromofuscus ATCC 49982 and successfully expressed in Escherichia coli BAP1 to produce the blue pigment at 3.93 g/l. To further improve the production of indigoidine, in this work, the direct biosynthetic precursor l-glutamine was fed into the fermentation broth of the engineered E. coli strain harboring Sc-IndC and Sc-IndB. The highest titer of indigoidine reached 8.81 ± 0.21 g/l at 1.46 g/l l-glutamine. Given the relatively high price of l-glutamine, a metabolic engineering technique was used to directly enhance the in situ supply of this precursor. A glutamine synthetase gene (glnA) was amplified from E. coli and co-expressed with Sc-indC and Sc-indB in E. coli BAP1, leading to the production of indigoidine at 5.75 ± 0.09 g/l. Because a nitrogen source is required for amino acid biosynthesis, we then tested the effect of different nitrogen-containing salts on the supply of l-glutamine and subsequent indigoidine production. Among the four tested salts including (NH₄)₂SO₄, NH₄Cl, (NH₄)₂HPO₄ and KNO₃, (NH₄)₂HPO₄ showed the best effect on improving the titer of indigoidine. Different concentrations of (NH₄)₂HPO₄ were added to the fermentation broths of E. coli BAP1/Sc-IndC+Sc-IndB+GlnA, and the titer reached the highest (7.08 ± 0.11 g/l) at 2.5 mM (NH₄)₂HPO₄. This work provides two efficient methods for the production of this promising blue pigment in E. coli.     

Enhancement of medium-chain-length polyhydroxyalkanoates biosynthesis from glucose by metabolic engineering in <Emphasis Type="Italic">Pseudomonas mendocina</Emphasis>

Objectives

To enhance the biosynthesis of medium-chain-length polyhydroxyalkanoates (PHA_MCL) from glucose in Pseudomonas mendocina NK-01, metabolic engineering strategies were used to block or enhance related pathways.

Results

Pseudomonas mendocina NK-01 produces PHA_MCL from glucose. Besides the alginate oligosaccharide biosynthetic pathway proved by our previous study, UDP-d-glucose and dTDP-l-rhamnose biosynthetic pathways were identified. These might compete for glucose with the PHA_MCL biosynthesis. First, the alg operon, galU and rmlC gene were deleted one by one, resulting in NK-U-1(?alg), NK-U-2 (?alg?galU), NK-U-3(alg?galU?rmlC). After fermentation for 36 h, the cell dry weight (CDW) and PHA_MCL production of these strains were determined. Compared with NK-U: 1) NK-U-1 produced elevated CDW (from 3.19 ± 0.16 to 3.5 ± 0.11 g/l) and equal PHA_MCL (from 0.78 ± 0.06 to 0.79 ± 0.07 g/l); 2) NK-U-2 produced more CDW (from 3.19 ± 0.16 to 3.55 ± 0.23 g/l) and PHA_MCL (from 0.78 ± 0.06 to 1.05 ± 0.07 g/l); 3) CDW and PHA_MCL dramatically decreased in NK-U-3 (1.53 ± 0.21 and 0.41 ± 0.09 g/l, respectively). Additionally, the phaG gene was overexpressed in strain NK-U-2. Although CDW of NK-U-2/phaG decreased to 1.29 ± 0.2 g/l, PHA titer (%CDW) significantly increased from 24.5 % up to 51.2 %.

Conclusion

The PHA_MCL biosynthetic pathway was enhanced by blocking branched metabolic pathways in combination with overexpressing phaG gene.

     

Medium Optimization for Improved Production of Dihydrolipohyl Dehydrogenase from <Emphasis Type="Italic">Bacillus sphaericus</Emphasis> PAD-91 in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Dihydrolipohyl dehydrogenase (DLD) is a FAD-dependent enzyme that catalyzes the reversible oxidation of dihydrolipoamide. Herein, we report medium optimization for the production of a recombinant DLD with NADH-dependent diaphorase activity from a strain of Bacillus sphaericus PAD-91. The DLD gene that consisted of 1413 bp was expressed in Escherichia coli BL21 (DE3), and its enzymatic properties were studied. The composition of production medium was optimized using one-variable-at-a-time method followed by response surface methodology (RSM). B. sphaericus DLD catalyzed the reduction of lipoamide by NAD⁺ and exhibited diaphorase activity. The molecular weight of enzyme was about 50 kDa and determined to be a monomeric protein. Recombinant diaphorase showed its optimal activity at temperature of 30 °C and pH 8.5. K _m and V _max values with NADH were estimated to be 0.025 mM and 275.8 U/mL, respectively. Recombinant enzyme was optimally produced in fermentation medium containing 10 g/L sucrose, 25 g/L yeast extract, 5 g/L NaCl and 0.25 g/L MgSO₄. At these concentrations, the actual diaphorase activity was calculated to be 345.0 ± 4.1 U/mL. By scaling up fermentation from flask to bioreactor, enzyme activity was increased to 486.3 ± 5.5 U/mL. Briefly, a DLD with diaphorase activity from a newly isolated B. sphaericus PAD-91 was characterized and the production of recombinant enzyme was optimized using RSM technique.     

Synthesis of natural variants and synthetic derivatives of the cyclic nonribosomal peptide luminmide in permeabilized <Emphasis Type="Italic">E. coli</Emphasis> Nissle and product formation kinetics

We used a recombinant, permeabilized E. coli Nissle strain harbouring the plu3263 gene cluster from Photorhabdus luminescens for the synthesis of luminmide type cyclic pentapeptides belonging to the class of nonribosomally biosynthesized peptides (NRP). Cells could be fully permeabilized using 1 % v/v toluene. Synthesis of luminmides was increased fivefold when 0.3 mM EDTA was added to the substrate mixture acting as an inhibitor of metal proteases. Luminmide formation was studied applying different amino acid concentrations. Apparent kinetic parameters for the synthesis of the main product luminmide A from leucine, phenylalanine and valine were calculated from the collected data. K _s ^app values ranged from 0.17 mM for leucine to 0.57 mM for phenylalanine, and r _max ^app was about 3 × 10^?8 mmol min^?1(g CDW)^?1). By removing phenylalanine from the substrate mixture, the formation of luminmide A was reduced tenfold while luminmide B was increased from 50 to 500 μg/l becoming the main product. Two new luminmides were synthesized in this study. Luminmide H incorporates tryptophan replacing phenylalanine in luminmide A. In luminmide I, leucine was replaced with 4,5-dehydro-leucine, a non-proteinogenic amino acid fed to the incubation mixture. Our study shows new opportunities for increasing the spectrum of luminmide variants produced, for improving production selectivity and for kinetic in vitro studies of the megasynthetases.     

Colony formation of highly dispersed <Emphasis Type="Italic">Microcystis aeruginosa</Emphasis> by controlling extracellular polysaccharides and calcium ion concentrations in aquatic solution

This study isolated extracellular polysaccharides (EPS) as a powder material from cyanobacterial blooms and the powdered EPS was used to trigger colony formation of dispersed unicellular M. aeruginosa by controlling EPS concentration in culture medium. The effect of Ca²⁺ ions on the colony formation of M. aeruginosa was also investigated, then the interaction between EPS and Ca²⁺ ions on colony formation was discussed. The results showed that the addition of the powdered EPS into the medium did not cause morphological changes of M. aeruginosa, suggesting that EPS alone would not induce the colony formation of M. aeruginosa. On the other hand, a high concentration of calcium ions (1000 mg/l) caused colony formation. When EPS and Ca²⁺ ions in the culture medium were adjusted to 200 and 1000 mg/l, respectively, the colony density, the average cell number per colony and the particle size of M. aeruginosa showed ca. 1.7–2.0 times greater values than those in the Ca²⁺ added medium. Calcium ion contributed to the aggregation of M. aeruginosa via crosslinked reaction with negatively charged M. aeruginosa cells, and the addition of EPS possessing negatively charged functional groups such as carboxy groups could enhance the reaction, promoting the crosslinked reaction between EPS and Ca²⁺ ions.     

Programmed cell death in the cyanobacterium <Emphasis Type="Italic">Microcystis aeruginosa</Emphasis> induced by allelopathic effect of submerged macrophyte <Emphasis Type="Italic">Myriophyllum spicatum</Emphasis> in co-culture system

This investigation demonstrates that programmed cell death (PCD) in a cyanobacterium, Microcystis aeruginosa, resulting from allelopathic stress induced by a submerged macrophyte, Myriophyllum spicatum, in a co-culture system. The hallmarks of PCD, caspase-3-like protease activity, DNA fragmentation, and destruction of cell ultrastructure, as well as intracellular PCD signaling radicals, reactive oxygen species (ROS), and nitric oxide (NO), were measured in M. aeruginosa cells co-cultured with M. spicatum for 7 days. The results showed a dose–response relationship between M. spicatum biomass and M. aeruginosa mortality. A caspase-3-like protease was activated and elevated from day 3. Thylakoid disintegration, cytoplasmic vacuolation, and fuzzy nuclear zone were observed by transmission electron microscopy, and distinct DNA fragmentation was detected in M. aeruginosa cells at a M. spicatum biomass of 6.0 g fresh weight (FW) L^?1 during the 7 days. Allelochemicals of total phenolic compounds (TPCs) were determined in co-culture water, and the concentration increased with increasing of M. spicatum biomass and co-culture time. Compared with the level of ROS production in the control group, a significant overproduction of ROS was detected in M. aeruginosa cells in the treatment group, and this was positively correlated with TPC concentration. Furthermore, the level of intracellular NO increased with the percent mortality of M. aeruginosa. The results indicated that a PCD pathway was induced in the cyanobacterium M. aeruginosa when co-cultured with the submerged macrophyte M. spicatum.     

Efficient <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of <Emphasis Type="Italic">Vigna mungo</Emphasis> using immature cotyledonary-node explants and phosphinothricin as the selection agent

Herbicide (Basta®)-tolerant Vigna mungo L. Hepper plants were produced using cotyledonary-node and shoot-tip explants from seedlings germinated in vitro from immature seeds. In vitro selection was performed with phosphinothricin as the selection agent. Explants were inoculated with Agrobacterium tumefaciens strain LBA4404 (harboring the binary vector pME 524 carrying the nptII, bar, and uidA genes) in the presence of acetosyringone. Shoot regeneration occurred for 6 wk on regeneration medium (MS medium with 4.44 μM benzyl adenine, 0.91 μM thidiazuron, and 81.43 μM adenine sulfate) with 2.4 mg/l PPT, explants being transferred to fresh medium every 14 d. After a period on elongation medium (MS medium with 2.89 μM gibberellic acid and 2.4 mg/l PPT), β-glucuronidase-expressing putative transformants were rooted in MS medium with 7.36 μM indolyl butyric acid and 2.4 mg/l PPT. β-Glucuronidase expression was observed in the primary transformants (T₀) and in the seedlings of the T₁ generation. Screening 128 GUS-expressing, cotyledonary-node-derived, acclimatized plants by spraying the herbicide Basta® at 0.1 mg/l eliminated nonherbicide-resistant plants. Southern hybridization analysis confirmed the transgenic nature of the herbicide-resistant plants. All the transformed plants were fertile, and the transgene was inherited by Mendelian genetics. Immature cotyledonary-node explants produced a higher frequency of transformed plants (7.6%) than shoot-tip explants (2.6%).     

Calcium regulates glutamate dehydrogenase and poly-γ-glutamic acid synthesis in <Emphasis Type="Italic">Bacillus natto</Emphasis>

Objective

To study the effect of Ca²⁺ on glutamate dehydrogenase (GDH) and its role in poly-γ-glutamic acid (γ-PGA) synthesis in Bacillus natto HSF 1410.

Results

When the concentration of Ca²⁺ varied from 0 to 0.1 g/l in the growth medium of B. natto HSF 1410, γ-PGA production increased from 6.8 to 9.7 g/l, while GDH specific activity and NH₄Cl consumption improved from 183 to 295 U/mg and from 0.65 to 0.77 g/l, respectively. GDH with α-ketoglutarate as substrate primarily used NADPH as coenzyme with a K _m of 0.08 mM. GDH was responsible for the synthesis of endogenous glutamate. The specific activity of GDH remained essentially unchanged in the presence of CaCl₂ (0.05–0.2 g/l) in vitro. However, the specific activity of GDH and its expression was significantly increased by CaCl₂ in vivo. Therefore, the regulation of GDH and PGA synthesis by Ca²⁺ is an intracellular process.

Conclusion

Calcium regulation may be an effective approach for producing γ-PGA on an industrial scale.

     

4-Vinylphenol production from glucose using recombinant <Emphasis Type="Italic">Streptomyces mobaraense</Emphasis> expressing a tyrosine ammonia lyase from <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

Objectives

To find a novel host for the production of 4-vinylphenol (4VPh) by screening Streptomyces species.

Results

The conversion of p-coumaric acid (pHCA) to 4VPh in Streptomyces mobaraense was evaluated using a medium containing pHCA. S. mobaraense readily assimilated pHCA after 24 h of cultivation to produce 4VPh. A phenolic acid decarboxylase, derived from S. mobaraense (SmPAD), was purified following heterologous expression in Escherichia coli. SmPAD was evaluated under various conditions, and the enzyme’s k_cat/K_m value was 0.54 mM ^?1 s^?1. Using intergenetic conjugation, a gene from Rhodobacter sphaeroides encoding a tyrosine ammonia lyase, which catalyzes the conversion of l-tyrosine to p-coumaric acid, was introduced into S. mobaraense. The resulting S. mobaraense transformant produced 273 mg 4VPh l^?1 from 10 g glucose l^?1.

Conclusion

A novel strain suitable for the production of 4VPh and potentially other aromatic compounds was isolated.

     

Cloning of <Emphasis Type="Italic">phaCAB</Emphasis> genes from thermophilic <Emphasis Type="Italic">Caldimonas manganoxidans</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis> for poly(3-hydroxybutyrate) (PHB) production

PHB biosynthesis pathway, consisting of three open reading frames (ORFs) that encode for β-ketothiolase (phaA _Cma , 1179 bp), acetoacetyl-CoA reductase (phaB _Cma , 738 bp), and PHA synthase (phaC _Cma , 1694 bp), of Caldimonas manganoxidans was identified. The functions of PhaA, PhaB, and PhaC were demonstrated by successfully reconstructing PHB biosynthesis pathway of C. manganoxidans in Escherichia coli, where PHB production was confirmed by OD₆₀₀, gas chromatography, Nile blue stain, and transmission electron microscope (TEM). The protein sequence alignment of PHB synthases revealed that phaC _Cma shares at least 60% identity with those of class I PHB synthase. The effects of PhaA, PhaB, and PhaC expression levels on PHB production were investigated. While the overexpression of PhaB is found to be important in recombinant E. coli, performances of PHB production can be quantified as follows: PHB concentration of 16.8 ± 0.6 g/L, yield of 0.28 g/g glucose, content of 74%, productivity of 0.28 g/L/h, and Mw of 1.41 MDa.     

Isolation of biosurfactant-producing <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> RS29 from oil-contaminated soil and evaluation of different nitrogen sources in biosurfactant production

An efficient biosurfactant-producing native Pseudomonas aeruginosa RS29 has been isolated from crude oil contaminated soil. Isolation was followed by optimization of different factors to achieve maximum production of biosurfactant in terms of surface tension reduction (STR) and emulsification index (E24). The isolated strain produced highest biosurfactant in the presence of glycerol after 48 h of incubation at 37.5°C, with pH range of 7–8 and at salinity <0.8% (w/v). The extent of STR and the E24 of medium with different nitrogen sources were investigated and found to be maximal for sodium nitrate (26.3 mN/m, E24?=?80%) and potassium nitrate (26.4 mN/m, E24?=?79%). The production of biomass by the designated strain was found to be maximal in ammonium-nitrate-containing medium as compared to the other nitrogen sources. A kinetic study revealed that biosurfactant production is positively correlated with growth of P. aeruginosa, and highest STR was achieved (27.0 mN/m) after 44 h of growth. The biosurfactant was produced as a primary metabolite and 6 g/L crude biosurfactant was extracted by chloroform:methanol (2:1). The critical micelle concentration of the biosurfactant was 90 mg/L. The absorption bands of the FTIR spectra confirmed the rhamnolipid nature of the biosurfactant. The biosurfactant was thermostable (up to 121°C for 15 min) and could withstand a wide range of pH (2–10) and NaCl concentration (2%–10% w/v). The extracted biosurfactant had good foaming and emulsifying activities and was of satisfactory quality in terms of stability (temperature, pH and salinity) and foaming activity.     

Biosynthesis of di-rhamnolipids and variations of congeners composition in genetically-engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

Objectives

To engineer Escherichia coli for the heterologous production of di-rhamnolipids, which are important biosurfactants but mainly produced by opportunistic pathogen Pseudomonas aeruginosa.

Results

The codon-optimized rhlAB and rhlC genes originating from P. aeruginosa and Burkholderia pseudomallei were combinatorially expressed in E. coli to produce di-rhamnolipids with varied congeners compositions. Genes involved in endogenous upstream pathways (rhamnose and fatty acids synthesis) were co-overexpressed with rhlAB–rhlC, resulting in variations of rhamnolipids production and congeners compositions. Under the shake-flask condition, co-overexpression of rfbD with rhlAB–rhlC increased rhamnolipids production (0.64 ± 0.02 g l^?1) than that in strain only expressing rhlAB–rhlC (0.446 ± 0.009 g l^?1), which was mainly composed of di-rhamnolipids congeners Rha–Rha–C₁₀–C₁₀.

Conclusion

Biosynthesis of di-rhamnolipids and variations of congeners composition in genetically engineered E. coli strains were achieved via combiniations of mono-/di-rhamnolipids synthesis modules and endogenous upstream modules.

     

Improvement of uridine production in <Emphasis Type="Italic">Bacillus subtilis</Emphasis> by metabolic engineering

Objectives

To construct a Bacillus subtilis strain for improved uridine production.

Results

The AAG_2846–2848 fragment of the pyrAB gene, encoding carbamoylphosphate synthetase, was deleted in B. subtilis TD246 leading to a 245% increase of uridine production and the conversion from glucose to uridine increased by 10.5%. Overexpression of the pyr operon increased the production of uridine by a further 31% and the conversion rate of glucose to uridine was increased by 18%. In addition, the blocking of arginine synthesis or disabling of glutamate dehydrogenase significantly enhanced the uridine production. The highest-producing strain, B. subtilis TD297, accumulated 11 g uridine/l with a yield of 240 mg uridine/g glucose in shake-flask cultivation.

Conclusion

This is the first report of engineered B. subtilis strains which can produce more than 11 g uridine/l, with a yield reaching 240 mg uridine/g glucose in shake-flask cultivation.

     

Engineering a glycerol utilization pathway in Corynebacterium glutamicum for succinate production under O<Subscript>2</Subscript> deprivation

Objective

To explore the glycerol utilization pathway in Corynebacterium glutamicum for succinate production under O₂ deprivation.

Result

Overexpression of a glycerol facilitator, glycerol dehydrogenase and dihydroxyacetone kinase from Escherichia coli K-12 in C. glutamicum led to recombinant strains NC-3G diverting glycerol utilization towards succinate production under O₂ deprivation. Under these conditions, strain NC-3G efficiently consumed glycerol and produced succinate without growth. The recombinant C. glutamicum utilizing glycerol as the sole carbon source showed higher intracellular NADH/NAD⁺ ratio compare with utilizing glucose. The mass conversion of succinate increased from 0.64 to 0.95. Using an anaerobic fed-batch fermentation process, the final strain produced 38.4 g succinate/l with an average yield of 1.02 g/g.

Conclusions

The metabolically-engineered strains showed an efficient succinate production using glycerol as sole carbon source under O₂ deprivation.

     

<Emphasis Type="Italic">Rhodococcus</Emphasis> bacteria as a promising source of oils from olive mill wastes

The accumulation of triacylglycerols (TAG) is a common feature among actinobacteria belonging to Rhodococcus genus. Some rhodococcal species are able to produce significant amounts of those lipids from different single substrates, such as glucose, gluconate or hexadecane. In this study we analyzed the ability of different species to produce lipids from olive oil mill wastes (OMW), and the possibility to enhance lipid production by genetic engineering. OMW base medium prepared from alperujo, which exhibited high values of chemical oxygen demand (127,000 mg/l) and C/N ratio (508), supported good growth and TAG production by some rhodococci. R. opacus, R. wratislaviensis and R. jostii were more efficient at producing cell biomass (2.2–2.7 g/l) and lipids (77–83% of CDW, 1.8–2.2 g/l) from OMW than R. fascians, R. erythropolis and R. equi (1.1–1.6 g/l of cell biomass and 7.1–14.0% of CDW, 0.1–0.2 g/l of lipids). Overexpression of a gene coding for a fatty acid importer in R. jostii RHA1 promoted an increase of 2.2 fold of cellular biomass value with a concomitant increase in lipids production during cultivation of cells in OMW. This study demonstrates that the bioconversion of OMW to microbial lipids is feasible using more robust rhodococal strains. The efficiency of this bioconversion can be significantly enhanced by engineering strategies.