- Open Access
Establishment of BmoR-based biosensor to screen isobutanol overproducer
© The Author(s) 2019
- Received: 8 October 2018
- Accepted: 31 January 2019
- Published: 7 February 2019
Isobutanol, a C4 branched-chain higher alcohol, is regarded as an attractive next-generation transport fuel. Metabolic engineering for efficient isobutanol production has been achieved in many studies. BmoR, an alcohol-regulated transcription factor, mediates a σ54-dependent promoter Pbmo of alkane monooxygenase in n-alkane metabolism of Thauera butanivorans and displays high sensitivity to C4–C6 linear alcohols and C3–C5 branched-chain alcohols. In this study, to achieve the high-level production of isobutanol, we established a screening system which relied on the combination of BmoR-based biosensor and isobutanol biosynthetic pathway and then employed it to screen isobutanol overproduction strains from an ARTP mutagenesis library.
Firstly, we constructed and verified a GFP-based BmoR-Pbmo device responding to the isobutanol produced by the host. Then, this screening system was employed to select three mutants which exhibited higher GFP/OD600 values than that of wild type. Significantly, GFP/OD600 of mutant 10 was 190.7 ± 4.8, a 1.4-fold higher value than that of wild type. Correspondingly, the isobutanol titer of that strain was 1597.6 ± 129.6 mg/L, 2.0-fold higher than the wild type. With the overexpression of upstream pathway genes, the isobutanol production from mutant 10 reached 14.0 ± 1.0 g/L after medium optimization in shake flask. The isobutanol titer reached 56.5 ± 1.8 g/L in a fed-batch production experiment.
This work screened out isobutanol overproduction strains from a mutagenesis library by using a screening system which depended on the combination of BmoR-based biosensor and isobutanol biosynthetic pathway. Optimizing fermentation condition and reinforcing upstream pathway could realize the increase of isobutanol production from the overproducer. Lastly, fed-batch fermentation of the mutant enhanced the isobutanol production to 56.5 ± 1.8 g/L.
Higher alcohols including n-butanol, isobutanol, 2-methyl-1-butanol (2-MB), 3-methyl-1-butanol (3-MB) and 2-phenylethanol have drew much attention as next-generation transport fuels because of their higher energy density, lower vapor pressure and lower hygroscopicity when compared with traditional biofuel such as ethanol . Microbial-based metabolic engineering, as an eco-friendly strategy, has been used to produce many value-added compounds from renewable resources [2–7]. Microbial production of higher alcohols also has been realized in many engineered hosts [3, 8–10]. The pathways for the biosynthesis of these alcohols were extended from 2-keto acids, the intermediates in amino acid biosynthetic pathways. Subsequently, 2-keto acids were converted to corresponding alcohols by the sequential catalysis through 2-keto acid decarboxylase (KDC) and alcohol dehydrogenase (ADH) [11, 12].
In general, modifying microorganism was an essential step in metabolic engineering. Microbial mutation breeding was a common strategy which included random mutagenesis by nitrosoguanidine (NTG) , ultraviolet treatment (UV)  or atmospheric and room temperature plasma (ARTP) [19, 20]. After mutagenesis of the host microorganism, the methods for screening mutant strains that overproduced the target molecules or their relevant precursors were desired. Traditional screening methods depended on high performance liquid chromatography (HPLC) or gas chromatograph (GC), which were time-consuming and limited by high cost and low efficiency. Biosensor, as an emerging tool, could respond to the specific metabolite and then dynamically regulate the whole metabolic pathway, leading to the balance of metabolic flux and the increase of the productivity of desired compound [21–26]. Biosensors were used in many studies to regulate the expressions of fluorescent proteins, which provided the detection signals for high-throughput screening [27–29]. Zhang et al. constructed NCgl0581 biosensor in C. glutamicum to screen out a high-producing l-serine strain that produced 34.78 g/L l-serine with a yield of 0.35 g/g sucrose, which were 35.9 and 66.7% higher than those of the parent strain . Liu et al. adopted the l-phenylalanine-specific transcription factor (TyrR) in E. coli to screen out a variant which could yield 1.8-fold higher l-phenylalanine when compared with the parent strain .
So far, using a strategy for screening high-level isobutanol production strains from the mutagenesis library has not been accomplished. BmoR, an alcohol-regulated transcription factor in n-alkane metabolism of Thauera butanivorans, mediated a σ54-dependent promoter Pbmo of alkane monooxygenase . The alcohol molecules could bind to BmoR (an enhancer-binding protein as hexamer) and the generated combination would remodel σ54-RNAP holoenzyme (Eσ54) closed complex and then activate the transcription initiation of promoter Pbmo . Dietrich et al. firstly used BmoR as a biosensor to respond to various alcohols and the results showed that BmoR exhibited high sensitivity to C4–C6 linear alcohols and C3–C5 branched-chain alcohols, of which n-butanol showed the broadest linear range of detection (from 100 μM to 40 mM). Then, BmoR-based biosensor was employed to screen out the strain with the highest conversion rate from 2-oxopentanoate to n-butanol in a RBS mutagenesis library .
For the purpose of acquiring the high-level isobutanol production strains, it is essential to acquire efficient host microorganisms. In this study, a biosensor-based strategy was designed for screening isobutanol overproduction strains. Isobutanol generated from glucose in hosts or absorbed from environment would bind to BmoR hexamer whose expression was driven by a constitutive promoter. The BmoR with isobutanol binding would activate Eσ54 closed complex and then induce the transcription initiation of promoter Pbmo. The whole response process was shown in Fig. 1. Firstly, we constructed a GFP-based BmoR-Pbmo biosensor system and testified its effectiveness. Then, this biosensor system combining with isobutanol biosynthetic pathway was introduced into the mutagenesis library to screen the isobutanol overproducers. As a result, the mutant which was screened out from the mutagenesis library could produce 14.0 ± 1.0 g/L isobutanol in shake flask and 56.5 ± 1.8 g/L isobutanol in a 3-L bioreactor. This work illustrated that BmoR-based biosensor could respond to intracellular isobutanol and could be used to screen isobutanol overproducers, which has the potential to be engineered to overproduce l-valine since the biosynthesis pathways of isobutanol and l-valine shared the same precursor 2-KIV.
Characterization of BmoR-P bmo biosensor via feeding isobutanol
GFP/OD600max and Km values of BmoR-based biosensor towards isobutanol, n-butanol and 3-MB
Dynamic range (GFP/OD600max)
197.8 ± 51.2
4.2 ± 0.3
336.8 ± 137
2.3 ± 1.1
200.7 ± 7.5
4.6 ± 1.1
Optimizing the copy number of BmoR-P bmo device in the isobutanol-producing strains
Establishment of the screening system
Screening high-level isobutanol production strains
Improvement of isobutanol production via strengthening the upstream pathway and optimizing yeast extract concentration
Production of isobutanol in a fed-batch bioreactor
In this work, we confirmed that the biosensor BmoR could respond to isobutanol and could be utilized as a basic element for screening isobutanol-overproducing strains. We assembled a BmoR-Pbmo system and then combined it with isobutanol biosynthetic pathway to construct a GFP-based screening strategy in E. coli. From an ARTP mutagenesis library, we screened out three strains which have ability to produce more isobutanol than wild type in the absence of overexpression of upstream pathway for precursor 2-KIV production. Besides, reinforcing the 2-KIV overproduction pathway and optimizing medium improved the isobutanol titer from a mutated host to 14.0 ± 1.0 g/L. Moreover, the titer of isobutanol reached 56.5 ± 1.8 g/L in a fed-batch production experiment.
Medium, strains and plasmids
Plasmids and strains used in this study
Plac-lacZα; colE1; ampr
PLlacO1; LacI; colA; kanr
PLlacO1; pSC101*; cmr
PLlacO1-kivd-adhA; colE1; ampr
PLlacO1-alsS-ilvC-ilvD; p15A; kanr
PbmoR-bmoR; Pbmo-gfp; colE1; ampr
PbmoR-bmoR; Pbmo-lacZα; colE1; ampr
PbmoR-bmoR; Pbmo-lacZα; pSC101*; cmr
PbmoR-bmoR; Pbmo-lacZα; PLlacO1-kivd-adhA; colE1; ampr
PbmoR-bmoR; Pbmo-lacZα; colA; cmr
PbmoR-bmoR; Pbmo-gfp; PLlacO1-kivd-adhA; colE1; ampr
PbmoR-bmoR; Pbmo-gfp; colA; cmr
PbmoR-bmoR; Pbmo-gfp; pSC101*; cmr
recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F′ proAB lacIqZΔM15 Tn10 (Tetr)]
ΔrhaBADLD78F′ [traD36 proABlacIqZΔM15 Tn10(Tet r )]
BW25113 (F′) with pYH1
BW25113 (F′) with pYH2
BW25113 (F′) with pSA69 and pYH4
BW25113 (F′) with pSA69, pSA65 and pYH5
BW25113 (F′) with pSA69, pSA65 and pYH3
BW25113 (F′) with pSA65 and pYH5
BW25113 (F′) with pSA65 and pYH10
Mutant 10 with pSA69
Plasmids pUC19 from TransGen Biotech, pYK  and pSA74  were used as templates to amplify genes lacZα, colA origin and pSC101* origin, respectively. Plasmids pSA65 and pSA69 were from previous study . Plasmid pSA65 was used as template to amplify colE1 origin. Gene gfp (accession number: AAX07425.1) was synthesized by OE-PCR. The PbmoR-bmoR and Pbmo were referenced from NCBI (accession number: AY093933.3). PbmoR-bmoR was synthesized by OE-PCR after codon optimization for E. coli and then ligated with colE1 origin, ampr and Pbmo-gfp via Gibson Assembly, generating plasmid pYH1. To construct plasmid pYH2, gfp of pYH1 was replaced with lacZα. Replacing colE1 origin and ampr of pYH2 with pSC101* origin and cmr, respectively, generated plasmid pYH3. To create plasmid pYH5, pSC101* origin of pYH3 was substituted with colA origin. PbmoR-bmoR, Pbmo-lacZα, PLlacO1-kivd-adhA which was amplified from pSA65, colE1 origin and ampr were assembled via Gibson Assembly to generate plasmid pYH4. Gene lacZα of pYH3, pYH4 and pYH5 were replaced with gfp, resulting in plasmids pYH11, pYH9 and pYH10, respectively. All the plasmids were sequenced by GENEWIZ company.
Extracellular confirmation of BmoR-P bmo biosensor based on GFP fluorescence
Escherichia coli BW25113 (F′) harboring plasmid pYH1 was pre-inoculated into 5 mL LB with 100 μg/mL ampicillin and then cultured at 37 °C overnight. Then, 50 μL of the seed culture was transferred into 950 μL fresh LB medium supplemented with different concentrations (0, 0.01, 0.1, 1, 10, 20, 40, 50 or 100 mM) of n-butanol, isobutanol and 3-MB, respectively, in 96-deep-well plates and the cultures were then left at 30 °C for 16 h. After that, GFP fluorescence and OD600 values were detected by microplate reader (BioTek Cytation 3). GFP fluorescence was measured using an excitation wavelength of 470 nm and an emission wavelength of 510 nm. GFP fluorescence value was normalized as GFP/OD600, and the background fluorescence of medium was subtracted. Km values were estimated with OriginPro8.5 through non-linear regression of the Hill1 equation.
Extracellular confirmation of BmoR-P bmo biosensor based on blue–white screening
Escherichia coli BW25113 (F′) harboring the biosensor plasmid pYH2 were cultured overnight in 5 mL LB medium with 100 μg/mL ampicillin, and then 50 μL of seed culture was transferred into 5 mL fresh LB medium with different concentrations of isobutanol (0, 0.01, 0.1, 1, 10, 20, 40, 50 or 100 mM) feeding when OD600 reached 0.2–0.4. Cultures were then left at 30 °C for 24 h, and then 10 μL of the seed culture was diluted 5000 times and spread on the LB plate with 100 μg/mL X-gal, different concentrations of isobutanol and associated antibiotics. The plates were placed in a 30 °C incubator for 72 h.
Verifying BmoR-P bmo biosensor response to isobutanol produced by cell based on blue–white screening
Plasmids with different copies (pYH4, pYH5 and pYH3, high-copy, medium-copy and low-copy-number, respectively) were introduced into isobutanol-producing strain E. coli BW25113 (F′) (pSA65/pSA69). The single colonies were inoculated into 5 mL LB medium with appropriate antibiotics and grew at 37 °C until OD600 reached around 1.0. Then, 10 μL of the seed culture was diluted 5000 times and spread on the M9 plate with 100 μg/mL X-gal, 0.1 mM IPTG and associated antibiotics. The plates were placed in a 30 °C incubator for 72 h.
ARTP mutagenesis and screening
The ARTP mutation system which could cause greater gene damage than traditional mutagenesis was employed to generate the mutation library . E. coli BW25113 (F′), as initial strain, was pre-incubated into 5 mL LB medium and cultured at 37 °C for 4 h to reach its logarithmic phase. Then, 10 μL of the culture was transferred to stainless steel minidisc and subsequently exposed to ARTP with 60-s irradiation. The mutated cells were recovered in 3 mL fresh LB medium at 37 °C for 6 h and then stored in 15% glycerol for screening. For screening, the cells in mutagenesis library were transformed with plasmids pSA65 and pYH10. The single colonies were pre-incubated into 5 mL LB medium with 100 μg/mL ampicillin and 25 μg/mL chloromycetin at 37 °C for 12 h. 50 μL of the seed culture was added into 1 mL M9 medium with 0.1 mM IPTG in 96-deep-well plates. All 96-deep-well plates with cultures were incubated at 30 °C for 20 h. The fluorescence and OD600 values were measured by the microplate reader.
Fermentation verification of the isobutanol overproduction strains in shake flask
The single colonies were pre-inoculated into 5 mL LB medium with associated antibiotics at 37 °C for 12 h. Then, 200 μL culture was inoculated into 20 mL M9 with 0.1 mM IPTG and various concentration of yeast extract in 250 mL screw cap conical flask and then left at 30 °C. Samples were taken every 12 h. Then, 200 μL of sample was added into 96-well plates for fluorescence and OD600 measurement. Production of alcohols was quantified by Agilent 6890 GC chromatograph equipped with flame ionization detector (Agilent Technologies, CA, USA). The separation of alcohols was carried out by a DB-FFAP capillary column (30 m × 0.32 mm × 0.25 μm; Agilent Technologies). For analysis of isobutanol, the GC oven temperature was initially held at 80 °C for 3 min, increased with a gradient of 115 °C/min until 230 °C, and kept at 230 °C for an additional 1 min. Nitrogen was used as the carrier gas. The injector and detector were maintained at 250 and 280 °C, respectively. Supernatant (1 μL) was sampled and injected at a split ratio of 1:30 and n-pentanol was used as internal standard.
Culture medium for fed-batch fermentation
To evaluate the isobutanol-producing potential of the mutated strain which was screened out from the mutation library, the fermentation was performed in a 3-L bioreactor with 2 L working volume. The culture medium containing 40 g/L glucose, 3 g/L (NH4)2SO4, 14.6 g/L K2HPO4, 4 g/L KH2PO4, 2.2 g/L sodium citrate, 8 g/L yeast extract, 1.25 g/L MgSO4·7H2O, 0.1 g/L ampicillin, 0.05 g/L kanamycin, 0.025 g/L chloromycetin and 1 mL/L trace metal solution was used for bioreactor fermentation. Trace metal solution contained 14.1 g EDTA, 2.5 g CoCl2·6H2O, 15 g MnCl2·4H2O, 1.5 g CuCl2·2H2O, 3 g H3BO3, 2.1 g Na2MoO4·2H2O, 33.8 g Zn(CH3COO)2·2H2O and 80 g FeCl3·6H2O per liter. During the cultivation period, 1.5 L stock solution containing 500 g/L glucose, 1.25 g/L MgSO4·7H2O, 0.1 g/L ampicillin, 0.05 g/L kanamycin, 0.025 g/L chloromycetin and 0.1 mM IPTG was fed to the batch culture.
Bioreactor culture conditions
The bioreactor was inoculated with 5% of overnight pre-culture and the cells grown at 37 °C with 1 vvm of air flow rate and 600 rpm of stirrer speed for 2 h. Then, 0.1 mM IPTG was added into the bioreactor and the temperature was changed to 30 °C to induce the expression of the enzymes in isobutanol production pathway. The pH was controlled at 6.8 by automatic addition of ammonia solution (25%). After induction of 12 h, the air flow rate was increased from 1 to 3 vvm in order to strip out isobutanol from the broth. The evaporated isobutanol was condensed by a condenser and subsequently the generated liquid isobutanol flowed into collection bottle A which was cooled with ice (Fig. 7). The residual uncondensed isobutanol was collected into bottle B and C which containing 800 mL water and were also cooled with ice (Fig. 7). The samples were taken to determinate the biomass, glucose concentration and isobutanol titer.
YH generated the idea. YH, ZC and HY designed the project. HY, ZC, NW and WH conducted the experiments. YZ and WZ directed the fed-batch fermentation experiments. YH, YY, HY and ZC analyzed the data. YH, HY and ZC wrote the manuscript. All authors read and approved the final manuscript.
The authors wish to thank Beijing Institute of Technology for the supply of microplate reader (BioTek Cytation 3) and Biotechnology Research Institute of Chinese Academy of Agricultural Sciences for the supply of fluorescence microscope (Nikon model Eclipse Ni-U) and bioreactors (Bioengineering AG).
The authors declare that they have no competing interests.
Availability of data and materials
All the data generated during the current study are included in the manuscript.
Consent for publication
Ethics approval and consent to participate
The authors would like to acknowledge financial support of the National Natural Science Foundation of China (Grant No. 21676026), the China Postdoctoral Science Foundation Funded Project (Grant No. 2018M630083) and Fundamental Research Funds for the Central Universities.
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