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.     

Production of recombinant Chikungunya virus envelope 2 protein in Escherichia coli

Chikungunya, a mosquito-borne viral disease caused by Chikungunya virus (CHIKV), has drawn substantial attention after its reemergence causing massive outbreaks in tropical regions of Asia and Africa. The recombinant envelope 2 (rE2) protein of CHIKV is a potential diagnostic as well as vaccine candidate. Development of cost-effective cultivation media and appropriate culture conditions are generally favorable for large-scale production of recombinant proteins in Escherichia coli. The effects of medium composition and cultivation conditions on the production of recombinant Chikungunya virus E2 (rCHIKV E2) protein were investigated in shake flask culture as well as batch cultivation of Escherichia coli. Further, the fed-batch process was also carried out for high cell density cultivation of E. coli expressing rE2 protein. Expression of rCHIKV E2 protein in E. coli was induced with 1 mM isopropyl-beta-thiogalactoside (IPTG) at ~23 g dry cell weight (DCW) per liter of culture and yielded an insoluble protein aggregating to form inclusion bodies. The final DCW after fed-batch cultivation was ~35 g/l. The inclusion bodies were isolated, solubilized in 8 M urea and purified through affinity chromatography to give a final product yield of ~190 mg/l. The reactivity of purified E2 protein was confirmed by Western blotting and enzyme-linked immunosorbent assay. These results show that rE2 protein of CHIKV may be used as a diagnostic reagent or for further prophylactic studies. This approach of producing rE2 protein in E. coli with high yield may also offer a promising method for production of other viral recombinant proteins.     

Plasmid pUPI126-encoded pyrrolnitrin production by Acinetobacter haemolyticus A19 isolated from the rhizosphere of wheat

An Acinetobacter species identified as A. haemolyticus A19 produces an antibiotic and the enzyme chitinase. The antibiotic produced by A. haemolyticus A19 was extracellular and inducible by co-cultivation with Klebsiella pneumoniae in the optimum ratio 2:1, respectively. pH 7, temperature 28 °C, and addition of 2 % (w/v) NaCl are the most suitable environmental conditions for production and activity of the antibiotic. The antibiotic was produced in the early stationary growth phase (48 h) of A. haemolyticus A19. It has a very broad spectrum of antimicrobial activity against plant and human pathogenic bacteria and fungi. The antibiotic was extracted with ethyl acetate and purified by column chromatography with further purification by preparative thin-layer chromatography. Yield of the antibiotic was 15 mg/l. The antibiotic was active at very low concentrations, for example 50 μg/ml, and was water-soluble. It was stable at room temperature for up to 7 days. ¹H NMR analysis revealed the antibiotic was a pyrrolnitrin. It was found that pyrrolnitrin production by A. haemolyticus A19 was encoded by plasmid pUPI126 of molecular weight 25.7 kb. Plasmid pUPI126 was transferred to E. coli HB101 at a frequency of 5 × 10^?5 per μg DNA. It was also conjugally transformed to E. coli HB101 rif ^r mutants at a frequency of 5.9 × 10^?8 per recipient cell. Plasmid pUPI126 was 100 % stable in Acinetobacter and 95 % stable in E. coli HB101. Transconjugants and transformants both produced the antibiotic. This is the first report of plasmid-mediated pyrrolnitrin production by A. haemolyticus A19 isolated from wheat rhizosphere.     

Engineering a <Emphasis Type="Italic">Streptomyces coelicolor</Emphasis> biosynthesis pathway into <Emphasis Type="Italic">Escherichia coli</Emphasis> for high yield triglyceride production

Background

Microbial lipid production represents a potential alternative feedstock for the biofuel and oleochemical industries. Since Escherichia coli exhibits many genetic, technical, and biotechnological advantages over native oleaginous bacteria, we aimed to construct a metabolically engineered E. coli strain capable of accumulating high levels of triacylglycerol (TAG) and evaluate its neutral lipid productivity during high cell density fed-batch fermentations.

Results

The Streptomyces coelicolor TAG biosynthesis pathway, defined by the acyl-CoA:diacylglycerol acyltransferase (DGAT) Sco0958 and the phosphatidic acid phosphatase (PAP) Lppβ, was successfully reconstructed in an E. coli diacylglycerol kinase (dgkA) mutant strain. TAG production in this genetic background was optimized by increasing the levels of the TAG precursors, diacylglycerol and long-chain acyl-CoAs. For this we carried out a series of stepwise optimizations of the chassis by 1) fine-tuning the expression of the heterologous SCO0958 and lpp β genes, 2) overexpression of the S. coelicolor acetyl-CoA carboxylase complex, and 3) mutation of fadE, the gene encoding for the acyl-CoA dehydrogenase that catalyzes the first step of the β-oxidation cycle in E. coli. The best producing strain, MPS13/pET28-0958-ACC/pBAD-LPPβ rendered a cellular content of 4.85% cell dry weight (CDW) TAG in batch cultivation. Process optimization of fed-batch fermentation in a 1-L stirred-tank bioreactor resulted in cultures with an OD_600nm of 80 and a product titer of 722.1 mg TAG L^-1 at the end of the process.

Conclusions

This study represents the highest reported fed-batch productivity of TAG reached by a model non-oleaginous bacterium. The organism used as a platform was an E. coli BL21 derivative strain containing a deletion in the dgkA gene and containing the TAG biosynthesis genes from S. coelicolor. The genetic studies carried out with this strain indicate that diacylglycerol (DAG) availability appears to be one of the main limiting factors to achieve higher yields of the storage compound. Therefore, in order to develop a competitive process for neutral lipid production in E. coli, it is still necessary to better understand the native regulation of the carbon flow metabolism of this organism, and in particular, to improve the levels of DAG biosynthesis.

     

Increased production of γ-lactones from hydroxy fatty acids by whole Waltomyces lipofer cells induced with oleic acid

Among several fatty acids tested, oleic acid was selected as the most efficient inducer for the production of 4-hydroxydodecanoic acid, a metabolite of β-oxidation, by Waltomyces lipofer. Cells were induced by incubation for 12 h in a medium containing 10 g l^?1 yeast extract, 10 g l^?1 peptone, 5 g l^?1 oleic acid, 1 g l^?1 glucose, and 0.05 % (w/v) Tween 80. The optimal reaction conditions for the production of γ-lactones by induced cells were pH 6.5, 35 °C, 200 rpm, 0.71 M Tris, 60 g l^?1 hydroxy fatty acid, and 20 g l^?1 cells. Non-induced cells produced 38 g l^?1 γ-dodecalactone from 60 g l^?1 10-hydroxystearic acid after 30 h, with a conversion yield of 63 % (w/w) and a productivity of 1.3 g l^?1 h^?1 under the optimized conditions, whereas induced cells produced 51 g l^?1 γ-dodecalactone from 60 g l^?1 10-hydroxystearic acid after 30 h, with a conversion yield of 85 % (w/w) and a productivity of 1.7 g l^?1 h^?1. The conversion yield and productivity of induced cells were 22 % and 1.3-fold higher, respectively, than those of non-induced cells. Induced cells also produced 28 g l^?1 γ-decalactone and 12 g l^?1 γ-butyrolactone from 60 g l^?1 12-hydroxystearic acid and 60 g l^?1 10-hydroxydecanoic acid, respectively, after 30 h. The concentration, conversion yield, and productivity of γ-dodecalactone and γ-decalactone are the highest reported thus far. This is the first study on the biotechnological production of γ-butyrolactone.     

The role of antioxidants enzymes of E. coli ASU3, a tolerant strain to heavy metals toxicity, in combating oxidative stress of copper

The current study describes the isolation and characterization of E. coli from wastewater that collected from El-Malah canal in Assiut, Egypt. Twelve isolates were investigated for heavy metal resistance by which one of them showed multiple metal resistances. Furthermore, the bacterium was identified as E. coli ASU3 according to biochemical tests and then, preserved at Assuit University Mycological Centre with accession number AUMC B83. It exhibited high minimal inhibitory concentrations for metals and antibiotic resistance. The order of metals toxicity to the bacterium was Cr⁶⁺ > Cu²⁺ > Co²⁺ > Pb²⁺ > Ni²⁺ > Cr³⁺ > Cd²⁺ > Zn²⁺. Total protein content of E. coli ASU3 decreased with the increase of copper concentration. Under exposure of different concentrations of copper, the induction of antioxidant enzymes such as catalase, peroxidase and ascorbate peroxidase was increased and these antioxidant enzymes can contribute to combating oxidative stresses.     

Fatty alcohol production in engineered E. coli expressing Marinobacter fatty acyl-CoA reductases

Although successful production of fatty alcohols in metabolically engineered Escherichia coli with heterologous expression of fatty acyl-CoA reductase has been reported, low biosynthetic efficiency is still a hurdle to be overcome. In this study, we examined the characteristics of two fatty acyl-CoA reductases encoded by Maqu_2220 and Maqu_2507 genes from Marinobacter aquaeolei VT8 on fatty alcohol production in E. coli. Fatty alcohols with diversified carbon chain length were obtained by co-expressing Maqu_2220 with different carbon chain length-specific acyl-ACP thioesterases. Both fatty acyl-CoA reductases displayed broad substrate specificities for C12–C18 fatty acyl chains in vivo. The optimized mutant strain of E. coli carrying the modified tesA gene and fadD gene from E. coli and Maqu_2220 gene from Marinobacter aquaeolei VT8 produced fatty alcohols at a remarkable level of 1.725 g/L under the fermentation condition.     

Enhanced production of the nonribosomal peptide antibiotic valinomycin in Escherichia coli through small-scale high cell density fed-batch cultivation

Nonribosomal peptides (NRPs), a large family of natural products, possess numerous pharmaceutically significant bioactivities. However, many native microbial producers of NRPs are not cultivable or have low production yields making mass production infeasible. The recombinant production of natural products in a surrogate host has emerged as a strategy to overcome these limitations. De novo recombinant production of the NRP antibiotic valinomycin in an engineered Escherichia coli host strain was established with the necessary biosynthetic pathway constituents from Streptomyces tsusimaensis. In the present study, the initially modest valinomycin yields could be significantly increased from 0.3 up to 2.4 mg L^?1 by switching from a batch to an enzyme-based fed-batch mode in shake flasks. A subsequent design of experiment-driven optimization of parallel fed-batch cultivations in 24-well plates with online monitoring of dissolved oxygen and pH led to valinomycin yields up to 6.4 mg L^?1. Finally, repeated glucose polymer feeding to enzyme-based high cell density cultivations in shake flasks resulted in cell densities of OD₆₀₀ >50 and a valinomycin titer of appr. 10 mg L^?1. This represents a 33-fold improvement compared to the initial batch cultivations and is the highest concentration of a nonribosomal peptide which has been produced in E. coli without feeding of specific precursors so far to our knowledge. Also, such a small-scale optimization under fed-batch conditions may be generally applicable for the development and scale-up of natural product production processes in E. coli.     

Engineered <Emphasis Type="Italic">E. coli</Emphasis> W enables efficient 2,3-butanediol production from glucose and sugar beet molasses using defined minimal medium as economic basis

Background

Efficient microbial production of chemicals is often hindered by the cytotoxicity of the products or by the pathogenicity of the host strains. Hence 2,3-butanediol, an important drop-in chemical, is an interesting alternative target molecule for microbial synthesis since it is non-cytotoxic. Metabolic engineering of non-pathogenic and industrially relevant microorganisms, such as Escherichia coli, have already yielded in promising 2,3-butanediol titers showing the potential of microbial synthesis of 2,3-butanediol. However, current microbial 2,3-butanediol production processes often rely on yeast extract as expensive additive, rendering these processes infeasible for industrial production.

Results

The aim of this study was to develop an efficient 2,3-butanediol production process with E. coli operating on the premise of using cost-effective medium without complex supplements, considering second generation feedstocks. Different gene donors and promoter fine-tuning allowed for construction of a potent E. coli strain for the production of 2,3-butanediol as important drop-in chemical. Pulsed fed-batch cultivations of E. coli W using microaerobic conditions showed high diol productivity of 4.5 g l^?1 h^?1. Optimizing oxygen supply and elimination of acetoin and by-product formation improved the 2,3-butanediol titer to 68 g l^?1, 76% of the theoretical maximum yield, however, at the expense of productivity. Sugar beet molasses was tested as a potential substrate for industrial production of chemicals. Pulsed fed-batch cultivations produced 56 g l^?1 2,3-butanediol, underlining the great potential of E. coli W as production organism for high value-added chemicals.

Conclusion

A potent 2,3-butanediol producing E. coli strain was generated by considering promoter fine-tuning to balance cell fitness and production capacity. For the first time, 2,3-butanediol production was achieved with promising titer, rate and yield and no acetoin formation from glucose in pulsed fed-batch cultivations using chemically defined medium without complex hydrolysates. Furthermore, versatility of E. coli W as production host was demonstrated by efficiently converting sucrose from sugar beet molasses into 2,3-butanediol.

     

Enhanced production of l-phenylalanine in Corynebacterium glutamicum due to the introduction of Escherichia coli wild-type gene aroH

Metabolic engineering is a powerful tool which has been widely used for producing valuable products. For improving l-phenylalanine (l-Phe) accumulation in Corynebacterium glutamicum, we have investigated the target genes involved in the biosynthetic pathways. The genes involved in the biosynthesis of l-Phe were found to be strictly regulated genes by feedback inhibition. As a result, overexpression of the native wild-type genes aroF, aroG or pheA resulted in a slight increase of l-Phe. In contrast, overexpression of aroF ^wt or pheA ^fbr from E. coli significantly increased l-Phe production. Co-overexpression of aroF ^wt and pheA ^fbr improved the titer of l-Phe to 4.46 ± 0.06 g l^?1. To further analyze the target enzymes in the aromatic amino acid synthesis pathway between C. glutamicum and E. coli, the wild-type gene aroH from E. coli was overexpressed and evaluated in C. glutamicum. As predicted, upregulation of the wild-type gene aroH resulted in a remarkable increase of l-Phe production. Co-overexpression of the mutated pheA ^fbr and the wild-type gene aroH resulted in the production of l-Phe up to 4.64 ± 0.09 g l^?1. Based on these results we conclude that the wild-type gene aroH from E. coli is an appropriate target gene for pathway engineering in C. glutamicum for the production of aromatic amino acids.     

Recruiting alternative glucose utilization pathways for improving succinate production

The phosphoenolpyruvate (PEP): carbohydrate phosphotransferase system (PTS) of Escherichia coli was usually inactivated to increase PEP supply for succinate production. However, cell growth and glucose utilization rate decreased significantly with PTS inactivation. In this work, two glucose transport proteins and two glucokinases (Glk) from E. coli and Zymomonas mobilis were recruited in PTS^? strains, and their impacts on glucose utilization and succinate production were compared. All PTS^? strains recruiting Z. mobilis glucose facilitator Glf had higher glucose utilization rates than PTS^? strains using E. coli galactose permease (GalP), which was suggested to be caused by higher glucose transport velocity and lower energetic cost of Glf. The highest rate obtained by combinatorial modulation of glf and glk _{E. coli} (2.13 g/L?h) was 81 % higher than the wild-type E. coli and 30 % higher than the highest rate obtained by combinatorial modulation of galP and glk _{E. coli} . On the other hand, although glucokinase activities increased after replacing E. coli Glk with isoenzyme of Z. mobilis, glucose utilization rate decreased to 0.58 g/L?h, which was assumed due to tight regulation of Z. mobilis Glk by energy status of the cells. For succinate production, using GalP led to a 20 % increase in succinate productivity, while recruiting Glf led to a 41 % increase. These efficient alternative glucose utilization pathways obtained in this work can also be used for production of many other PEP-derived chemicals, such as malate, fumarate, and aromatic compounds.     

Model-assisted formate dehydrogenase-O (fdoH) gene knockout for enhanced succinate production in Escherichia coli from glucose and glycerol carbon sources

Succinic acid is an important platform chemical that has broad applications and is been listed as one of the top twelve bio-based chemicals produced from biomass by the US Department of Energy. The metabolic role of Escherichia coli formate dehydrogenase-O (fdoH) under anaerobic conditions in relation to succinic acid production remained largely unspecified. Herein we report, what are to our knowledge, the first metabolic fdoH gene knockout that have enhanced succinate production using glucose and glycerol substrates in E. coli. Using the most recent E. coli reconstruction iJO1366, we engineered its host metabolism to enhance the anaerobic succinate production by deleting the fdoH gene, which blocked H⁺ conduction across the mutant cell membrane for the enhanced succinate production. The engineered mutant strain BMS4 showed succinate production of 2.05 g l^?1 (41.2-fold in 7 days) from glycerol and .39 g l^?1 (6.2-fold in 1 day) from glucose. This work revealed that a single deletion of the fdoH gene is sufficient to increase succinate production in E. coli from both glucose and glycerol substrates.     

Enhancing isomaltulose production by recombinant Escherichia coli producing sucrose isomerase: culture medium optimization containing agricultural wastes and cell immobilization

Isomaltulose is a structural isomer of sucrose commercially used in food industries. In this work, recombinant Escherichia coli producing sucrose isomerase (SIase) was used to convert sucrose into isomaltulose. To develop an economical industrial medium, untreated cane molasses (10.63 g l^?1), yeast extract (25.93 g l^?1), and corn steep liquor (10.45 g l^?1) were used as main culture compositions for SIase production. The relatively high SIase activity (14.50 ± 0.11 U mg DCW^?1) was obtained by the recombinant cells. To the best of our knowledge, this is the first investigation on SIase production by engineered E. coli using untreated cane molasses. The recombinant E. coli cells expressing the SIase gene were immobilized in calcium alginate gel in order to improve the efficiency of recycling. The immobilization was most effective with 2 % (w/v) sodium alginate and 3 % (w/v) calcium chloride. The optimal initial biomass for immobilization was 20 % (w/v, wet wt.), with a hardening time of 8 h for cell immobilization. The immobilized E. coli cells exhibited good stability for 30 batches with the productivity of 0.45 g isomaltulose g pellet^?1 h^?1. A continuous isomaltulose formation process using a column reactor remained stable for 40 days with 83 ± 2 % isomaltulose yield, which would be beneficial for economical production of isomaltulose.     

Carotenoid and lipid production by the autotrophic microalga Chlorella protothecoides under nutritional, salinity, and luminosity stress conditions

Today microalgae represent a viable alternative source for high-value products. The specie Chlorella protothecoides (Cp), heterotrophically grown, has been widely studied and provides a high amount of lutein and fatty acids (FA) and has a good profile for biodiesel production. This work studies carotenoid and FA production by autotrophic grown Cp. Cp was grown until the medium’s nitrogen was depleted, then diluted in NaCl solution, resulting in nutritional, luminosity, and salinity stresses. Different NaCl concentrations were tested (10, 20, 30 g/L) at two different dilutions. After dilution, a color shifting from green to orange-red was noticed, showing carotenoid production. The best production of both carotenoids and FA was attained with a 20 g/L NaCl solution. The total carotenoid content was 0.8 % w/w (canthaxanthin (23.3 %), echinenone (14.7 %), free astaxanthin (7.1 %), and lutein/zeaxanthin (4.1 %)). Furthermore, the total lipid content reached 43.4 % w/w, with a FA composition of C18:1 (33.64 %), C16:0 (23.30 %), C18:2 (11.53 %), and less than 12 % of C18:3, which is needed to fulfill the biodiesel quality specifications (EN 14214).     

Modification of glycolysis and its effect on the production of l-threonine in Escherichia coli

High concentrations of acetate, the main by-product of Escherichia coli (E. coli) high cell density culture, inhibit bacterial growth and l-threonine production. Since metabolic overflux causes acetate accumulation, we attempted to reduce acetate production by redirecting glycolysis flux to the pentose phosphate pathway by deleting the genes encoding phosphofructokinase (pfk) and/or pyruvate kinase (pyk) in an l-threonine-producing strain of E. coli, THRD. pykF, pykA, pfkA, and pfkB deletion mutants produced less acetate (9.44 ± 0.83, 3.86 ± 0.88, 0.30 ± 0.25, and 6.99 ± 0.85 g/l, respectively) than wild-type THRD cultures (19.75 ± 0.93 g/l). THRDΔpykF and THRDΔpykA produced 11.05 and 5.35 % more l-threonine, and achieved a 10.91 and 5.60 % higher yield on glucose, respectively. While THRDΔpfkA grew more slowly and produced less l-threonine than THRD, THRDΔpfkB produced levels of l-threonine (102.28 ± 2.80 g/l) and a yield on glucose (0.34 g/g) similar to that of THRD. The dual deletion mutant THRDΔpfkBΔpykF also achieved low acetate (7.42 ± 0.81 g/l) and high l-threonine yields (111.37 ± 2.71 g/l). The level of NADPH in THRDΔpfkA cultures was depressed, whereas all other mutants produced more NADPH than THRD did. These results demonstrated that modification of glycolysis in E. coli THRD reduced acetate production and increased accumulation of l-threonine.     

A homomeric geranyl diphosphate synthase-encoding gene from <Emphasis Type="Italic">Camptotheca acuminata</Emphasis> and its combinatorial optimization for production of geraniol in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Geranyl diphosphate (GPP), the unique precursor for all monoterpenoids, is biosynthesized from isopentenyl diphosphate and dimethylallyl diphosphate via the head-to-tail condensation reaction catalyzed by GPP synthase (GPPS). Herein a homomeric GPPS from Camptotheca acuminata, a camptothecin-producing plant, was obtained from 5′- and 3′-rapid amplification of cDNA ends and subsequent overlap extension and convenient PCR amplifications. The truncate CaGPPS was introduced to replace ispA of pBbA5c-MevT(CO)-MBIS(CO, ispA), a de novo biosynthetic construct for farnesyl diphosphate generation, and overexpressed in Escherichia coli, together with the truncate geraniol synthase-encoding gene from C. acuminata (tCaGES), to confirm CaGPPS-catalyzed reaction in vivo. A 24.0 ± 1.3 mg L^?1 of geraniol was produced in the recombinant E. coli. The production of GPP was also validated by the direct UPLC-HRMS^E analyses. The tCaGPPS and tCaGES genes with different copy numbers were introduced into E. coli to balance their catalytic potential for high-yield geraniol production. A 1.6-fold increase of geraniol production was obtained when four copies of tCaGPPS and one copy of tCaGES were introduced into E. coli. The following fermentation conditions optimization, including removal of organic layers and addition of new n-decane, led to a 74.6 ± 6.5 mg L^?1 of geraniol production. The present study suggested that the gene copy number optimization, i.e., the ratio of tCaGPPS and tCaGES, plays an important role in geraniol production in the recombinant E. coli. The removal and addition of organic solvent are very useful for sustainable high-yield production of geraniol in the recombinant E. coli in view of that the solubility of geraniol is limited in the fermentation broth and/or n-decane.     

n-Butanol Production from Acid-Pretreated Jatropha Seed Cake by Clostridium acetobutylicum

n-Butanol fermentation using Clostridium strains suffers from low titers due to the inability of the strains to tolerate n-butanol. The current study demonstrates a process to get high titer of n-butanol in a single batch mode from the renewable feedstock jatropha seed cake by employing Clostridium acetobutylicum. Chemical mutagenesis was done for improvement of the strain for better n-butanol tolerance and production. Optimization of the parameters resulted in 13.2 g L^?1 of n-butanol in 120 h using acid-treated jatropha seed cake hydrolysate (7 %?w/v) in anaerobic sugar medium. The process was scaled up to 15 L level, yielding 18.6 g L^?1 of n-butanol in 72 h. The strain was found to be tolerant up to 30 g L^?1 n-butanol under optimized conditions. The n-butanol tolerance was accompanied by over-expression of the stress response protein, GroEL, change in fatty acid profile, and ability to accumulate rhodamine 6G in the strain. The study has a significant impact on economically producing n-butanol from biomass.     

Regulation of poly-(R)-(3-hydroxybutyrate-co-3-hydroxyvalerate) biosynthesis by the AtoSCDAEB regulon in phaCAB + Escherichia coli

AtoSC two-component system (TCS) upregulates the high-molecular weight poly-(R)-3-hydroxybutyrate (PHB) biosynthesis in recombinant phaCAB ⁺ Escherichia coli strains, with the Cupriavidus necator phaCAB operon. We report here that AtoSC upregulates also the copolymer P(3HB-co-3HV) biosynthesis in phaCAB ⁺ E. coli. Acetoacetate-induced AtoSC maximized P(3HB-co-3HV) to 1.27 g/l with a 3HV fraction of 25.5 % wt. and biopolymer content of 75 % w/w in a time-dependent process. The atoSC locus deletion in the ?atoSC strains resulted in 4.5-fold P(3HB-co-3HV) reduction, while the 3HV fraction of the copolymer was restricted to only 6.4 % wt. The ?atoSC phenotype was restored by extrachromosomal introduction of AtoSC. Deletion of the atoDAEB operon triggered a significant decrease in P(3HB-co-3HV) synthesis and 3HV content in ?atoDAEB strains. However, the acetoacetate-induced AtoSC in those strains increased P(3HB-co-3HV) to 0.8 g/l with 21 % 3HV, while AtoC or AtoS expression increased P(3HB-co-3HV) synthesis 3.6- or 2.4-fold, respectively, upon acetoacetate. Complementation of the ?atoDAEB phenotype was achieved by the extrachromosomal introduction of the atoSCDAEB regulon. Individual inhibition of β-oxidation and mainly fatty acid biosynthesis pathways by acrylic acid or cerulenin, respectively, reduced P(3HB-co-3HV) biosynthesis. Under those conditions, introduction of atoSC or atoSCDAEB regulon was capable of upregulating biopolymer accumulation. Concurrent inhibition of both the fatty acid metabolic pathways eliminated P(3HB-co-3HV) production. P(3HB-co-3HV) upregulation in phaCAB ⁺ E. coli by AtoSC signaling through atoDAEB operon and its participation in the fatty acids metabolism interplay provide additional perceptions of AtoSC critical involvement in E. coli regulatory processes towards biotechnologically improved polyhydroxyalkanoates biosynthesis.     

Analytical limits of four β-glucuronidase and β-galactosidase-based commercial culture methods used to detect Escherichia coli and total coliforms

Colilert^® (Colilert), Readycult^® Coliforms 100 (Readycult), Chromocult^® Coliform agar ES (Chromocult), and MI agar (MI) are β-galactosidase and β-glucuronidase-based commercial culture methods used to assess water quality. Their analytical performance, in terms of their respective ability to detect different strains of Escherichia coli and total coliforms, had never been systematically compared with pure cultures. Here, their ability to detect β-glucuronidase production from E. coli isolates was evaluated by using 74 E. coli strains of different geographic origins and serotypes encountered in fecal and environmental settings. Their ability to detect β-galactosidase production was studied by testing the 74 E. coli strains as well as 33 reference and environmental non-E. coli total coliform strains. Chromocult, MI, Readycult, and Colilert detected β-glucuronidase production from respectively 79.9, 79.9, 81.1, and 51.4% of the 74 E. coli strains tested. These 4 methods detected β-galactosidase production from respectively 85.1, 73.8, 84.1, and 84.1% of the total coliform strains tested. The results of the present study suggest that Colilert is the weakest method tested to detect β-glucuronidase production and MI the weakest to detect β-galactosidase production. Furthermore, the high level of false-negative results for E. coli recognition obtained by all four methods suggests that they may not be appropriate for identification of presumptive E. coli strains.