Characterization of Endogenous and Reduced Promoters for Oxygen

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Characterization of endogenous and reduced promoters for oxygen-limited processes using Escherichia coli Alvaro R. Lara, Karim E. Jaén, Juan Carlos Sigala, Martina Mühlmann, Lars Regestein, and Jochen Büchs ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.6b00233 • Publication Date (Web): 07 Oct 2016 Downloaded from http://pubs.acs.org on October 8, 2016

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ACS Synthetic Biology

Characterization of endogenous and reduced promoters for oxygen-limited processes using Escherichia coli Alvaro R. Lara1*, Karim E. Jaén1, Juan-Carlos Sigala1, Martina Muehlmann2, Lars Regestein2, Jochen Büchs2** 1

Departamento de Procesos y Tecnología, Universidad Autónoma Metropolitana-Cuajimalpa. Av. Vasco de Quiroga 4871, Santa Fe, C.P. 05348, Mexico City, México

2

RWTH Aachen University, AVT - Biochemical Engineering, Worringer Weg 1, 52074 Aachen, Germany

*Corresponding author. E-mail: [email protected] **Corresponding author. E-mail: [email protected]

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ABSTRACT. Oxygen limitation can be used as a simple environmental inducer for the expression of target genes. However, there is scarce information on the characteristics of microaerobic promoters potentially useful for cell engineering and synthetic biology applications. Here, we characterized the Vitreoscilla hemoglobin promoter (Pvgb) and a set of microaerobic endogenous promoters in Escherichia coli. Oxygen-limited cultures at different maximum oxygen transfer rates were carried out. The FMN-binding fluorescent protein (FbFP), which is a non-oxygen dependent marker protein, was used as a reporter. Fluorescence and fluorescence emission rates under oxygen-limited conditions were the highest when FbFP was under transcriptional control of PadhE, Ppfl and Pvgb . The lengths of the E. coli endogenous promoters were shortened by 60 %, maintaining their key regulatory elements. This resulted in improved promoter activity in most cases, particularly for PadhE, Ppfl and PnarK. Selected promoters were also evaluated using an engineered E. coli strain expressing Vitreoscilla hemoglobin (VHb). The presence of the VHb resulted in a better repression using these promoters under aerobic conditions, and increased the specific growth and fluorescence emission rates under oxygen-limited conditions. These results are useful for the selection of promoters for specific applications and for the design of modified artificial promoters.

KEYWORDS: microaerobic promoters, oxygen-limited cultures, reduced promoters, Vitreoscilla hemoglobin, FbFP expression, microbioreactors


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Successful development of expression systems rely on the availability of well characterized promoters with suitable properties. For industrial applications, it is desirable that promoters can be induced by mechanisms that do not rely on the addition of a chemical agent1, 2, shifts of pH or temperature3. Oxygen limitation is an attractive means of inducing the expression of genes of interest. Current biotechnological processes are preponderantly based on aerobic cultures, which have the advantage of maximal energy generation per mole of carbon source. However, oxygen transfer rate (OTR) is often a constraint for bioreactor operation and scale-up, particularly if high celldensities are sought. Therefore, oxygen-limited processes may be a cost-effective option for industrial applications, having the advantage of requiring lower energy inputs and heat generation4, 5. Biopolymers like polyhydroxybutyrate (PHB)6 and alginate7 can be produced under oxygen limitation at relatively high levels. Moreover, several small molecules, including organic acids, ethanol and Lalanine, are efficiently produced under anaerobic conditions8. Furthermore, it has been demonstrated that the anaerobic production of a variety of molecules is feasible from a thermodynamics perspective4. Assembly of efficient synthetic networks requires fine tuning and balancing of enzymatic activities9. For that means, promoters that respond at different activity levels to oxygen limitation are needed. These can be provided by studying the properties of endogenous promoters6, 10, searching for heterologous promoters with activity in the chassis to be used for production11, and manipulating the sequence of the promoter12. In this study, the properties of a group of endogenous microaerobic promoters of Escherichia coli were studied and compared to a constitutive heterologous promoter. The constitutive promoter used was Pkat, which controls the expression of the aminoglycoside phosphotransferase gen (kat). The microaerobic promoters were selected from previous reports that have shown their utility and responsiveness to oxygen depletion. Namely, promoters of ethanol dehydrogenase (PadhE), dimethyl sulfoxide reductase (PdmsA), nitrite reductase (PnirB) and pyruvate formate-lyase (Ppfl) genes have been used for the production of PHB6, 13. The focA-pfl operon in E. coli contains the genes coding for a formate transporter (focA) and pfl. Each gene is controlled by different promoters and they are expressed at different levels upon oxygen limitation14. To compare the behavior of both promoters, PfocA was also included in the present study. The promoter of a nitrate/nitrite transporter (Pnar) has been used for expression of β-galactosidase15 under oxygenlimited conditions and for designing sensors for oxygen limitations in cultures of E. coli16. The promoter of the Vitreoscilla hemoglobin gene (Pvgb) is active in E. coli and has been used for expressing β-galactosidase17 and prourokinase18 under oxygen-limited conditions. Due to different expressed products, culture media and cultivation conditions in the previously mentioned publications, a comparison of different promoters is difficult. Furthermore, the characterization of 3 ACS Paragon Plus Environment


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such promoters has not yet been performed systematically. The activity of the different promoters could be influenced by the oxygen availability, which is a result not only of the dissolved oxygen tension (DOT), but also of the OTRmax. This has not been taken into account in previous studies. Here, the endogenous promoters of E.coli and Pvgb were synthesized and cloned into the plasmid pUC57kan. All the assemblies shared common characteristics like a ribosome binding site (Shine Dalgarno sequence, SD), a spacer region of 8 bases previous to the start codon and a terminator sequence (Figure 1). As a reporter molecule, the FMN-binding fluorescent protein (FbFP) was used. FbFP does not require oxygen for maturation and fluorescence emission19,

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; therefore, it is a

suitable model and reporter protein for induction by oxygen limitation. All the promoters used in the present study are controlled by the FNR (fumarate and nitrate reductase) protein (source: Regulon DB database, www.regulondb.ccg.unam.mx). On its active form FNR is a DNA-binding homodimer in which each monomer contains an oxygen-labile [4Fe-4S]2+ cluster. Such clusters promoter the FNR dimerization and enhance site-specific DNA binding21. Destabilization of such clusters by O2 results in two monomers containing [3Fe-4S]1+ clusters. In presence of O2, the monomers lose their F-S cluster, and are unable to bind DNA and therefore to function as transcriptional activators. The monomers are further transformed to the apoprotein. The overall reaction is as follows22 (equation 1): FNR-[4Fe-4S]2+ + O2→ [2Fe-2S]2+ + Fe3+ + Fe2+ + 2S2- + O2 •¯ + apoFNR

(1)

Superoxide is transformed to O2, which results in a signal amplification for O2 sensing by FNR22. Under microaerobic conditions, FNR is readily activated. It has been estimated that upon a transition from aerobic to oxygen-limited conditions, 95 % of the present FNR becomes active in 3.2 min23, and that its activity peaks in 15 min24. These characteristic times are short enough for efficient induction of microbial cultures. Chelators like ferene can make Fe a strong oxidant able to transform sulfide ion to sulfur atoms25. However, typical components of culture media are not expected to influence the activity of FNR. Therefore, FNR is a good O2 sensor for controlling oxygen-limited gene expression. The different promoters were evaluated in microtiter plates with online monitoring of pH, DOT, scattered light (for biomass growth) and fluorescence emission. Oxygen transfer characteristics of such type of microbioreactors have been previously analyzed by Funke et al26. These calculations enable a previous estimation of the maximum oxygen transfer capacity (OTRmax) which can be provided by the reactor system for the desired experimental conditions. Based on these defined conditions and online measurements, scale up and reproduction of any results in shake flasks is simply applicable27. The activity of the different promoters was related not only to fluorescence emission but also to the specific growth rate, and compared with the function of a constitutive 4 ACS Paragon Plus Environment

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promoter for a better characterization28, 29. The successful application of microaerobic processes will also depend on the availability of strains with better performance under oxygen limitation30-32. Therefore, based on the results, selected promoters were also evaluated in an E. coli strain expressing the Vitreoscilla hemoglobin, which is widely reported to improve microbial performance under oxygen-limited conditions33. Altogether, the results provide useful information for selection and sequence shortening of microaerobic promoters, as well as some insight of their use in engineered E. coli strains. RESULTS AND DISCUSSION The different promoters were synthesized and assembled in pUC57kan to express FbFP. While FNR control is common to all the inducible promoters used, the microaerobic metabolism is controlled by several other regulatory elements in E. coli, which are not common for all the tested promoters. Figure 1 depicts common positive regulation by IHF and ArcA and for some endogenous promoters; positive or negative regulation can be exerted by NarL and CRP/AMP, depending on the promoter. Pvgb is also positively controlled by FNR and ArcA in E. coli34.

Figure 1. Assembly of the expression system cloned in pUC57Kan. a: All the studied promoters, except Pkat (constitutive promoter) are under transcriptional control of the FNR protein (green indicates positive control). Some of them (PfocA, Ppfl, Pvgb) are also positively controlled by ArcA (Aerobic Regulatory Control protein), while NarL and CRP/AMP exert negative (red color indicates negative control) or positive control in different promoters. For all the assembles, the consensus Shine-Dalgarno (SD) sequence, an 8 nucleotide spacer, and the rrnbT1 terminator were used for all the constructions. Reduced versions of the promoters are indicated by the subscript red. b: Length of the sequences cloned in pUC57Kan. The reduced versions of the promoters contain only the key sequences for regulation and transcription. The complete sequences cloned in pUC57 are given as Supporting Information. 5 ACS Paragon Plus Environment


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Characterization of the promoters under aerobic conditions. Aerobic conditions can be considered as non-inducing conditions for the promoters studied. However, it is known that promoters from fermentative pathways display a basal expression level35. Therefore, it is necessary to determine the activity of the promoters under aerobic conditions. Cultures were performed at relatively high OTRmax (ca. 65 mmol L-1 h-1) using 48-baffled microtiter plates (Flower Plates®), low filling volumes (VL = 800

µL) and high agitation rates (n = 1100 rpm). The growth profiles are shown in Figure 2. In all cases, DOT was always above 30 %, which confirms that the cultures were completely aerobic (Fig. 2a, 2b) and that growth was only limited by glucose. The strains displayed different lag phases when bearing the different promoters. The lag phase for the strains bearing PadhE, PadhEred, Pnark, Pnarkred, Pkat, and Pvgb lasted for approximately 1 h (Fig. 2e and 2f). In the case of PdmsA, PdmsAred, PfocA, PfocAred, PnirB, PnirBred, Ppfl, Ppflred, the lag phase lasted for nearly 2 h (Fig. 2e and 2f).

The DOT decreased during the cultures in parallel to pH (Fig. 2c, 2d), as expected from the accumulation of acidic byproducts from overflow metabolism and the consumption of nutrients for biomass synthesis. In all cases the exponential growth phase was accompanied by a fast drop of DOT and pH. Growth cessation coincided with the raise of DOT and pH, indicating assimilation of acidic byproducts at low oxygen demand. The fluorescence emission of FbFP is shown in Figures 2g and 2h. In the case of the constitutive promoter (Pkat), the fluorescence emission increased in parallel to biomass (Fig. 2e and 2f), reaching nearly 38 AU by the end of the culture. In comparison, all the endogenous promoters are related with lower FbFP expression during the first 2-6 h of culture (Fig. 2e). The highest FbFP fluorescence values were achieved using PadhE (ca. 25 AU) and PnirB (ca. 15 AU), while the lowest using Pvgb (ca. 5 AU). Shortening sequences of promoters had different consequences on each one. Namely, reduction of PdmsA, PnirB, and PfocA increased the fluorescence emission 2, 3 and 5-fold, respectively, compared to the native versions (Fig. 2e and 2f). While size reduction had nearly no effect on the final fluorescence for PnarK and Ppfl, the reduced version of PadhE resulted in a final fluorescence 3.6 times lower than that of the native promoter (Fig. 2e and 2f). The use of FbFP fluorescence to monitor promoter activity requires that its fluorescence is a reliable online reporter of transcription and translation. For this purpose, fluorescence should be directly proportional to FbFP concentration. The expressed FbFP should mature fast, remain stable and be degraded at constant rates. The latter factors are reflected in a constant specific fluorescence. Mukherjee and coworkers showed that FbFP specific fluorescence under control of Plac decreased over exponential growth when IPTG concentrations of 0.1 mM and higher were used. When IPTG was added at 0.01 mM, the specific FbFP fluorescence was constant35.


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Figure 2. Aerobic cultures (OTRmax ca. 65 mmol L-1 h-1) of E. coli BL21 expressing the FMN binding fluorescent protein (FbFP) under control of the different natural (left column) or reduced (right column) promoters. Online monitoring of DOT (a, b), pH (c, d), cell growth by scattered light (e, f) and FbFP fluorescence (g, h) data are shown. Culture conditions: 48-well Flower Plate®, VL = 800 µL, n = 1100 rpm, d0 = 3 mm, mineral medium buffered with MOPS (0.2 M) plus 5 g L-1 of glucose. 7 ACS Paragon Plus Environment


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The same authors hypothesized that the drop of specific fluorescence could occur due to a high burden on cellular flavin resources and low availability of FMN compared to the strongly expressed FbFP, especially attributed to the employed carbon source (glycerol, referred to as a “poor” carbon source)35. In contrast, Drepper and coworkers reported that the specific FbFP fluorescence was nearly constant through the cell growth when using a constitutive promoter of moderate activity (PaphII) and glucose as the carbon source19. In the present study, nearly constant specific FbFP fluorescence was observed during the cell growth (values reported in Table 1), in agreement with data from Drepper and coworkers19 and the use of microaerobic promoters, whose activity resulted low, as described below.

A closer examination of the promoter activity if bioprocess applications are sought should be linked to the specific growth rate (µ) of the host and the biomass in the culture. Table 1 shows the values of µ, specific fluorescence intensity (per biomass unit) and the specific fluorescence emission rate (qF) of each aerobic culture during the exponential growth phase. In the case of the native promoters, there was no general correlation between the size of the promoter, µ and the accumulated biomass. For instance, cells expression under control of Pkat led to the highest fluorescence levels and also the host displayed the highest µ (Fig. 2e, Table 1). In contrast, when bearing the Pvgb, the growth rate and biomass accumulation were similar, while the fluorescence values considerably lower than that of the cells bearing Pkat (Fig. 2e, Table 1). The specific fluorescence intensity was considerably higher using the Pkat than any other promoter (Table 1). The length reduction of the promoters also had different consequences. The specific fluorescence decreased to one-half, and µ increased 15 % for PadhEred, compared to PadhE (Table 1). In contrast, the specific fluorescence increased notoriously in the case of PdmsAred, PfocAred, and PnirBred, while for Ppfl neither µ, nor the specific fluorescence showed changes (Table 1). The value of qF combines µ and fluorescence emission and can be a better parameter for promoter comparison. Since even during fully aerobic conditions some FbFP fluorescence was detected for the microaerobic promoters, qF is also a parameter to measure the basal (uninduced) promoter activity. However, in the case of non-induced conditions, it should be taken into account that low qF values can result from a low µ, which may be undesirable from the bioprocess point of view. The lowest qF values were obtained for PdmsA, Ppfl and Ppflred. However, not only specific fluorescence, but also µ were low for these promoters. In comparison, PadhEred and Pvgb display a relatively low specific fluorescence, while µ was high (Table 1). Therefore, at this point, Ppfl, Pvgb, Ppflred and PadhEred allowed


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high growth rates and low FbFP expression, which are desired characteristics under aerobic conditions. Table 1. Promoter activity characterization in aerobic cultures. The specific growth rate (µ), specific fluorescence intensity and specific fluorescence emission rate (qF)a were calculated for each culture during exponential growth. Average values are reported. ± denotes the experimental error between duplicates. Promoter

µ

(h-1)

Specific fluorescence intensity (AU AU-1)

qF (AU AU-1 h-1)

kat 0.80 ± 0.07 2.00 ± 0.05 1.69 ± 0.13 adhE 0.67 ± 0.06 0.70 ± 0.05 0.49 ± 0.05 dmsA 0.57 ± 0.03 0.19 ± 0.00 0.11 ± 0.01 focA 0.59 ± 0.00 0.24 ± 0.00 0.14 ± 0.00 narK 0.71 ± 0.06 0.48 ± 0.01 0.34 ± 0.03 nirB 0.56 ± 0.01 0.57 ± 0.01 0.32 ± 0.00 pfl 0.63 ± 0.03 0.21 ± 0.01 0.13 ± 0.01 vgb 0.74 ± 0.02 0.28 ± 0.02 0.21 ± 0.02 adhE red 0.77 ± 0.03 0.33 ± 0.01 0.25 ± 0.01 dmsA red 0.60 ± 0.02 0.34 ± 0.00 0.20 ± 0.01 focA red 0.57 ± 0.01 0.85 ± 0.03 0.50 ± 0.01 narK red 0.71 ± 0.05 0.55 ± 0.03 0.40 ± 0.03 nirB red 0.50 ± 0.01 1.61 ± 0.03 0.80 ± 0.00 pfl red 0.60 ± 0.02 0.21 ± 0.00 0.13 ± 0.00 a qF was determined as the slope in the plot of FbFP fluorescence emission (F-Fo) versus scattered light intensity (I-Io) as a measure of biomass.

Characterization of the promoters under oxygen limitation at low OTRmax. Oxygen limitation should induce the expression of FbFP for all the promoters studied, except Pkat. However, the promoters can show different sensitivity to the DOT36, 37 . Moreover, the rate at which oxygen is delivered to the broth is a relevant parameter, particularly if a bioprocess is being scaled-up using microaerobic promoters. As a first approach, the activity of the promoters was evaluated at low OTRmax using round well plates. Under the chosen experimental conditions (VL = 1200 µL, n = 650 rpm), the OTRmax is approximately 10 mmol L-1 h-1

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. The culture profiles are shown in Figure 3. The lag phase lasted

for less than 2 h for all the strains (Fig. 2e and 2f). After about 4 h, all the cultures were oxygenlimited (Fig. 3a and 3b) and DOT dropped to values close to zero. The oxygen-limited phase lasted for around 4 h. The pH fell down to ca. 6.5 in all cultures and started to increase after nearly 7 h of culture (Fig. 3c and 3d). This coincided with a transient increase of DOT, which afterwards fell down again for around 1 h and then rose to 100 % (Fig. 3a and 3b). Such transient DOT increase coincident with pH raise indicates glucose depletion. The subsequent DOT decrease may indicate the



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consumption of byproducts (acetate, ethanol, formate, lactate, succinate) that still require some oxygen to be fully oxidized and that cause oxygen limitation due to the low OTRmax27. The cell growth displayed a similar pattern for all cultures and the attained biomass was also similar (Fig. 3e and 3f). The fluorescence emission using Pkat was akin to the case of aerobic culture, except that it took longer to achieve its plateau (Fig. 2e and 3e). In general, the expression associated with the native microaerobic promoters seemed to be well repressed under the first 4 h and fluorescence started to increase later on (Fig. 3g), coincident with oxygen depletion (Fig. 3a). This is in agreement with the fast response of FNR as oxygen sensor, as discussed above and reflects the fast maturation of FbFP, which occurs in less than 3 min35. The maximum fluorescence emitted using native promoters was similar to that obtained under aerobic conditions. Exceptions are PnarK and Pvgb, that displayed an increase of 67 and 245 %, respectively, compared to aerobic cultures (Fig. 2g and 3g). In the case of the shortened promoters, PfocA and PnirB started to express FbFP shortly after 2h of culture, when aerobic conditions still prevailed (Fig. 3b and 3h). Transcription in which the other promoters are involved, is repressed until ca. 4 h (Fig. 3h). Fluorescence emitted using PadhEred and PnarKred were 50 % higher than the obtained in aerobic cultures (Fig. 2h and 3h). Despite the relatively high fluorescence emitted using PnirBred (Fig. 3h), it was 18 % lower than in the case of aerobic cultures (Fig. 2h). The fluorescence reached when using the other reduced promoters was not greater than in the case of aerobic cultures. Most of the cultures under oxygen-limited conditions reached lower levels of scattered light compared to aerobic cultures. This is due to the less efficient energy generation and formation of byproducts under oxygen limitation. Therefore, a comparison of specific yield and rate allows a better comparison of the promoters. Such parameters, calculated during the oxygen-limited growth, are shown in Table 2. Compared to aerobic growth, µ decreased in general by 50 % during oxygenlimited growth. Oxygen limitation in cultures using Pkat diminished the specific fluorescence in 37 %, compared to aerobic conditions (Tables 1 and 2). This may be attributed to a less efficient transcriptional and/or translational activity, due to the mentioned metabolic limitations. In comparison with aerobic cultures, oxygen limitation did not influence the specific fluorescence intensity when PnirB, PfocAred and PnarKred, were used, and even resulted in a lower specific fluorescence in the case of PnirBred (Table 2). All the other promoters induced the microaerobic expression of FbFP at different yields. Relative to aerobic conditions, Pvgb, Ppfl, and Ppflred displayed the greater induction ratios (Table 2). With the exception of this group of promoters, the qF under oxygen limitation were lower than those of aerobic conditions. This implies that the impact on µ was higher than the increase of fluorescence. Reducing the size of the native promoters improved µ for PadhEred, PdmsAred, 10 ACS Paragon Plus Environment

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PnarKred, and Ppflred, compared to the native versions (Table 2). However, positive impacts of size reduction on the specific fluorescence intensity and qF were observed only for PdmsA and PfocA (Table 2).

Figure 3. Oxygen-limited cultures of E. coli BL21 expressing the FMN binding fluorescent protein (FbFP) under control of the different natural (left column) or reduced (right column) microaerobic promoters at low OTRmax (ca. 10 mmol L-1 h-1). Online monitoring of DOT (a, b), pH (c, d), cell growth by scattered light (e, f) and FbFP fluorescence data are shown. Culture conditions: 48-well round well plate, VL = 1200 µL, n = 650 rpm, d0 = 3 mm, mineral medium buffered with MOPS (0.2 M) plus 5 g L-1 of glucose. 11 ACS Paragon Plus Environment


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Table 2. Promoter activity characterization in oxygen-limited cultures at low OTRmax (ca. 10 mmol L1 -1 h , 5 g/L glucose). The specific growth rate (µ), specific fluorescence intensity and specific fluorescence emission rate (qF)a were calculated for each culture during the period of oxygen-limited growth. Average values are reported. ± denotes the experimental error between duplicates.

Promoter

µ

(h-1)

Specific fluorescence intensity (AU AU-1)

qF (AU AU-1 h-1)

Induction ratiob

kat 0.47 ± 0.02 1.26 ± 0.07 0.59 ± 0.10 0.63 adhE 0.33 ± 0.01 0.87 ± 0.05 0.29 ± 0.02 1.24 dmsA 0.38 ± 0.00 0.29 ± 0.01 0.11 ± 0.00 1.53 focA 0.43 ± 0.01 0.32 ± 0.01 0.14 ± 0.01 1.33 narK 0.35 ± 0.01 0.58 ± 0.02 0.19 ± 0.00 1.21 nirB 0.38 ± 0.01 0.58 ± 0.04 0.22 ± 0.01 1.02 pfl 0.35 ± 0.02 0.28 ± 0.05 0.10 ± 0.03 1.33 vgb 0.33 ± 0.01 0.67 ± 0.03 0.22 ± 0.00 2.39 adhE red 0.36 ± 0.01 0.37 ± 0.00 0.13 ± 0.00 1.12 dmsA red 0.44 ± 0.01 0.39 ± 0.01 0.17 ± 0.00 1.15 focA red 0.34 ± 0.01 0.89 ± 0.05 0.28 ± 0.02 1.05 narK red 0.40 ± 0.00 0.55 ± 0.03 0.21 ± 0.01 1.00 nirB red 0.32 ± 0.01 1.39 ± 0.13 0.44 ± 0.02 0.86 pfl red 0.40 ± 0.00 0.31 ± 0.01 0.12 ± 0.01 1.48 a qF was determined as the slope in the plot of FbFP fluorescence emission (F-Fo) versus scattered light intensity (I-Io) as a measure of biomass. b Induction ratio was calculated as the specific fluorescence intensity under aerobic conditions divided by the corresponding value under oxygenlimited conditions.

Characterization of the promoters under oxygen limitation at high OTRmax. The effect of the different promoters on the FbFP expression was also measured in oxygen-limited cultures at higher OTRmax and glucose concentration (10 g L-1) than the previous experiment. This is useful for having a more detailed characterization of the promoter performance under process relevant conditions. The growth profiles are depicted in Figure 4. During the first 4-5 h of the experiment, the cultures behaved similar to the aerobic cultures, and two subgroups with similar profiles of DOT, pH and growth were seen for original and reduced promoters (Fig. 3 and 4). The strains displayed different lag phases when bearing the different promoters. The lag phase for the strain bearing Pvgb was lasted around 1 h, while for strains bearing PadhE, PadhEred, Pnark, Pnarkred, and Pkat, it lasted for approximately 1 h (Fig. 4e and 4f). In the case of PdmsA, PdmsAred, PfocA, PfocAred, PnirB, PnirBred, Ppfl, Ppflred, the length of the lag phase was almost 3 h (Fig. 4e and 4f). In all cases, DOT raised after approximately 2 h, although not as fast as in aerobic cultures. Opposite to oxygen-limited cultures at low OTRmax, no second fall of DOT was observed, which indicates that OTR was high enough to sustain the assimilation of fermentative byproducts. pH reached minimum values of approximately 5.8 (Fig. 4c and 4d). 12 ACS Paragon Plus Environment

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Figure 4. Oxygen-limited cultures of E. coli BL21 expressing FbFP under control of the different natural (left column) or reduced (right column) microaerobic promoters at high OTRmax (ca. 45 mmol L-1 h-1). Online monitoring of DOT (a, b), pH (c, d), cell growth by scattered light (e, f) and FbFP fluorescence (g, h) data are shown. Culture conditions: 48-well Flower Plate®, VL = 1200 µL, n = 1100 rpm, d0 = 3 mm, mineral medium buffered with MOPS (0.2 M) plus 10 g L-1 of glucose.



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This is the lowest value for all the experiments performed, and is attributed to a higher accumulation of acidic byproducts due to the higher glucose concentration used. Yet, it is not expected that this pH value would affect the growth of E. coli38-40. In most cases, the scattered light signal continued increasing after DOT and pH signals stabilized (Fig. 4e and 4f). This can be attributed to morphological changes (like cell size) during the stationary phase, which interfere with the scattered light readings41. Because such phenomenon was only observed in this group of cultures, it is possible that the low pH values may have played a role on it. In all cases, the FbFP fluorescence reached a plateau after DOT stabilization (fig. 4e and 4f). The FbFP fluorescence reached values higher to the previous cultures under oxygen-limiting conditions (Fig. 3 and Fig 4). The influence of the promoters on the expression of FbFP and on growth was characterized during the aerobic and oxygen-limited phases of the cultures. Figure 5 shows the calculated parameters. The transition to oxygen-limited conditions reduced the growth rate (Fig. 5a), being the strains bearing PadhE and Pvgb the most strongly affected (Fig. 5a). The relatively short period of oxygen limitation was enough to induce the expression of FbFP with most of the promoters (Fig. 5b), except with PnirB, PnirBred and PfocAred. In most cases, the specific fluorescence intensity after dissolved oxygen depletion reached values similar to those under oxygen-limited conditions at low OTRmax (Fig. 5b, Table 2). Interestingly, the specific fluorescence intensity for Ppflred under oxygen-limited conditions was 32 % higher (0.54 ± 0.03 AU AU-1) at high than at low OTRmax (Table 2). Moreover, the specific fluorescence under oxygen limitations for PnarKred and Ppflred increased 46 % (0.76 ± 0.05 and 0.54 ± 0.03 AU AU-1, respectively) at high OTRmax, compared to low OTRmax (Table 2). The strong reduction of growth rate after transition to oxygen-limited conditions resulted in low qF values. Only PdmsA, PfocA, Ppfl, Ppflred and Pvgb exhibited and increase of qF after oxygen depletion (Fig. 5c). In a previous report, the effects of different “oxygen levels” on Pvgb were studied. Different shaking frequencies were applied to generate different OTRs at DOT = 0 % in shake flasks11. The authors reported that after 2 h of inoculation, the promoter reached its maximum, followed by a decrease to basal level. Such results are in contrast with the data presented in Fig. 3 and 4, where FbFP fluorescence increased parallel to biomass during oxygen limitation. However, no online DOT data were shown for the experiments in shake-flasks. Moreover, such experiments were performed in unbuffered lysogeny broth11, and therefore wide pH changes (not monitored) could also have affected the Pvgb activity. Khosla and Bailey17 reported an increase of 2.5-fold of Pvgb under oxygen-limitation, compared to aerobic cultures, which is in close agreement with the results shown in Table 2. Pnar has been evaluated for recombinant β-galactosidase production42. High induction levels of up to 157-fold were reported using nitrate to enhance β-galactosidase expression in complex medium. However, the expression level associated with PnarK is remarkably low, compared to the mentioned report. Several 14 ACS Paragon Plus Environment

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other promoters from Table 2 have been used for PHB production6, 13 and no specific information about promoter activity was provided.

Figure 5. Promoter activity characterization in oxygen-limited cultures at high OTRmax (ca. 45 mmol L-1 h-1). The specific growth rate (µ), specific FbFP fluorescence intensity and specific FbFP fluorescence emission rate (qF) were calculated during aerobic (grey bars) or oxygen-limited (white bars) growth. Error bars show the experimental deviation between duplicate cultures.



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Characterization of selected promoters under oxygen low OTRmax and 10 g L-1 glucose. Based on the most desirable characteristics for microaerobic promoters (low expression under aerobic conditions, high activity under oxygen limitations, low impact on growth rate), a group of promoters was selected for further evaluation. Selected promoters include Pvgb, Ppfl, PadhEred, Ppflred, PnarKred. Despite its impact on growth rate, PadhE was also included because it displayed the highest fluorescence yield in experiments under oxygen limitation and high OTRmax (Fig. 5). In order to create conditions of stronger induction (higher cell-densities and low OTRmax), glucose was added at 10 g L-1. Culture profiles are shown in Figure 6. Cultures became oxygen-limited around 4 h post-inoculation and remained at DOT = 0 % air sat. for approximately 6 h (Fig. 6a and 6b). As in previous cultures, pH decreased through the cultures and increased shortly before the transient DOT increase (Fig. 6c and 6d). Later on, DOT reached 100 % and pH remained relatively constant, indicating the total consumption of the initial glucose. The maximum biomass attained was similar for all promoters (approx. 35 AU), except for Pvgb, which reached nearly 30 AU (Fig. 6e and 6f). Although in general the biomass attained was around 60 % higher than that using 5 g L-1 of glucose and low OTRmax (Fig. 6e and 6f), the fluorescence intensity in cultures using 10 g L-1 doubled the obtained using 5 g L-1 (Fig. 3g, 3h, 6g and 6h). Particularly, in the case of Ppfl, the fluorescence using 10 g L-1 of glucose and low OTRmax was 3-fold that of cultures using 5 g/L of glucose and low OTRmax (Fig. 3g, 3h, 6g and 6h). When using a low OTRmax, 10 g L-1 of glucose had a stronger effect on µ than using 5 g L-1, as shown in Figure 6a. This can be attributed to a lower availability of oxygen per cell and possibly to a higher accumulation of fermentative byproducts. Cultures using PadhE and Ppfl showed the lowest growth rate under oxygen-limited conditions, followed by cultures using PnarKred and Ppflred. Cultures using Pvgb and PadhEred displayed the highest µ (Fig. 7a). Despite growth inhibition, the specific fluorescence emission doubled that of cultures using only 5 g/L of glucose, except in the case of PnarKred (Fig. 7b and Table 3). Fluorescence yield was higher for PadhE and Pvgb, while it was similar for the other promoters (Fig. 7b). Interestingly, the use of Pvgb resulted in the highest qF, which is a result of higher µ and fluorescence yield, followed by PadhE. While the size reduction of PadhE resulted in higher µ, the comparatively low fluorescence yields obtained resulted in a lower qF (Fig. 7c). On the contrary, the size reduction of Ppflred resulted in a relatively small increase of µ and fluorescence yield, contributing to a higher qF (Fig. 7c).


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Figure 6. Oxygen-limited cultures of E. coli BL21 expressing the FbFP under control of the selected natural (left column) and reduced (right column) microaerobic promoters. Online monitoring of DOT (a, b), pH (c, d), cell growth by scattered light (e, f) and FbFP fluorescence emission (g, h) data are shown. Culture conditions: 48-well round well plate, VL = 1200 µL, n = 650 rpm, d0 = 3 mm (OTRmax ca. 10 mmol L-1 h-1), mineral medium buffered with MOPS (0.2 M) plus 10 g L-1 of glucose.



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Figure 7. Promoter activity characterization using 10 g L-1 of glucose and low OTRmax (ca. 10 mmol L1 -1 h ). The specific growth rate (µ), specific FbFP fluorescence intensity and specific fluorescence emission rate (qF) were calculated during oxygen-limited growth.

Oxygen-limited expression of FbFP using selected promoters and engineered E. coli. The studied promoters may have potential applications for oxygen-limited bioprocesses. This can be beneficial for high cell-density cultures, which are the standard for industrial applications. However, such conditions strongly impacted the growth rate, which may point out to a generalized limited biosynthetic capacity. Expression of Vitreoscilla hemoglobin (VHb) is well known to ameliorate the physiological effects of oxygen limitation in cultures. Although the exact mechanism of action of VHb is still unknown, it has been proposed that it improves the oxygen delivery to the cytochromes, generally resulting in higher growth rates under oxygen limitation33. Therefore, an E. coli expressing the VHb was used as a host to explore the possibility of using engineered strains with the promoters studied. To avoid effects of plasmid copy number and increased plasmid size, the VHb gene was inserted into the chromosome and expressed constitutively, as described in the Methods section. The engineered strain, named here as E. coli BL21-VHb, was cultured under aerobic and oxygenlimited conditions using 5 g L-1 of glucose and low OTRmax as in previous cultures, which allow direct comparison with the experiments using the wild-type strain. The growth profiles, compared to those 18 ACS Paragon Plus Environment

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of the wild-type strains, are shown in the Supporting Information file 1. Data summarizing promoter characterization are shown in Table 3. The aerobic expression of VHb increased the growth rate between 12 and 45 % for the different genetic constructions, compared to wild-type strain cultures (Tables 1 and 3). Exceptions were cultures of the strains bearing Pkat and Pvgb, in which the growth was similar to that of the wild-type cultures (Tables 1 and 3). It has been demonstrated that aerobic expression of VHb increases the growth rate of E. coli K-derivatives with higher activity of the tricarboxylic acid (TCA) cycle43. The glyoxylate shunt is active in E. coli BL2144, which may cause the observed increase of growth rate when VHb is present. Interestingly, the specific fluorescence emission was between 14 and 24 % lower for the microaerobic promoters when VHb was expressed under aerobic conditions, compared to wild-type cultures, while the fluorescence using Pkat remained approximately the same (Tables 1 and 3).

Table 3. Promoter activity characterization in cultures of E. coli BL21-VHb. The specific growth rate (µ), specific fluorescence intensity and specific fluorescence emission rate (qF)a were calculated for each culture during aerobic or oxygen-limited growth. Average values are reported. ± denotes the experimental error between duplicates.

Promoter

µ

(h-1) kat adhE adhE red narK red pfl pfl red vgb

0.85 ± 0.05 0.78 ± 0.02 0.86 ± 0.00 0.90 ± 0.00 0.87 ± 0.01 0.80 ± 0.03 0.70 ± 0.02

Specific fluorescence qF intensity (AU AU-1) (AU AU-1 h-1) Aerobic conditions 1.87 ± 0.01 1.59 ± 0.08 0.56 ± 0.03 0.44 ± 0.03 0.28 ± 0.01 0.24 ± 0.01 0.45 ± 0.01 0.40 ± 0.01 0.18 ± 0.01 0.16 ± 0.00 0.17 ± 0.00 0.14 ± 0.01 0.22 ± 0.01 0.15 ± 0.01 Oxygen-limited conditions

Induction ratiob kat 0.51 ± 0.01 1.34 ± 0.04 0.68 ± 0.01 0.72 adhE 0.41 ± 0.03 0.73 ± 0.03 0.30 ± 0.01 1.30 adhE red 0.48 ± 0.03 0.50 ± 0.01 0.24 ± 0.02 1.79 narK red 0.49 ± 0.03 0.77 ± 0.05 0.37 ± 0.00 1.71 pfl 0.46 ± 0.03 0.37 ± 0.02 0.17 ± 0.02 2.06 pfl red 0.47 ± 0.01 0.33 ± 0.01 0.15 ± 0.00 1.94 vgb 0.58 ± 0.01 0.51 ± 0.03 0.29 ± 0.02 2.32 a qF was determined as the slope in the plot of FbFP fluorescence emission (F-FO) versus scattered light intensity (I-IO) as a measure of biomass. b Induction ratio was calculated as the specific fluorescence intensity under aerobic conditions divided by the corresponding value under oxygenlimited conditions.



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The physiological effects of aerobic expression of VHb in E. coli are nearly unknown. However, it may be hypothesized that VHb contributes to a more oxidized state under aerobic conditions, which could increase the amount of FNR molecules on its oxidized form, better repressing the microaerobic promoters. In fact, it has been shown that VHb can detoxify superoxide in a reaction involving its heme pocket45, 46: VHb-[Fe3+] + O2 •¯ → VHb-[Fe2+] + O2

(2)

Therefore, this reaction could enhance the regulation by FNR, as can be seen in equation 1. Nevertheless, with the present information, this cannot be confirmed. Data in Table 3 show that in general, VHb expression is a viable strategy to improve aerobic growth and contributes to better repression of transcription related with microaerobic promoters under aerobic conditions. When the engineered strain was cultured under oxygen-limited conditions, the positive effect of VHb on growth rate was more evident. Compared to oxygen-limited cultures of the wild-type strain (Table 1), VHb expression increased µ between 18 and 75 %, depending on the genetic construction (Table 3). The specific fluorescence also increased when VHb was expressed, compared to wild-type cultures, except for PadhE and Pvgb (Tables 1 and 3), in which it decreased slightly. Nevertheless, VHb expression under oxygen-limited conditions increase qF for all promoters, compared to oxygen limited cultures of the wild-type strain (Tables 1 and 3). Moreover, the induction ratio for the selected promoters was higher when VHb was present, except for Pvgb. The greater increase of induction ratio was observed for Ppfl, PnarKred and Ppflred (Tables 1 and 3). Such increase in induction ratio and qF is in agreement with higher recombinant protein production observed when VHb is present under oxygen-limited conditions29. This can be attributed to a higher energy generation due to the probable effect of VHb on the respiratory chain of E. coli29. VHb can interact with subunit I of cytochrome o (reaction site for oxygen binding) and subunit A of cytochrome d (reaction site for O2 reduction in respiration). Whether this results in a higher ATP production is unclear, since the reported studies show inconsistent results, probably due to different experimental settings46. However, different studies coincide in a strong decrease of NADH concentration in E. coli as result of VHb expression. This is well in agreement with the possible role of VHb to enhance aerobic respiration46. The increased ATP synthesis that should result from this could then be readily consumed for biomass and FbFP synthesis, which may explain the increased specific fluorescence. Since oxygen-limited metabolism generates less ATP per molecule of glucose consumed than aerobic respiration, it can be expected that extra supply of ATP will increase the biosynthetic capacity of the cell under such conditions. The decrease on fluorescence yield for Pvgb cannot be interpreted straightforward. It may be possible that VHb play a role on the regulation of Pvgb to inhibit its own expression when a given amount of VHb is present. This should be clarified in future research. 20 ACS Paragon Plus Environment

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The results presented here provide data for the selection of promoters for oxygen-limited processes. In contrast to prior studies, all results were validated by online measured signals for DOT, pH, scattered light and fluorescence. The cultures were performed in microtiter plates, which are well characterized screening systems with respect to achievable maximum oxygen transfer rates OTRmax. This allows a better insight in the role of the promoters and in information that is more suitable for culture design and operation. The reduction of length of the promoters had, in general, positive results. This is particularly relevant if several genes will be overexpressed, individually or as operon, because smaller genetic constructions are generally preferred by the host. Evaluation of genetic elements in strains that are designed for large-scale conditions is also highly desirable since early stages of product and process development. Here, it was shown that VHb is a valuable strategy to improve microaerobic processes beyond its well-known effects: VHb expression conducted to better repression of FbFP with microaerobic promoters under aerobic conditions, while enhanced their function under oxygen-limited conditions. There were exceptions to this observation, which deserve further investigation. The interesting performance of Pvgb indicates that other heterologous promoters may yield good results in E. coli.

METHODS Strains. Escherichia coli strain BL21 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. The strain E. coli BL21 VHb was created by chromosomal insertion of the gene coding for the Vitreoscilla stercoraria hemoglobin (vgb) according to the methodology proposed by Sabido, et al.47. Briefly, the vgb gene (GenBank: L21670.1) was codon optimized for expression in E. coli using the free access Optimizer tool program (http://genomes.urv.es/OPTIMIZER/). The optimized gene was synthesized by GeneScript, cloned in pUC18, and then subcloned in pLoxGentrc between the NcoI and EcoRI sites to yield pLoxGentrc-vgb. In such construction, the start codon of the vgb gene is located downstream the trc promoter. The DNA region comprising the trc promoter, vgb and gentamicin-resistance genes flanked by Lox sequences was PCR amplified from the pLoxGentrc-vgb plasmid using the high-fidelity polymerase Phusion (ThermoScientific, CA, USA) and oligonucleotides AF and 7R. The employed cycle and oligonucleotides sequences are provided in the Supporting information file 1. The resulting 2487 nt product, that contains 50 nt regions homologous to the genes lacI and lacZ, was used to transform E.coli BL21 (DE3) bearing the plasmid was pKD46, in which the Red system proteins were already induced with arabinose. This way, the PCR product was integrated into the chromosome by 21 ACS Paragon Plus Environment


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homologous recombination between lacI and lacZ. The integration was confirmed by resistance to gentamicin and the absence of blue coloration in colonies grown in X-Gal plus 1 mM IPTG LB plates. PCR tests demonstrated successful integration of synthetic vgb gene. The interruption of lacI and lacZ and the introduction of the trc promoter yield the expression of vgb constitutive.

Parts synthesis and assembly. Endogenous microaerobic promoters were chosen from a literature survey. Sequences and main regulatory characteristics were obtained from RegulonDB database (www.regulondb.ccg.unam.mx). Sequence for Pvgb was taken from the NCBI database (accession number M30794). Shortened promoters were constructed eliminating the sections that were not necessary for regulatory mechanisms based on Regulon DB database. A ribosome binding site (RBS) (Shine-Dalgarno sequence) and a spacer region of 8 bases were added previous to the start codon. FbFP sequence from Bacillus subtilis 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. The complete sequences were synthesized and cloned in pUC57kan by GenScript (Piscataway, NJ, USA). The sequence of the promoters used is provided in the Supporting information.

Culture media. 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 elsewhere35. Glucose was added at final concentration of 5 or 10 g L-1, as described for each experiment. Kanamycin sulfate was used in all the cultures at a concentration of 50 µg mL-1.

Culture conditions. For pre-culture development, 100 µL of cryopreserved cells were used to inoculate 10 mL of TB and grown at 30 °C in Erlenmeyer flask 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 mineral medium with glucose was added at final concentrations of 5 or 10 g L-1. The 22 ACS Paragon Plus Environment

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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. Two types of plates were used, which allow relatively low or high OTR, based on the well geometry: round wells (MTP-R48-BOH, Lot 1402, m2p Labs, Beasweiler, Germany) or Flower Plates® (MTP-48-BOH, Lot 1515, m2p Labs, Beasweiler, Germany), respectively. Plates were sealed with a hydrophobic porous rayon sterile sealing film (AeraSeal, Excel Scientific, Victorville, CA, USA). Cultures were performed at 37 °C, 85 % humidity, shaking diameter of 3 mm, and shaking frequency as stated for each experiment. Depending on the experiment, the culture volume per well was 800 or 1200 µL. Biomass was monitored by scattered light (λex = 620 nm; gain: 20). Fluorescence was used to monitor DOT (λex = 520 nm; λem = 600 nm; gain: 83), pH (λex = 485 nm; λem = 530 nm; gain: 45) and FbFP (λex = 450 nm; λem = 492 nm; gain: 90). The OTRmax values were taken from Funke et al., 200922. All cultures were performed by duplicate.

Data analysis. The initial data of scattered light and fluorescence intensity were subtracted from the measured data, since these have been attributed to diverse factors like media background, filling volume or type of microtiter plate41, while some FbFP is expressed during the growth of the precultures. Parameters for promoter characterization (µ, specific fluorescence emission and qF) were determined during exponential growth phase. Specific fluorescence emission was determined as the slope in the plot of fluorescence emission (F-F0) versus scattered light intensity (I-I0) data points. qF was calculated as the product of µ multiplied by the specific fluorescence emission. For calculation of the parameters under oxygen-limited conditions, the data points used were those after 1 h of reaching a DOT < 10 % and previous to the raise of the DOT signal.

Supporting Information.

Figure S1. Aerobic cultures of the E. coli strains BL21 and BL21 VHb. Figure S2. Aerobic cultures of the E. coli strains BL21 and BL21 VHb. Figure S3. Oxygen-limited cultures of the E. coli strains BL21 and BL21 VHb. Figure S4. Oxygen-limited cultures of the E. coli strains BL21 and BL21 VHb. Table S1. Oligonucleotides used for generating PCR integration product. Table S2. Thermocycler program used for generating the integration product. Table S3. Complete sequences cloned in the plasmid pUC57kan used in this study.



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Nomenclature Abbreviations d0 Shaking diameter (mm) DOT Dissolved oxygen tension (% air saturation) FbFP FMN binding fluorescent protein n shaking frequency (rpm) qF Specific fluorescence emission rate (AU AU-1 h-1) OTR Oxygen transfer rate (mmol L-1 h-1) VL Volume of the liquid phase (µL)

Genes (protein expressed) adhE ethanol dehydrogenase dmsA dimethyl sulfoxide reductase focA formate transporter kat aminoglycoside phosphotransferase nark nitrate/nitrite transporter nirB nitrite reductase pfl pyruvate-formate lyase vgb Vitreoscilla hemoglobin

Symbols

λem Emmision wavelength [nm] λex Excitation wavelength [nm]


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µ Specific growth rate (h-1)

Acknowledgements This work was supported by CONACyT grants 256617, 264460 and 248926.

Author information Corresponding Authors E-mail: [email protected]; Tel: 52 55 58146501 [email protected]; 49 241 80 24633

Author´s Contributions ARL conceived the project, performed the cultures and data analyses. KEJ and JC Sigala designed the shortened sequences. MM helped on the set-up of the BioLector for high-throughput cultures. LR and JB contributed in the design of the experiments and general data interpretation. All the authors participated in preparing the manuscript.

Notes The authors declare no competing financial interests.

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For Table of Contents use only

Title: "Characterization of endogenous and reduced promoters for oxygen-limited processes using Escherichia coli"

Authors: Lara, Alvaro; Jaén, Karim; Sigala, Juan Carlos; Mülhman, Martina; Regestein, Lars; Büchs, Jochen

Graphical abstract


Characterization of Endogenous and Reduced Promoters for Oxygen

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