Production of poly(3-hydroxybutyrate) from inexpensive substrates

Two inexpensive substrates, starch and whey were used to produce poly(3-hydroxybutyrate) (PHB) in fed-batch cultures of Azotobacter chroococcum and recombinant Escherichia coli, respectively. Oxygen limitation increased PHB contents in both fermentations. In fed-batch culture of A. chroococcum, cell concentration of 54 g l⁻¹ with 46% PHB was obtained with oxygen limitation, whereas 71 g l⁻¹ of cell with 20% PHB was obtained without oxygen limitation. The timing of PHB biosynthesis in recombinant E. coli was controlled using the agitation speed of a stirred tank fermentor. A PHB content of 80% could be obtained with oxygen limitation by increasing the agitation speed up to only 500 rpm.     

Fate of Escherichia coli O145 present naturally in bovine slurry applied to vegetables before harvest,after washing and simulated wholesale and retail distribution

Aims

To determine the fate of Escherichia coli on vegetables that were processed through commercial wash treatments and stored under simulated retail conditions at 4°C or wholesale at fluctuating ambient temperatures (0–25°C, dependent on season).

Methods and Results

Bovine slurry that was naturally contaminated with E. coli O145 was applied without dilution or diluted 1:10 using borehole water to growing potatoes, leeks or carrots. Manure was applied 1 week prior to harvest to simulate a near‐harvest contamination event by manure deposition or an application of contaminated water to simulate a flooding event or irrigation from a contaminated water source. At harvest, crops were contaminated at up to 2 log cfu g^?1. Washing transferred E. coli into the water of a flotation tank used for potato washing and did not completely remove all traces of contamination from the crop. Manure‐contaminated potatoes were observed to contain 0·72 cfu E. coli O145 g^?1 after processing and retail storage. Manure‐contaminated leeks harboured 0·73–1·55 cfu E. coli O145 g^?1 after washing and storage. There was no cross‐contamination when leeks were spray washed. Washing in an abrasive drum resulted in less than perfect decontamination for manure‐contaminated carrots. There were five post‐distribution isolations from carrots irrigated with contaminated water 24 h prior to harvest.

Conclusions

Standard commercial washing and distribution conditions may be insufficient to reliably control human pathogenic E. coli on fresh produce.

Significance and Impact

Previous speculation that the cause of a UK foodborne disease outbreak was soil from imperfectly cleaned vegetables is plausible.     

High cell density media for <Emphasis Type="Italic">Escherichia coli</Emphasis> are generally designed for aerobic cultivations – consequences for large-scale bioprocesses and shake flask cultures

Background  

For the cultivation of Escherichia coli in bioreactors trace element solutions are generally designed for optimal growth under aerobic conditions. They do normally not contain selenium and nickel. Molybdenum is only contained in few of them. These elements are part of the formate hydrogen lyase (FHL) complex which is induced under anaerobic conditions. As it is generally known that oxygen limitation appears in shake flask cultures and locally in large-scale bioreactors, function of the FHL complex may influence the process behaviour. Formate has been described to accumulate in large-scale cultures and may have toxic effects on E. coli.     

Engineering an efficient secretion of leech carboxypeptidase inhibitor in Escherichia coli

Background  

Despite advances in expression technologies, the efficient production of heterologous secreted proteins in Escherichia coli remains a challenge. One frequent limitation relies on their inability to be exported to the E. coli periplasm. However, recent studies have suggested that translational kinetics and signal sequences act in concert to modulate the export process.     

The constancy of global regulation across a species: the concentrations of ppGpp and RpoS are strain-specific in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background  

Sigma factors and the alarmone ppGpp control the allocation of RNA polymerase to promoters under stressful conditions. Both ppGpp and the sigma factor σ^S (RpoS) are potentially subject to variability across the species Escherichia coli. To find out the extent of strain variation we measured the level of RpoS and ppGpp using 31 E. coli strains from the ECOR collection and one reference K-12 strain.     

Bacteriocin synthesis in uropathogenic and commensal <Emphasis Type="Italic">Escherichia coli</Emphasis>: colicin E1 is a potential virulence factor

Background  

Bacteriocin production is an important characteristic of E. coli strains of human origin. To date, 26 colicin and 9 microcin types have been analyzed on a molecular level allowing molecular detection of the corresponding genes. The production incidence of 29 bacteriocin types and E. coli phylogroups were tested in a set of 361 E. coli strains isolated from human urinary tract infections (UTI) and in 411 control strains isolated from feces of patients without bacterial gut infection.     

Oxygen limitation modulates pH regulation of catabolism and hydrogenases, multidrug transporters, and envelope composition inEscherichia coli K-12

Background  

InEscherichia coli, pH regulates genes for amino-acid and sugar catabolism, electron transport, oxidative stress, periplasmic and envelope proteins. Many pH-dependent genes are co-regulated by anaerobiosis, but the overall intersection of pH stress and oxygen limitation has not been investigated.     

Persistence of enterohaemorrhagic and nonpathogenic E. coli on spinach leaves and in rhizosphere soil*

Aims: Survival of Escherichia coli O157:H7 and nonpathogenic E. coli on spinach leaves and in organic soil while growing spinach in a growth chamber was investigated. Methods and Results: Spinach plants were maintained in the growth chamber at 20°C (14 h) and 18°C (10 h) settings at 60% relative humidity. Five separate inocula, each containing one strain of E. coli O157:H7 and one nonpathogenic E. coli isolate were applied to individual 4‐week‐old spinach plants (cultivar ‘Whale’) grown in sandy soil. Leaf and soil inocula consisted of 100 μl, in 5 μl droplets, on the upper side of leaves resulting in 6·5 log CFU plant^?1 and 1 ml in soil, resulting in 6·5 log CFU 200 g^?1 soil per plant. Four replicates of each plant shoot and soil sample per inoculum were analysed on day 1 and every 7 days for 28 days for E. coli O157:H7 and nonpathogenic E. coli (by MPN) and for heterotrophic plate counts (HPC). Escherichia coli O157:H7 was not detected on plant shoots after 7 days but did survive in soil for up to 28 days. Nonpathogenic E. coli survived up to 14 days on shoots and was detected at low concentrations for up to 28 days. In contrast, there were no significant differences in HPC from days 0 to 28 on plants, except one treatment on day 7. Conclusions: Escherichia coli O157:H7 persisted in soil for at least 28 days. Escherichia coli O157:H7 on spinach leaves survived for less than 14 days when co‐inoculated with nonpathogenic E. coli. There was no correlation between HPC and E. coli O157:H7 or nonpathogenic E. coli. Significance and Impact of the Study: The persistence of nonpathogenic E. coli isolates makes them possible candidates as surrogates for E. coli O157:H7 on spinach leaves in field trials.     

Impact of the <Emphasis Type="Italic">rpoS</Emphasis> genotype for acid resistance patterns of pathogenic and probiotic <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background  

Enterohemorrhagic E. coli (EHEC), a subgroup of Shiga toxin (Stx) producing E. coli (STEC), may cause severe enteritis and hemolytic uremic syndrome (HUS) and is transmitted orally via contaminated foods or from person to person. The infectious dose is known to be very low, which requires most of the bacteria to survive the gastric acid barrier. Acid resistance therefore is an important mechanism of EHEC virulence. It should also be a relevant characteristic of E. coli strains used for therapeutic purposes such as the probiotic E. coli Nissle 1917 (EcN). In E. coli and related enteric bacteria it has been extensively demonstrated, that the alternative sigma factor σ^S, encoded by the rpoS gene, acts as a master regulator mediating resistance to various environmental stress factors.     

Production of biohydrogen by recombinant expression of [NiFe]-hydrogenase 1 in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background  

Hydrogenases catalyze reversible reaction between hydrogen (H₂) and proton. Inactivation of hydrogenase by exposure to oxygen is a critical limitation in biohydrogen production since strict anaerobic conditions are required. While [FeFe]-hydrogenases are irreversibly inactivated by oxygen, it was known that [NiFe]-hydrogenases are generally more tolerant to oxygen. The physiological function of [NiFe]-hydrogenase 1 is still ambiguous. We herein investigated the H₂ production potential of [NiFe]-hydrogenase 1 of Escherichia coli in vivo and in vitro. The hya A and hya B genes corresponding to the small and large subunits of [NiFe]-hydrogenase 1 core enzyme, respectively, were expressed in BL21, an E. coli strain without H₂ producing ability.     

Identifying the mechanism of Escherichia coli O157:H7 survival by the addition of salt in the treatment with organic acids

Aim

In this study, the effects of the addition of salt to treatment with acids (one of several organic acids and salt in various solutions including rich or minimal broth, buffer, or distilled water) on the reduction of Escherichia coli O157:H7 were investigated. The protein expression profiles corresponding to acid stress (acetic acid) with or without salt addition were studied using a comparative proteomic analysis of E. coli O157:H7.

Methods and Results

When acetic, lactic, or propionic acid was combined with 3% NaCl, mutually antagonistic effects of acid and salt on viability of E. coli O157:H7 were observed only in tryptone and yeast extract broth. After exposure to acetic acid alone or in combination with salt, approximately 851 and 916 protein spots were detected, respectively. Analysis of 10 statistically significant differentially expressed proteins revealed that these proteins are mainly related to energy metabolism.

Conclusions

When we compared protein expression of E. coli O157:H7 treated with acetic acid and the combination of the acid and salt, the differentially expressed proteins were not related to acid stress‐ and salt stress‐inducible proteins such as stress shock proteins.

Significance and Impact of the Study

According to these results, the increased resistance of E. coli O157:H7 to acetic acid after the addition of salt may not be the result of synthesis of proteins related to these phenomena; therefore, further research needs to be conducted to identify the mechanism of the mutually antagonistic effect of some organic acids and salt.     

Role of the ArcAB two-component system in the resistance of Escherichia coli to reactive oxygen stress

Background  

The global regulatory system ArcAB controls the anaerobic growth of E. coli, however, its role in aerobic conditions is not well characterized. We have previously reported that ArcA was necessary for Salmonella to resist reactive oxygen species (ROS) in aerobic conditions.     

Production of <Emphasis Type="SmallCaps">l</Emphasis>-phenylalanine from glycerol by a recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis>

The production of l-phenylalanine is conventionally carried out by fermentations that use glucose or sucrose as the carbon source. This work reports on the use of glycerol as an inexpensive and abundant sole carbon source for producing l-phenylalanine using the genetically modified bacterium Escherichia coli BL21(DE3). Fermentations were carried out at 37°C, pH 7.4, using a defined medium in a stirred tank bioreactor at various intensities of impeller agitation speeds (300–500 rpm corresponding to 0.97–1.62 m s⁻¹ impeller tip speed) and aeration rates (2–8 L min⁻¹, or 1–4 vvm). This highly aerobic fermentation required a good supply of oxygen, but intense agitation (impeller tip speed ~1.62 m s⁻¹) reduced the biomass and l-phenylalanine productivity, possibly because of shear sensitivity of the recombinant bacterium. Production of l-phenylalanine was apparently strongly associated with growth. Under the best operating conditions (1.30 m s⁻¹ impeller tip speed, 4 vvm aeration rate), the yield of l-phenylalanine on glycerol was 0.58 g g⁻¹, or more than twice the best yield attainable on sucrose (0.25 g g⁻¹). In the best case, the peak concentration of l-phenylalanine was 5.6 g L⁻¹, or comparable to values attained in batch fermentations that use glucose or sucrose. The use of glycerol for the commercial production of l-phenylalanine with E. coli BL21(DE3) has the potential to substantially reduce the cost of production compared to sucrose- and glucose-based fermentations.     

Development of a combined immunochromatographic lateral flow assay for accurate and rapid Escherichia coli O157:H7 detection

Escherichia coli O157:H7 is an important pathogenic Bacterium that threatens human health. A convenient, sensitive and specific method for the E. coli O157:H7 detection is necessary. We developed two pairs of monoclonal antibodies through traditional hybridoma technology, one specifically against E. coli O157 antigen and the other specifically against E. coli H7 antigen. Using these two pairs of antibodies, we developed two rapid test kits to specifically detect E. coli O157 antigen and E. coli H7 antigen, respectively. The detection sensitivity for O157 positive E. coli is 1 × 10³ CFU per ml and for H7 positive E. coli is 1 × 10⁴ CFU per ml. Combining these two pairs of antibodies together, we developed a combo test strip that can specifically detect O157: H7, with a detection sensitivity of 1 × 10⁴ CFU per ml, when two detection lines are visible to the naked eye. This is currently the only rapid detection reagent that specifically detects O157: H7 by simultaneously detecting O157 antigen and H7 antigens of E. coli. Our product has advantages of simplicity and precision, and can be a very useful on-site inspection tool for accurate and rapid detection of E. coli O157:H7 infection.     

Physiological relation between respiration activity and heterologous expression of selected benzoylformate decarboxylase variants in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background  

The benzoylformate decarboxylase (BFD) from Pseudomonas putida is a biotechnologically interesting biocatalyst. It catalyses the formation of chiral 2-hydroxy ketones, which are important building blocks for stereoselective syntheses. To optimise the enzyme function often the amino acid composition is modified to improve the performance of the enzyme. So far it was assumed that a relatively small modification of the amino acid composition of a protein does not significantly influence the level of expression or media requirements. To determine, which effects these modifications might have on cultivation and product formation, six different BFD-variants with one or two altered amino acids and the wild type BFD were expressed in Escherichia coli SG13009 pKK233-2. The oxygen transfer rate (OTR) as parameter for growth and metabolic activity of the different E. coli clones was monitored on-line in LB, TB and modified PanG mineral medium with the Respiratory Activity MOnitoring System (RAMOS).     

Herd prevalence of Enterobacteriaceae producing CTX‐M‐type and CMY‐2 β‐lactamases among Japanese dairy farms

Aims

To determine the herd prevalence of Enterobacteriaceae producing CTX‐M‐type extended‐spectrum β‐lactamases (ESBLs) among 381 dairy farms in Japan.

Methods and Results

Between 2007 and 2009, we screened 897 faecal samples using BTB lactose agar plates containing cefotaxime (2 μg ml^?1). Positive isolates were tested using ESBL confirmatory tests, PCR and sequencing for CTX‐M, AmpC, TEM and SHV. The incidence of Enterobacteriaceae producing CTX‐M‐15 (n = 7), CTX‐M‐2 (n = 12), CTX‐M‐14 (n = 3), CMY‐2 (n = 2) or CTX‐M‐15/2/14 and CMY‐2 (n = 4) in bovine faeces was 28/897 (3·1%) faecal samples. These genes had spread to Escherichia coli (n = 23) and three genera of Enterobacteriaceae (n = 5). Herd prevalence was found to be 20/381 (5·2%) dairy farms. The 23 E. coli isolates showed clonal diversity, as assessed by multilocus sequence typing and pulsed‐field gel electrophoresis. The pandemic E. coli strain ST131 producing CTX‐M‐15 or CTX‐M‐27 was not detected.

Conclusions

Three clusters of CTX‐M (CTX‐M‐15, CTX‐M‐2, CTX‐M‐14) had spread among Japanese dairy farms.

Significance and Impact of the Study

This is the first report on the prevalence of multidrug‐resistant CTX‐M‐15–producing E. coli among Japanese dairy farms.     

Study of the Action of Se and Cu on the Growth Metabolism of <Emphasis Type="Italic">Escherichia coli</Emphasis> by Microcalorimetry

The biological effect of Se and Cu²⁺ on Escherichia coli (E. coli) growth was studied by using a 3114/3236 TAM Air Isothermal Calorimeter, ampoule method, at 37°C. From the thermogenesis curves, the thermokinetic equations were established under different conditions. The kinetics showed that a low concentration of Se (1–10 μg/mL) promoted the growth of E. coli, and a high concentration of Se (>10 μg/mL) inhibited the growth, but the Cu²⁺ was always inhibiting the growth of E. coli. Moreover, there was an antagonistic or positive synergistic effect of Se and Cu²⁺ on E. coli in the different culture medium when Se was 1–10 μg/ml and Cu²⁺ was 1–20 μg/ml. There was a negative synergistic effect of Se and Cu²⁺ on E. coli when Se was higher than 10 μg/ml and Cu²⁺ was higher than 20 μg/ml. The antagonistic or synergistic effect between Se and Cu²⁺ on E. coli was related to the formation of Cu–Se complexes under the different experimental conditions chosen.     

Evaluation of Petrifilm™ EC method for enumeration of E. coli from soil

Aims: To evaluate the suitability of commercially available Petrifilm? EC plates for enumeration of Escherichia coli from soil. Methods and Results: A confirmed E. coli strain isolated from liquid swine manure was inoculated into sterilized sandy clay loam and loam soils at the concentrations of 10², 10³, 10⁵ CFU g^?1 of soil. The efficiency of recovery on Petrifilm? EC plates for soils spiked with E. coli was compared with standard membrane filtration techniques on m‐FC basal medium supplemented with 3‐bromo‐4‐chloro‐5‐indoyl‐β‐d ‐glucopyranoside (BCIG) and most probable numbers (MPN) techniques in E. coli medium with 4‐methylumbelliferyl‐β‐d ‐glucuronide (EC‐MUG) broth. Petrifilm? EC and m‐FC (BCIG) methods were then assessed for the ability to recover E. coli from field soils applied with swine manure. No significant differences (P > 0·05) were observed between Petrifilm? EC, m‐FC (BCIG) and MPN methods for the recovery of E. coli from spiked samples, irrespective of soil type. However, recovery of E. coli from manure‐applied field soil samples showed a significant difference (P < 0·05) between the Petrifilm? EC method and the m‐FC method in enumerating E. coli possibly as a result of false positives on m‐FC. Conclusion: The Petrifilm? EC method is suitable for the enumeration of E. coli from soil with a detection limit of 10 CFU g^?1 soil. Significance and Impact of the Study: The commercially available Petrifilm? EC method is comparatively low cost, easy to use method for the enumeration of E. coli from soil without the need for further confirmation tests.     

Enhanced <Emphasis Type="SmallCaps">l</Emphasis>-phenylalanine production by recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> BR-42 (pAP-B03) resistant to bacteriophage BP-1 via a two-stage feeding approach

The l-phenylalanine (l-Phe) production by Escherichia coli WSH-Z06 (pAP-B03) was frequently prevented by bacteriophage BP-1 infestation. To cope with the bacteriophage BP-1 problem for an improved l-Phe production, one bacteriophage BP-1-resistant mutant, E. coli BR-42, was obtained from 416 mutant colonies of E. coli WSH-Z06 after N-methyl-N’-nitro-N-nitrosoguanidine (NTG) mutagenesis by selection for resistance to bacteriophage BP-1. The recombinant E. coli BR-42-carrying plasmid pAP-B03 had a high capacity in l-Phe production and a remarkable tolerance to 1 × 10¹⁰ pfu (plaque-forming unit)/ml bacteriophage stock. For an enhanced l-Phe production by E. coli BR-42 (pAP-B03), the effects of different feeding strategies including pH–stat, constant rate feeding, linear decreasing rate feeding, and exponential feeding on l-Phe production were investigated; and a two-stage feeding strategy, namely exponential feeding at μ _set = 0.18 h⁻¹ in the first 20 h and a following linear varying rate feeding with F = (−0.55 × t + 18.6) ml/h, was developed to improve l-Phe production. With this two-stage feeding approach, a maximum l-Phe titer of 57.63 g/l with a high l-Phe productivity (1.15 g/l/h) was achieved, which was 15% higher than the highest level (50 g/l) reported so far according to our knowledge. The recombinant E. coli BR-42 (pAP-B03) is a potential l-Phe over-producer in substantial prevention of bacteriophage BP-1 infestation compared to its parent strain WSH-Z06 (pAP-B03).     

A fed‐batch based cultivation mode in Escherichia coli results in improved specific activity of a novel chimeric‐truncated form of tissue plasminogen activator

Aims

A novel chimeric‐truncated form of tissue‐type plasminogen activator (t‐PA) with improved fibrin affinity and resistance to PAI was successfully produced in CHO expression system during our previous studies. Considering advantages of prokaryotic expression systems, the aim in this study was to produce the novel protein in Escherichia coli (BL21) strain and compare the protein potency in batch and fed‐batch processes.

Methods and Results

The expression cassette for the novel t‐PA was prepared in pET‐28a(+). The E. coli expression procedure was compared in traditional batch and newly developed fed batch, EnBase^® Flo system. The protein was purified in soluble format, and potency results were identified using Chromolize t‐PA Assay Kit. The fed‐batch fermentation mode, coupled with a Ni‐NTA affinity purification procedure under native condition, resulted in higher amounts of soluble protein, and about a 30% of improvement in the specific activity of the resulted recombinant protein (46·66 IU mg^?1) compared to traditional batch mode (35·8 IU mg^?1).

Conclusions

Considering the undeniable advantages of expression in the prokaryotic expression systems such as E. coli for recombinant protein production, applying alternative methods of cultivation is a promising approach. In this study, fed‐batch cultivation methods showed the potential to replace miss‐folded formats of protein with proper folded, soluble form with improved potency.

Significance and Impact of the Study

Escherichia coli expression of recombinant proteins still counts for nearly 40% of marketed biopharmaceuticals. The major drawback of this system is the lack of appropriate post‐translational modifications, which may cause potency loss/decline. Therefore, applying alternative methods of cultivation as investigated here is a promising approach to overcome potency decrease problem in this protein production system.