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. 2025 Feb 24;122(6):1598–1607. doi: 10.1002/bit.28958

Sigma Factors as Potential Targets to Enhance Recombinant Protein Expression

Laura Pohlen 1, Emily García 2, Luz María Martínez 2, Noemí Flores 2, Jochen Büchs 3, Guillermo Gosset 2,✉, Alvaro R Lara 4,✉
PMCID: PMC12067039  PMID: 39994955

ABSTRACT

The transcriptional factors control the expression of many genes and represent an important layer of complexity in cell factories. However, the effect of individual sigma factor deletions from a biomanufacturing perspective has not been addressed. In this contribution, growth, green fluorescence protein (GFP) expression, and oxygen consumption of Escherichia coli BW25113 strains with individual inactivation of each sigma factor were characterized under various conditions. Specific growth rate, specific GFP fluorescence, and fluorescence emission rates were compared in a mineral media and in lysogeny broth at two temperatures. rpoD has been reported to be lethal for E. coli; however, the evaluated rpoD mutant did not exhibit major growth defects in the mineral medium. This is attributed to the presence of a second copy of rpoD in this strain. GFP was expressed at three different induction levels in a mineral and LB media. The fliA mutant was the best producer in the mineral medium, whereas the rpoD mutant overperformed the other strains in LB medium. This suggests that a lower rpoD gene dosage is positive for the performance of the cell factory in a complex medium. In cultures at 20°C, the rpoS mutant exhibited the greatest recombinant expression. To our knowledge, this is the first systematic study evaluating the potential of sigma factor deletion for improving recombinant protein production.

Keywords: microbial engineering, minimal cells, oxygen transfer, sigma factors

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1. Introduction

Bacterial RNA polymerases require subunits, named sigma (σ) factors to recognize specific promoters and start the transcription process. In Escherichia coli, the “housekeeping” and essential σ70 (rpoD) controls the expression of many genes expressed during exponential growth (Shimada et al. 2014). Alternative σ‐factors have been described in E. coli, σ54 (rpoN), σ38 (rpoS), σ32 (rpoH), σ28 (fliA), σ24 (rpoE, which is an essential gene), and σ19 (fecI). Cho and coworkers reconstructed a genome‐scale network of the σ‐factors and reported 4724 σ‐factor specific promoters (Cho et al. 2014). While the σ‐factors control the expression of many genes, whether such genes are dispensable for the cell factory under production conditions remains largely unstudied, but there are some reports suggesting that σ‐factor deletions can be useful to improve cellular or production performance. For instance, RpoS is known as a general stress response or stationary phase transcriptional factor that controls the expression of approx. 500 genes (Gottesman 2019). It has been shown that the deletion of rpoS increases the production of recombinant protein in chemostat cultures of E. coli at low dilution rates (Chou et al. 1996), as well as putrescine and 1‐pronanol in fed‐batch cultures (Qian et al. 2009; Jun Choi et al. 2012). Sigma factor RpoH has been used as chaperone to increase the production of lipoxygenase in E. coli (Pang et al. 2022). When E. coli is exposed to heterogeneous substrate availability, similar to the conditions in an industrial scale bioreactor, the expression level of thousands of genes changes. Löffler and coworkers showed that 89% of the differentially expressed genes under heterogeneous substrate availability were under control of sigma factors (Löffler et al. 2016). The genes controlled by rpoS were particularly affected by heterogeneous conditions. Bafna‐Rührer and coworkers cultured E. coli rpoS and fliA mutants under short periods of glucose starvation and found that the rpoS deletion improved the biomass yield, but fliA deletion worsened it (Bafna‐Rührer et al. 2024).

Despite the physiological relevance of the σ‐factors, there is not, to the best of our knowledge, a systematic study on the effect of individual knockouts on the growth and expression of recombinant protein under different culture conditions. Here, the performance of the individual σ‐factors mutants was characterized in a mineral medium and in lysogeny broth (LB), at 20°C and 37°C in microbioreactors with online measurement of the oxygen transfer rate (OTR), biomass, and NADH or GFP fluorescence signals. Such experimental setting allows detailed online data acquisition, and the information obtained is equivalent to the obtained in larger cultures. It has been demonstrated that cultures in microtiter plates can be successfully scaled to stirred bioreactors of several L volumes by choosing the appropriate scale‐up criterion (Seletzky et al. 2007; Kensy et al. 2009; Funke et al. 2010; Wewetzer et al. 2015; Ihling et al. 2020).

The impact of individual sigma factor deletion was first evaluated in a buffered mineral medium supplemented with 4 g L−1 glucose. The strains were cultured at 37°C, except for the rpoH mutant, which did not grow at this temperature. The growth profiles for the strains were similar, exhibiting a clear relation between biomass accumulation, OTR, and NADH fluorescence (Figure 1a). Notwithstanding, the NADH fluorescence remained constant during the stationary phase for all strains except for the rpoS mutant, in which a slow but steady increase was observed until 18 h of culture. Possibly, the lack of control of genes related to the stationary phase promoted some metabolic activity using intracellularly available metabolite pools, affecting the redox state of the cell. Apart from the fliA and fecI mutants, which grew at the same rate as the WT, the other sigma factors deletions decreased the growth rate between 13% and 30% (Figure 1b). Biomass synthesis was similar for all the strains, with small differences for rpoN and rpoS mutants (Figure 1b). The specific NADH fluorescence intensity was 25% lower for the rpoD and rpoS mutants, which suggests a decreased energy metabolism in these genotypes. The accumulated oxygen transferred to the liquid was lower for all the mutants except rpoN (Figure 1b). This can be related to decreased protein formation in the mutants, which results in ATP savings. In turn, deletion of the rpoN gene affects nitrogen metabolism by limiting the uptake of ammonium into the cell, which may explain the reduced growth of this mutant. Thus, the rpoN mutants may perceive nitrogen limitation, increasing the activity of the glutamine synthetase, which increases the ATP demand by 14%, compared to nitrogen‐excess conditions (Reitzer and Schneider 2001). This may contribute to an oxygen demand similar to that of the wild‐type, and higher than the rest of the mutants. Interestingly, the accumulated oxygen transfer rate for the fecI and fliA mutants decreased by 18% and 11%, respectively, compared to the WT strain, despite all three exhibiting similar growth rates (Figure 1b). The lower oxygen consumption by the fliA mutant is probably the consequence of the absence of flagellum synthesis and flagellum rotation, which are highly energy‐demanding processes (Schavemaker and Lynch 2022). This may result in less energy being required and, thus, less oxygen consumed by the cells. However, the reasons for reduced oxygen consumption by the fecI mutant remain elusive.

Figure 1.

Figure 1

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(a) Culture profile of the untransformed strains in a mineral medium. Biomass concentration was calculated from the online monitored scattered light intensity. The oxygen transfer rate and NADH fluorescence were also monitored. Shaded bands indicate the standard deviation between replicates. (b) Main culture parameters. Specific growth rate (µ) and NADH fluorescence were calculated over the exponential growth phase. * Indicates significant difference (n = 4; p < 0.05), compared to the WT strain.

The growth of the rpoD mutant was unexpected, since σ70 is considered an essential gene (Goodall et al. 2018). Examination of the mutant genome revealed the presence of a second copy of the rpoD gene (Supporting Information S1: Figure S1), in agreement with a previous report for strain BW25113 (Yamamoto et al. 2009). The rpoD expression, relative to the housekeeping gene ihfB was determined for the mutant and WT strains by RT‐qPCR. The results are depicted in Supporting Information S1: Figure S2. The rpoD expression level in the mutant strain was approx. 20% lower than in the WT (n = 7, p < 0.07). The expression of the duplicate rpoD appears to be insufficient to retain the growth rate at the same value as the WT in the mineral medium (Figure 1b). Because of the essentiality of rpoD, no further attempt to delete the second copy was made in this work. On the contrary, the mutant strain with only one rpoD copy was used for the following experiments.

The expression of recombinant protein was tested in the mineral medium within all strains using the green fluorescence protein (GFP) as a model. The gfp gene was coded in a high copy‐number plasmid under transcriptional control of the lac promoter. GFP expression was induced with 0.1, 0.2, or 0.3 mM IPTG. Culture profiles are shown in Figure 2. For a given strain, a higher GFP fluorescence is related to a lower maximal OTR (Figure 2a), which can be connected to a higher metabolic burden (Ladner et al. 2017; Mühlmann et al. 2018). The growth rate of the WT and rpoS mutant strains was not affected by the induction level (Figure 2b). In contrast, the growth rate of the other mutant strains using 0.3 mM IPTG was lower than the corresponding value at 0.1 mM IPTG (Figure 2b). In comparison to the WT strain, the growth rates of the rpoD, rpoS, and fliA mutants were considerably lower. The accumulated biomass was affected slightly, showing only small decreases for mutants other than rpoN (Figure 2b). Remarkably, the deletion of sigma factor genes resulted in strongly decreased GFP expression, compared to the WT, being fliA the only exception. The fliA mutant attained approx. Three and fourfold higher maximal and specific GFP fluorescence, respectively, than the WT strain. Moreover, despite the growth rate reduction of the fliA mutant, the specific GFP fluorescence emission rate in cultures of this mutant was between 1.8 and 3‐fold greater than for the WT strain (Figure 2b). When induced with 0.2 and 0.3 mM IPTG, all the mutants exhibited higher accumulated oxygen transfer rates compared to the corresponding values of the WT (Figure 2b). Interestingly, the greatest accumulated oxygen transfer rate was seen in cultures of the rpoS mutant induced with 0.2 mM IPTG, even though under such conditions, this strain grew slower, produced less biomass, and expressed less GFP than the other strains (Figure 2b). This results in higher stoichiometric oxygen demand to oxidize glucose to CO2 for cellular maintenance during the longer fermentation times of this strain. In contrast, in cultures of the fliA mutant induced with 0.2 mM IPTG, the accumulated oxygen transfer was the lowest among all the mutants, while the GFP‐specific fluorescence and emission rate was the highest (Figure 2b).

Figure 2.

Figure 2

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(a) Culture profile of the strains bearing the plasmid pV21 in mineral medium at 37°C. GFP expression was induced with 0.1, 0.2, or 0.3 mM IPTG from inoculation. Biomass concentration was calculated from online monitored scattered light intensity. The oxygen transfer rate and GFP fluorescence were also monitored. Shaded bands indicate the standard deviation between replicates. (b) Main culture parameters using 0.1 (blue bars), 0.2 (green bars), or 0.3 (grey bars) mM IPTG. Specific growth rate (µ), GFP fluorescence, and GFP fluorescence emission rate (qF) were calculated over the exponential growth phase. * Indicates significant difference (n = 4; p < 0.05), compared to the WT strain under the same conditions.

There is no clear relationship between the observed parameters. In general, the effects were more evident for the rpoS and fliA mutant strains. The positive effect observed in the fliA mutant can be attributed to better resource stewardship, due to less energy spent in motility‐related protein synthesis. Similar effects have been observed by deleting genes coding for the transcriptional dual regulator FlhC, which is the main regulator of flagellum synthesis and swarming migration (Han et al. 2021; Lara et al. 2024). Ziegler and coworkers also reported an increase in the production of a recombinant protein in a strain lacking fliA and some operons related to motility (Ziegler et al. 2021). In the case of the rpoS mutant, it is known that flagellar synthesis is upregulated, when the rpoS gene is deleted (Dong and Schellhorn 2009), which may partially explain the growth rate reduction and accumulated oxygen transfer increase.

Recombinant protein expression at low temperatures can increase the production of correctly folded proteins in E. coli (San‐Miguel et al. 2013). The use of expression systems engineered to induce protein expression in response to low temperature, is attractive to attain high expression levels (Lin et al. 2024). Therefore, a set of cultures at 20°C in the mineral medium was carried out, which included the rpoH mutant. As explained above, increasing the IPTG concentration from 0.2 to 0.3 mM did not increase the GFP fluorescence in cultures at 37°C. Therefore, for the cultures at 20°C, only 0.1 and 0.2 mM IPTG were used. The OTR values in cultures at 20°C were very low, compared to cultures at 37°C (Figures 2a and 3b). This may be due to the low oxygen consumption rate of the cells, as the growth at 20°C was slow (some cultures lasted up to 50 h, Figure 3a). The strains exhibited prolonged lag phases, lasting between 10 and 20 h (Figure 3a). The GFP fluorescence increased parallel to cell growth. In cultures of the rpoS mutant, both the GFP fluorescence and biomass signals strongly increased immediately after the OTR decrease, indicating glucose exhaustion (Figure 3b). This could be related to the downregulation of stationary phase genes in this mutant, which in turn may release cellular resources for further GFP expression. The specific growth rate was severely reduced in the rpoD, rpoS, and rpoH mutants, compared to the WT (Figure 3b). With exception of the rpoN mutant, all the other strains attained higher biomass than the WT (Figure 3b). The maximal GFP fluorescence was similar in cultures of the WT and rpoN and fliA mutant strains, while for the fecI mutant, it was 20% lower than for the WT under the two induction levels (Figure 3b). In contrast, GFP expression was remarkably higher in the rpoD, rpoH, and rpoS mutant strains than in the WT (Figure 3b).

Figure 3.

Figure 3

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(a) Culture profile of the strains bearing the plasmid pV21 in mineral medium at 20°C. GFP expression was induced with 0.1 or 0.2 mM IPTG from inoculation. Biomass concentration was calculated from the online monitored scattered light intensity. The oxygen transfer rate and GFP fluorescence were also monitored. Shaded bands indicate the standard deviation between replicates. (b) Main culture parameters using 0.1 (blue bars), or 0.2 (green bars) mM IPTG. Specific growth rate (µ), GFP fluorescence, and GFP fluorescence emission rate (qF) were calculated over the exponential growth phase. * Indicates significant difference (n = 4; p < 0.05), compared to the WT strain under the same conditions.

Particularly, the GFP fluorescence in cultures of the rpoS mutant was up to 19‐fold higher than in cultures of the WT strain. Changing the IPTG concentration from 0.1 to 0.2 mM increased GFP fluorescence by 23% in cultures of the rpoS mutant strain, whereas no relevant changes were observed for the other strains. The rpoS mutant exhibited the highest specific GFP fluorescence yield and fluorescence emission rate, which were up to nine and fourfold greater, respectively, than those of the WT strain. At 20°C, E. coli cells experience a cold shock, which lowers cell growth and biomass yields, as can be seen comparing the results of Figure 2 with those of Figure 3. The sigma factor σS is essential for the response to cold shock, as it is involved in this stress response and activates genes that code for membrane proteins to strengthen them (White‐Ziegler et al. 2008). A deletion of the rpoS gene leads to a lower growth of the cells (Figure 3b), which are more sensitive to the cold shock due to the lack of membrane support. However, at 20°C, the highest GFP fluorescence emission rate (q F ) can be observed for this mutant. The suppressed cold stress response appears to provide additional metabolic space for GFP expression. Therefore, the rpoS deletion seems to be an attractive option for improving recombinant protein expression at low temperatures in E. coli.

The specific GFP fluorescence was threefold higher in the rpoD mutant than in the WT strain (Figure 3b). It has been reported that mutations in the sequence of rpoD reduces its capacity to bind to the RNA polymerase core, creating conditions similar to reduced levels of rpoD expression (Jishage et al. 2002). A mutation in the sequence of rpoD led to a fourfold higher expression of a eukaryotic integral membrane protein at 20°C in batch mode. The authors also reported higher biomass formation in fed‐batch cultures in shake flask (Tomatis et al. 2019). In agreement with such information, the lower expression of rpoD in the mutant strain used in the present study, also resulted in a substantial increase of recombinant protein expression and, therefore, is an interesting target to increase the production of difficult‐to‐express proteins.

GFP expression was also evaluated in the widely used LB medium, at 37°C and with 0.1 and 0.2 mM IPTG. The results are shown in Figure 4. No growth of the rpoH mutant was observed, and consequently, this strain is excluded from Figure 4. The OTR time profile suggests the exhaustion of several nutrients during cell growth, which agrees with the complex composition of this culture medium (Figure 4a). Like in previous experiments, the GFP fluorescence increased proportionally to the cell growth (Figure 4a). The growth rate was lower in all mutants and induction levels, except for the rpoN mutant with 0.1 and 0.2 mM IPTG, and the fecI mutant with 0.1 mM IPTG (Figure 4b). The individual deletion of the sigma factors resulted in lower attained biomass, except in the case of the rpoN mutant. The unaffected growth rate and biomass synthesis of the rpoN mutant suggest that the genes controlled by this sigma factor are less relevant in the rich LB than in the mineral media, in which the growth rate of the rpoN mutant was noticeably lower than that of the WT (Figures 1, 2, 3). Furthermore, the biomass attained in cultures of the rpoN mutant was very similar to those of the WT, which contrasts with the rest of the mutant strains, that exhibited lower biomass values (Figure 4b). In contrast, only the rpoD, rpoS, and fliA mutants reached maximal GFP fluorescence values greater than the WT. During the exponential growth phase, the specific GFP fluorescence of the fecI and fliA mutants was approx. 20% lower than the corresponding value for the WT. The rpoN mutant achieved a specific GFP fluorescence value 50% higher than that of the WT. Remarkably, the specific GFP fluorescence of mutant strains rpoS and rpoD were approx. Three and ninefold greater than the achieved by the WT (Figure 4b). Consequently, the specific GFP fluorescence emission rate of the mutant strain rpoD was approx. Sevenfold higher compared to the WT (Figure 4b). In general, the accumulated oxygen transferred in cultures of the mutant strains was slightly but significantly (p < 0.05) lower than that of the WT (Figure 4b). The lowest value was observed in cultures of the mutant strain rpoD, which was approx. 20% lower than that of cultures of the WT. The higher GFP expression and lower oxygen consumption render the rpoD mutant an excellent option for protein expression in a rich medium. A reason for the superior performance of this strain could be related to a lower proteomic burden in the rich LB medium, due to lesser expression of the proteins controlled by the transcriptional factor RpoD. For instance, McKenna and coworkers created a library of rpoD gene mutations and found that the most truncated mutant led to strongly increased expression of a full‐length antibody at 30°C in a rich medium, although the exact reasons for this were not clarified (McKenna et al. 2019). Further studies are required to fine‐tune the expression level of rpoD as a strategy to optimize recombinant protein expression in rich media.

Figure 4.

Figure 4

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(a) Culture profile of the strains bearing the plasmid pV21 in LB medium at 37°C. GFP expression was induced with 0.1 or 0.2 mM IPTG from inoculation. Biomass concentration was calculated from the online monitored scattered light intensity. The oxygen transfer rate and GFP fluorescence were also monitored. Shaded bands indicate the standard deviation between replicates. (b) Main culture parameters using 0.1 (blue bars), or 0.2 (green bars) mM IPTG. Specific growth rate (µ), GFP fluorescence, and GFP fluorescence emission rate (qF) were calculated over the exponential growth phase. * Indicates significant difference (n = 4; p < 0.05), compared to the WT strain under the same conditions.

Taken together, the results show that deletion of sigma factors is a useful tool to improve the production of recombinant protein in E. coli, since they clearly affect the expression levels, growth rates, and oxygen consumption. As this study shows, the best target for deletion will depend on the culture conditions like temperature and media, and should be decided depending on the application. Namely, recombinant protein expression at low temperatures is benefited by rpoS, rpoH, and rpoD knockouts. At 37°C, rpoD deletion showed the best results in LB, while in a mineral medium, the fliA was a superior producer.

2. Materials and Methods

2.1. Strains and Plasmid

The K‐12 derivative BW25113 Escherichia coli was used as wild type (WT). The mutans strains with individual interruptions of the genes rpoD, rpoN, rpoS, fecI, and fliA, were obtained from the Keio Collection (Baba et al. 2006). The gene rpoH was interrupted using the method described by Datsenko and Wanner (2000), except that the mutant strain was incubated at 20°C. The strains were transformed with the high copy number plasmid pV21, which contains the gene of the GFP under transcriptional control of the lac promoter, and a gene conferring spectinomycin resistance (Velazquez et al. 2022). Transformed and untransformed strains were plated in LB‐agar in Petri dishes and grown at 37°C for 14–18 h, except for the rpoH mutant, which was grown at 20°C for 48 h. One colony per strain was selected and grown in Terrific Broth (TB) medium at 20°C (for the rpoH mutant) or 37°C (for all the other strains) until reaching an optical density at 600 nm (OD600) of approximately 6–8. Then, 0.9 mL of such culture broth was mixed with a sterile solution of glycerol (80% v/v) and immediately frozen at −80°C. The genotypes of the mutant strains were confirmed by PCR amplification of the involved gene using the oligomers described in the Supplementary File. To further confirm the PCR product of the ΔrpoD strain, 6 colonies were sampled and subjected to PCR amplification.

2.2. Media Composition

All chemicals applied for media preparation were of analytical grade and purchased from Carl Roth GmbH, if not stated otherwise. The composition of LB was (in g L−1): yeast extract, 5; tryptone, 10; NaCl, 5. The composition of TB was (in g L−1): yeast extract, 24; tryptone, 20; glycerol, 4. The mineral medium used was modified from a previous report (Wilms et al. 2001). The mineral medium composition was (in g L−1): (NH4)2SO4, 6.5; NH4Cl, 0.5; K2HPO4, 3.0; Na2SO4, 2.0; MgSO4 • 7H2O, 0.5; thiamine hydrochloride 0.01; MOPS, 41.85; plus 1 mL of trace elements solution per L of medium. Spectinomycin was purchased from Sigma Aldrich. The initial pH was adjusted to 7.4 by adding 0.2 M NaOH. The trace elements solution was prepared and autoclaved separately. The composition of the trace element solution was (in g L−1): ZnSO4• 7H2O, 0.54; CuSO4 • 5H2O, 0.48; MnSO4 • H2O, 0.30; CoCl2 • 6H2O, 0.54; FeCl3 • 6H2O, 41.76; CaCl2 • 2H2O, 1.98; Na2EDTA • 2H2O, 33.4. Glucose was added from a separately autoclaved solution at a concentration of 500 g L−1, to final concentrations of 4 g L−1 for cultures of the untransformed strains, or 3.5 g L−1 for cultures of the plasmid‐bearing strains. For all cultures of plasmid‐bearing cells, the media were supplemented with 0.1 g L−1 spectinomycin. GFP expression was induced with isopropyl β‐D‐1‐thiogalactopyranoside (IPTG) added before inoculation at final concentrations of 0.1, 0.2, or 0.3 mM.

2.3. Microbioreactor Cultures

The strains were characterized in microbioreactor cultures in lysogeny broth (LB) or in the mineral medium supplemented with 4 g L−1 glucose. For cultures in LB, a preculture was performed by transferring 0.05 mL of the cryopreserved cells to 8 mL of LB medium contained in a 250 mL Erlenmeyer flask. For cultures in the mineral medium, 0.05 mL of the cryopreserved cells were transferred to 8 mL of TB medium contained in a 250 mL Erlenmeyer flask. The cells were grown at 20 (for the rpoH mutant) or 37°C (for all other strains), and 350 rpm in an orbital shaker of 50 mm shaking diameter for 6 h. For the main cultures in mineral medium, 0.05 mL from the first preculture in TB medium were transferred to 8 mL of mineral medium contained in a 250 mL Erlenmeyer flask and cultured at 20 (for the rpoH mutant) or 37°C (for all other strains), and 350 rpm in an orbital shaker of 50 mm shaking diameter until mid‐exponential growth phase (approx. 14–16 h). The obtained cells were washed with 0.9% sterile NaCl solution and used to inoculate the main cultures. Microbioreactor cultures were carried out in 48‐round transparent bottom wells microtiter plates (MTP) (Beckman Coulter) with a liquid volume of 0.75 mL per well, an initial OD600 of 0.2 units, at 20 or 37°C, 1000 rpm and 3 mm shaking diameter (Climo‐Shaker ISF1‐X, Kuhner). The shaken monitoring system, named µRAMOS‐BioLector combination, is constructed in‐house, and enables measurement of the OTR in every individual well of the MTP based on the oxygen partial pressure and oxygen‐dependent emission of fluorescence sensors (Flitsch et al. 2016), and synchronized online measurement of fluorescence and scattered light (Ladner et al. 2016). The MTPs were covered with a gas‐permeable polyolefin sealing foil (HJ‐Bioanalytik GmbH), to reduce evaporation and prevent contaminations. Cell growth was monitored by measuring the scattered light (ScL) intensity at a wavelength of 620 nm. Biomass concentration was calculated from the ScL readings using an experimental correlation for E. coli (Kensy et al. 2009). The wavelengths for excitation and fluorescence emission of GFP were 480 and 507 nm, while for NADH were 340 and 460 nm, respectively.

2.4. rpoD Gene Transcription Level

Triplicate cultures of the WT and rpoD mutant strains were carried out in the mineral medium at 37°C as explained above. Samples were taken during the exponential growth of the cells. RNA extraction, purification, RT‐qPCR conditions, and relative transcription level calculation are described in detail elsewhere (Aguilar et al. 2012).

2.5. Data Analysis

The data from fluorescence and scattered light (ScL) intensities are represented as the corresponding reading minus the lowest reading (usually obtained during the first 20 min of culture). The specific fluorescence (SF) was calculated as the mean of the fluorescence divided by the ScL readings in every time point during the period involved. Specific yields were calculated by the proper mass balances over the time periods involved. The specific fluorescence emission rate was calculated by multiplying the specific growth rate by the specific GFP fluorescence intensity. Accumulated oxygen transfer was calculated by integration of the area under the curve in the plot of OTR versus time using the Integration gadget of the Origin software (OriginLab Corporation). For comparison between two groups means, unpaired Student's t‐tests were performed.

Author Contributions

A. R. Lara, G. Gosset, and J. Büchs: conceptualization. L. Pohlen, E. García, N. Flores, L. M. Martínez, A. R. Lara, and G. Gosset: experimental design, planning, execution, data acquisition, analysis. A. R. Lara, G. Gosset, and J. Büchs: funding acquisition. A. R. Lara, G. Gosset, J. Büchs, L. Pohlen, E. García, N. Flores, and L. M. Martínez: writing – review and editing.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supporting information.

BIT-122-1598-s001.docx (843KB, docx)

Acknowledgments

This work was supported by CONACyT grant A1‐S‐8646.

Contributor Information

Guillermo Gosset, Email: guillermo.gosset@ibt.unam.mx.

Alvaro R. Lara, Email: alvaro.lara@bce.au.dk.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supporting information.

BIT-122-1598-s001.docx (843KB, docx)

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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