- Letters to the Editor
- Open Access
Evaluation of microbial globin promoters for oxygen-limited processes using Escherichia coli
© The Author(s). 2017
- Received: 1 March 2017
- Accepted: 26 September 2017
- Published: 13 November 2017
Oxygen-responsive promoters can be useful for synthetic biology applications, however, information on their characteristics is still limited. Here, we characterized a group of heterologous microaerobic globin promoters in Escherichia coli. Globin promoters from Bacillus subtilis, Campylobacter jejuni, Deinococcus radiodurans, Streptomyces coelicolor, Salmonella typhi and Vitreoscilla stercoraria were used to express the FMN-binding fluorescent protein (FbFP), which is a non-oxygen dependent marker. FbFP fluorescence was monitored online in cultures at maximum oxygen transfer capacities (OTRmax) of 7 and 11 mmol L−1 h−1. Different FbFP fluorescence intensities were observed and the OTRmax affected the induction level and specific fluorescence emission rate (the product of the specific fluorescence intensity multiplied by the specific growth rate) of all promoters. The promoter from S. typhi displayed the highest fluorescence emission yields (the quotient of the fluorescence intensity divided by the scattered light intensity at every time-point) and rate, and together with the promoters from D. radiodurans and S. coelicolor, the highest induction ratios. These results show the potential of diverse heterologous globin promoters for oxygen-limited processes using E. coli.
- Microaerobic promoters
- Oxygen-limited cultures
- Globin promoters
- FbFP expression
Oxygen limitation can easily occur in high cell-density cultures due to technical and economic constraints that limit mass transfer in bioreactors. Oxygen limitation is commonly undesirable in cultures of E. coli because it causes strong unwanted metabolic deviations. However, operating the bioreactor at maximum oxygen transfer capacities (OTRmax) would be advantageous from an economy standpoint. By modifying the metabolism of E. coli, it is possible to decrease the amount of byproducts formation and to improve the biomass yield and growth rate under microaerobic conditions . Consequently, oxygen-limited bioprocesses could be an interesting option for the synthesis of valuable molecules, using self-inducible promoters that trigger transcription upon oxygen limitation. The development of such processes will require the availability of characterized promoters for the assembly of synthetic pathways. Oxygen-responsive promoters could also be applied as biosensors to detect oxygen-limited zones in bioreactors. We have previously characterized the performance of homologous oxygen sensitive promoters of E. coli and the promoter of the Vitreoscilla stercoraria hemoglobin (P vgb ) in oxygen-limited cultures . From a group of 14 promoters evaluated, P vgb showed interesting characteristics like good repression under aerobic conditions and the highest induction ratio. This suggests that heterologous globin promoters could be viable tools for driving oxygen responsive gene expression in E. coli. Koskenkorva and coworkers  searched globin promoters from Bacillus subtilis (P Bs ), Campylobacter jejuni (P Cj ), Deinococcus radiodurans (P Dr ), Streptomyces coelicolor (P Sc ), and Salmonella typhi (P St ). The promoters were isolated and cloned in a plasmid to express chloramphenicol acetyl transferase (CAT) in E. coli. When cultured in shake flasks at low shaking frequency (150 rpm), maximum CAT activity was reported for all promoters after 2 h of culture, and decreased afterwards . Despite the relevance of such results, further characterization of the promoters under defined conditions is required. Namely, the cultures were performed in complex medium without pH and dissolved oxygen tension (DOT) monitoring. Furthermore, the OTR was not reported, and the dynamics of CAT expression in cultures not shown. Synthetic biology applications require standardized and well characterized parts. In this context, the effect of environmental conditions on the promoter activity is of prime relevance, particularly if bioprocess applications are sought. In the present contribution, the abovementioned promoters were synthesized and used to express the FMN binding fluorescent protein (FbFP). FbFP is an adequate reporter because of its fast activation independent from oxygen . The assembly included the Shine-Dalgarno sequence and 8 bp spacer region as in our previous report , which allows a direct comparison of the results. Oxygen-limited cultures were performed in round well microtiter plates with optodes for pH and DOT monitoring using a chemically defined medium. Two filling volumes (1500 and 2400 μL per well) were used, which result in OTRmax values of ca. 11 and 7 mmol L−1 h−1, respectively . Expression of FbFP under control of the constitutive promoter P kat (which controls the expression of the aminoglycoside phosphotransferase gene kat), was used as a control to assess the effect of OTRmax in a constitutive expression system.
During the oxygen-limited period the fluorescence yields were similar for cultures at OTRmax of 7 or 11 mmol L−1 h−1 for all the promoters. The fluorescence yields for P St and P vgb were noticeably higher than for the rest of the globin promoters. These results differ from the previous report from Koskenkorva and coworkers , who found that the P Dr displayed the highest activity. These differences could be related to genetic factors and culture conditions. First, RBS used in this work and the reported by Koskenkorva and coworkers  are different. Also, the use of different 5′ UTR sequences and/or reporter genes as compared with these authors could lead to differences in regulation or apparent promoter strength through unwanted interactions on different levels of expression [10, 11]. Concerning the culture conditions, the studies by Koskenkorva et al.  were performed using LB medium, and an E. coli K12 strain, which could produce different results. Moreover, cultures were carried out in unbuffered medium , and therefore strong pH fluctuations are expected . However, pH values were not informed by the authors. In the present study, the maximum fluorescence emission yields were reached during the phase of DOT raise. The maximum fluorescence emission yield was greater in cultures at OTRmax ca. 11 mmol L−1 h−1 than in cultures at OTRmax ca. 7 mmol L−1 h−1 for most promoters, except for P St and P vgb . In all cases, the fluorescence yield were relatively stable after oxygen raise when the OTRmax was ca. 7 mmol L−1 h−1, but rapidly decreased at OTRmax ca. 11 mmol L−1 h−1. Again, P St and P vgb were the exceptions, as fluorescence yields decreased fast after the point of DOT raise (Fig. 4e and f).
The highest specific fluorescence emission rate under oxygen-limited conditions was observed for P St (Fig. 5c). In cultures at OTRmax ca. 11 mmol L−1 h−1 the specific fluorescence emission rate increased during the oxygen-limited phase, compared to the aerobic phase for the different promoters, except for P vgb (Fig. 5d). The result for P vgb is coincident with a previous study under similar conditions . The specific fluorescence emission rates under oxygen-limited conditions were greater at OTRmax ca. 11 mmol L−1 h−1, compared to those obtained at OTRmax ca. 7 mmol L−1 h−1. This is most probably a result of the limited resources for energy generation and biomass synthesis under oxygen-limitation.
The data set presented here provides useful information for the selection of oxygen sensitive promoters for particular designs. Severe oxygen limitation (OTRmax ca. 7 mmol L−1 h−1) seems to negatively affect the activity of most of the globin promoters studied. Nevertheless, cell engineering strategies aimed at improving the metabolic performance and energy generation by aerobic respiration of E. coli under oxygen-limited conditions can increase the specific fluorescence emission rate . Altogether, the information shown contributes to expand the toolbox for synthetic biology applications under bioprocessing conditions. For example, it opens the possibility to explore further combinations of these promoters with other reporter genes, 5’UTR and RBS sequences [10, 11].
Escherichia coli strain BL21 (DE3) was used as expression host. E. coli BL21 was transformed with each plasmid and conserved at −80 °C in a solution of 40% v/v glycerol.
Parts synthesis and assembly
The globin promoters used correspond to the reported by Koskenkorva and co-workers . The sequences were obtained from the NCBI database and are detailed, together with their accession number, in the Additional file 1. A ribosome binding site (RBS) (Shine-Dalgarno sequence) and a spacer region of 8 bases were added previous to the start codon. FbFP sequence was taken from Evocatal (Düsseldorf, Germany, Cat. No.: 2.1.030) and the rrnb T1 terminator was added downstream. All the sequences were flanked by a HindIII restriction sequence and cloned in the same orientation (5′-3′). The complete sequences were synthesized and cloned in pUC57kan by GenScript (Piscataway, NJ, USA).
Precultures were grown in terrific broth (TB) consisting of 12 g L−1 tryptone, 24 g L−1 yeast extract, 12.54 g L−1, K2HPO4, 2.31 g L−1, KH2PO4, and 5 g L−1 glycerol. The main cultures were carried out using a mineral medium supplemented with 3-(N-morpholino)-propanesulfonic acid (MOPS) at a final concentration of 0.2 M, described elsewhere  and the pH was adjusted to 7.4 prior to sterilization. Glucose was added at final concentration of 5 g L−1. Kanamycin sulfate was used in all the cultures at a concentration of 50 μg mL−1.
For pre-culture development, 100 μL of cryopreserved cells were used to inoculate 10 mL of TB and grown at 30 °C in 250 mL Erlenmeyer flasks shaken at a frequency of 300 rpm with a shaking diameter of 50 mm for 8 h. 1 mL of this culture was transferred to 250 mL Erlenmeyer flasks containing 50 mL of the mineral medium. The cells were grown at 37 °C and shaking frequency of 300 rpm for 6–8 h. This time corresponds to the exponential growth phase, and the absorbance of the broth (measured at 600 nm) was around 2.0. This culture was used to inoculate the microbioreactors at an initial absorbance of 0.1 units. Microbioreactor cultures were performed using the BioLector system (m2p Labs, Beasweiler, Germany), which allows online measurement of cell growth, DOT, pH and fluorescence as indicator of FbFP expression using 48 round wells plates (MTP-R48-BOH, Lot 1402, m2p Labs, Beasweiler, Germany). Plates were sealed with a hydrophobic porous rayon sterile sealing film (AeraSeal, Excel Scientific, CA, USA). Cultures were performed at 37 °C, 85% humidity, shaking diameter of 3 mm, and shaking frequency of 700 rpm. Depending on the experiment, the culture volume per well was 1500 or 2400 μL. Biomass was monitored by scattered light (λ ex = 620 nm; gain: 20). Fluorescence was used to monitor DOT (λ ex = 520 nm; λ em = 600 nm; gain: 83), pH (λ ex = 485 nm; λ em = 530 nm; gain: 45) and FbFP (λ ex = 450 nm; λ em = 492 nm; gain: 90). The OTRmax values were taken from Funke et al. 2009 . All the experiments included three technical replicates.
The initial data of scattered light and fluorescence intensity were subtracted from the measured data. Parameters for promoter characterization were determined during the aerobic or oxygen-limited phases. Specific fluorescence intensity was determined as the slope in the plot of fluorescence intensity (F-F0) versus scattered light intensity (I-I0) data points. The specific fluorescence emission rate was calculated as the product of μ multiplied by the specific fluorescence intensity. Fluorescence emission yields were calculated dividing the FbFP fluorescence intensity by the scattered light intensity of each time-point. For calculating the parameters under aerobic conditions, data corresponding to 2–4 h of culture were used for both OTRmax conditions, except for P vgb , for which data from 1 to 2.5 h (OTRmax ca. 7 mmol L−1 h−1) and 1–3.5 h (OTRmax ca. 11 mmol L−1 h−1) were used. For oxygen-limited conditions at OTRmax ca. 7 mmol L−1 h−1, the data from 4 to 7.5 (P kat ), 4.9–8.7 (P Bs ), 4–8.5 (P Cj ), 4.2–8.9 (P Dr and P Sc ), 4.9–9.4 (P St ) and 2.6–8.7 (P vgb ) h of culture were used. For calculating the parameters in cultures under oxygen-limited conditions at OTRmax ca. 11 mmol L−1 h−1, the data from 4 to 7.5 (P kat ), 4.9–8.7 (P Bs ), 4.5–8.2 (P Cj ), 5.2–8.9 (P Dr and P Sc ), 6.4–9.8 (P St ) and 3.5–7.9 (P vgb ) h of culture were used.
ArcA Component A of the Aerobic respiratory control protein (the response regulator component)
CRP Cyclic AMP receptor protein
d 0 Shaking diameter (mm)
DOT Dissolved oxygen tension (% air saturation)
FbFP FMN binding fluorescent protein
FNR Fumarate and nitrate reductase (transcriptional activator)
n shaking frequency (rpm)
q F Specific fluorescence intensity rate (AU AU−1 h−1)
OTR Oxygen transfer rate (mmol L−1 h−1)
VL Volume of the liquid phase (μL)
λ em Emission wavelength [nm]
λ ex Excitation wavelength [nm]
μ Specific growth rate (h−1)
This work was supported by CONACyT grants 256,617, 264,460 and 248,926.
Availability of data and materials
The authors declare that the data supporting the findings of this study are available within the article and its additional file.
Promoters sequences used to control the expression of FbFP.
ARL conceived the project, performed the cultures and data analyses. KEJ and JCS contributed to the design of the expression systems. LR and JB contributed in the design of the experiments and general data interpretation. All the authors participated in preparing the manuscript. All authors read and approved the final manuscript.
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