- Open Access
Enhancement of 3-hydroxypropionic acid production from glycerol by using a metabolic toggle switch
© Tsuruno et al. 2015
Received: 26 July 2015
Accepted: 17 September 2015
Published: 5 October 2015
3-hydroxypropionic acid (3-HP) is an important platform for the production of C3 chemicals, including acrylic acid, methyl acrylate, and acrylamide. Microbial production of 3-HP is mainly due to glycerol metabolism. In this study, in order to improve microbial 3-HP production, we applied a metabolic toggle switch for controlling the glycerol metabolism to redirect the excess metabolic flux of central metabolic pathway toward an exogenous 3-HP producing pathway in Escherichia coli.
The metabolic toggle switch enables conditional repression of the expression of a target gene during the fermentation. We individually performed conditional repression of glpK, tpiA, and gapA, which are involved in glycerol metabolism. The conditional repression of glpK and tpiA was not effective for 3-HP production under our experimental conditions. However, gapA conditional repression contributed to improve 3-HP production (titer, 54.2 ± 1.5 mM; yield, 32.1 ± 1.3 %) compared with that for the wild type strain. Additional deletion of endogenous yqhD, which is responsible for the production of a major byproduct, 1,3-propandiol, further increased 3-HP production (titer, 67.3 ± 2.1 mM; yield, 51.5 ± 3.2 %). The titer and yield were 80 and 94 % higher than those of the wild type strain, respectively. The obtained 3-HP yield from glycerol is comparable with the highest yield ever reported for microbial 3-HP production using glycerol as a sole carbon source. The measurement of intracellular metabolites showed the metabolic toggle switch successfully controlled the metabolic flux.
The conditional repression of gapA by using the metabolic toggle switch combined with deletion of endogeneous yqhD increased 3-HP production approximately twofold from glycerol. This result indicates the metabolic toggle switch can be applied in various bio-production using diverse substrates.
Growing concerns regarding the depletion of fossil resources and environmental sustainability have led to an increased demand for the development of bio-based chemicals and fuel production using renewable feedstock by microorganisms. Recently, in several cases, an exogenous synthetic pathway introduced in non-native hosts sufficiently produced various alcohols, acids, and chemicals [1–6]. Escherichia coli, in particular, has mainly been used as the host organism for bio-production using this synthetic pathway, due to its well-understood metabolism and potential for easy genetic manipulation . In many studies, conventional metabolic engineering, such as gene deletion which defects genes on chromosome responsible for competing pathway by homologous recombination, was applied to increase metabolic flux toward the synthetic pathway for improving the titer and yield of the final products [1, 8]. In many cases, the introduced synthetic pathway for carbon-based products such as alcohols or fatty acids competes with central carbon metabolism processes, including glycolysis and the tricarboxylic acid (TCA) cycle, for carbon sources. However, the deletion approach is unsuitable for enzymes that catalyze central carbon metabolism, as the deletion leads to growth defects or severely decreased growth of the host strain under certain conditions . Therefore, conventional metabolic engineering strategies may not be feasible in the case of the deletion of such genes for improvement of bio-production. There is therefore a need for other strategies that increase the metabolic flux to the objective products rather than that to a central carbon metabolite, thus avoiding the growth defect during production.
To overcome this issue, there have recently been several attempts to control metabolic flux by modulating the expression levels of enzymes involved in the central metabolic pathway [10–13]. We previously developed a metabolic toggle switch (MTS) to control the metabolic flux by using a genetic toggle switch that functions as a genetic circuit composed of two repressor proteins and two repressible promoters in E. coli [14, 15]. For isopropanol production, we performed conditional repression of citrate synthase (EC 18.104.22.168) encoded by gltA, which catalyzes the condensation reaction of one molecule of acetyl-CoA and one molecule of oxaloacetate to one molecule of citrate, in the middle of fermentation by addition of isopropyl β-D-1-thiogalactopyranoside (IPTG). The conditional repression of gltA inhibited the metabolic flux from glycolysis to the TCA cycle and resulted in accumulation of acetyl-CoA. This acetyl-CoA accumulation resulted in increased titer and yield of isopropanol (derived from acetyl-CoA) and avoided the severe growth defect observed using the conventional gene deletion . This encouraged the use of MTS for other bio-production processes employing a synthetic pathway to improve productivity. So far, studies controlling metabolic flux have been mainly focused on glucose metabolism. However, there has been no report of controlling the metabolic flux using substrates other than glucose, such as xylose or glycerol.
3-Hydroxypropionic acid (3-HP) is a three carbon non-chiral carboxylic acid that is often used as a platform for the production of several kinds of commercially important compounds, such as acrylic acid, methyl acrylate, acrylamide, ethyl 3-HP, malonic acid, propiolactone, and acrylonitrile . Because of its usefulness, 3-HP was selected by the US Department of Energy as one of the top value-added chemicals produced with biomass . 3-HP is produced through a two-step reaction from glycerol as follows: the first step is the conversion of glycerol to 3-hydroxypropionaldehyde (3-HPA), catalyzed by vitamin B12–dependent glycerol dehydratase, and the second step is the conversion of 3-HPA to 3-HP, catalyzed by aldehyde dehydrogenase. Recently, the growing demand for the production of biodiesel has led to a reduction in the price of crude glycerol, since glycerol is a major byproduct of biodiesel production [18, 19]. Therefore, glycerol has been regarded as an attractive carbon feedstock for 3-HP production.
To date, efforts to increase microbial 3-HP production have been made using engineered E. coli with an introduced synthetic pathway as a host organism. Mohan et al. optimized the fermentation conditions such as the pH, working volume, and initial glycerol concentration . Rathnasingh et al. adjusted the expression level of enzymes in the synthetic pathway and demonstrated that the alternative enzyme exhibited higher activity . Several recent studies focused on increasing the metabolic flux from glycerol toward 3-HP by the deletion of enzymes involved in glycerol metabolism [22, 23]. However, the deletion of several genes resulted in poor cell growth  and some of the deleted strains required glucose coupled with glycerol as an additional substrate for cell growth . Thus, an alternative approach to improve 3-HP production that avoids severe growth defect without using an additional substrate other than glycerol is required.
Under aerobic conditions, glycerol is converted to glyceraldehyde 3-phosphate, one of the intermediates of glycolysis, through glycerol-3-phosphate and dihydroxyacetone phosphate. Each reaction in the pathway is catalyzed by four enzymes: glycerol kinase, glycerol-3-phosphate dehydrogenase, triosephosphate isomerase, and glyceraldehyde-3-phosphate dehydrogenase encoded by glpK (EC 22.214.171.124), glpD (EC 126.96.36.199), tpiA (EC 188.8.131.52), and gapA (EC 184.108.40.206), respectively. Thus, we speculated that the conditional repression of these enzymes engaged in glycerol metabolism could direct the excess metabolic flux of the central metabolic pathway toward the introduced synthetic pathway for 3-HP production.
Here, we applied a MTS that involved conditional repression of glpK, tpiA or gapA and a synthetic metabolic pathway for 3-HP production in order to increase the titer and yield of 3-HP by redirecting metabolic flux from the central metabolic pathway toward 3-HP production. Conditional repression of gapA effectively increased 3-HP productivity, and prevented severe growth inhibition. Moreover, additional deletion of yqhD, which catalyzes the reaction for producing a major byproduct of 1,3-propanediol (1,3-PDO), further increased the titer and yield of 3-HP.
Results and discussion
3-HP production from glycerol via the synthetic metabolic pathway in E. coli
Bacterial strains and plasmids used in this study
E. coli strains
lacIq rrnBT14 ΔlacZWJ16 hsdR514 ΔaraBADAH33 ΔrhaBADLD78
JW0336 removed Kanr
TA1015/pTA216, pTA1065, pTA1196
TA2207/pTA216, pTA1196, pTA1277
TA2791/pTA216, pTA1196, pTA1393
BW25113 ΔgapA, Kanr
Gifted by Dr. Nakahigashi
TA367 removed Kanr
TA2732 ΔlacI/pTA216, pTA1335, Kanr
TA2793 removed Kanr
TA2814 ΔyqhD/pTA216, pTA1335
Ampr, Cmr, FLP+, λcI857+, Repts
pSC101*, Cmr, P lacI q ::lacI
ColE1, Kanr, PllacO1::dhaB1, dhaB2, dhaB3, gdrA, gdrB, araE
p15A, Kanr, PllacO1::tetR, PltetO1::gapA.LAA
p15A, Specr, PllacO1::tetR, PltetO1::MCS
ColE1, Kanr, PllacO1::dhaB1, dhaB2, dhaB3, gdrA, gdrB PllacO1::araE
p15A, Specr, PllacO1::tetR, PltetO1::gltA.LAA
p15A, Specr, PllacO1::tetR, PltetO1::tpiA.LAA
p15A, Specr, PllacO1::tetR, PltetO1::gapA.LAA
Effect of the conditional repression of glpK on 3-HP production
Glycerol kinase (glpK) catalyzes the conversion of glycerol to glycerol-3-phosphate, which enters glycolysis via dihydroxyacetone phosphate (DHAP) (Fig. 1). Deletion of glpk should result in increased yield of 3-HP from glycerol, but the deletion caused growth defects in E. coli cultured in M9 medium containing glycerol as the sole carbon source . We therefore speculated that conditional repression of glpK using MTS was an appropriate strategy for achieving a high titer and yield of 3-HP, and would reduce the metabolic flux towards the central metabolic pathway.
Effect of tpiA conditional repression on 3-HP production
In order to distribute a considerable amount of metabolic flux toward the central metabolic pathway, 3-HP production was initiated using TA2945, a 3-HP producing synthetic pathway and MTS strain with tpiA conditional repression (Figs. 1, 2; Table 1). Glycerol-3-phosphate dehydrogenase (GlpD) encoded by glpD is responsible for the conversion of glycerol-3-phosohate to DHAP (Fig. 1). Although this reaction is the immediate downstream of the reaction catalyzed by glycerol kinase (glpK), there is no metabolic pathway that catalyzes the glycerol-3-phosphate toward central metabolic pathway except for the reaction responsible for GlpD. We speculated that the conditional repression of glpD could not be expected a remarkable improvement of 3-HP production compared with the case of glpK. On the other hand, triosephosphate isomerase encoded by tpiA catalyzes the conversion of DHAP to glyceraldehyde-3-phosphate (GAP), such that the flux dispensed from DHAP can be converted to pyruvate by the methylglyoxal pathway via methylglyoxal, lactaldehyde, and lactate (Fig. 1). This methylglyoxal pathway is normally inactive ; however, high concentrations of DHAP can induce its activity .
Interestingly, TA2945 produced higher concentrations of 1,3-PDO (Fig. 5d). Particularly, 3-HP and 1,3-PDO concentration achieved by TA2945_0 h were 15.4 ± 1.4 and 40.9 ± 6.4 mM, respectively, showing that a large proportion of 3-HPA was not converted to 3-HP but was instead converted to 1,3-PDO (Fig. 1). This is similar to the results of a previous report on 3-HP production using a tpiA deletion strain . NAD+ is required for both the conversion of 3-HPA to 3-HP and of lactate to pyruvate in the methylglyoxal pathway (Fig. 1). In addition, the reduced metabolic flux toward the central metabolic pathway would prevent NAD+ generation. Accordingly, the intracellular level of NAD+ would be insufficient for 3-HP production resulting in increased metabolic flux from 3-HPA to 1,3-PDO.
Effect of gapA conditional repression on 3-HP production
To further evaluate the benefit of conditional repression for higher 3-HP production with distribution of some amount of metabolic flux to the central metabolic pathway, TA2821, a strain with gapA conditional repression due to an MTS having the 3-HP producing synthetic pathway, was constructed and tested for 3-HP production (Fig. 2; Table 1). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) encoded by gapA is responsible for the conversion of GAP to 1,3-bisphosphoglycerate (1,3-BPG) (Fig. 1).
In contrast to the results observed with the TA2945 tpiA conditional repression strain, detectable amounts of lactate were not observed during the fermentation of TA2821. In addition, the profiles of growth and glycerol consumption between TA2945 and TA2821 were relatively different (Figs. 5a, b, 6a, b). These results suggest that glycerol metabolism toward the central metabolic pathway in the gapA conditional repression strain did not depend on the methylglyoxal pathway. It was reported that E. coli strains deficient in GAPDH activity did not grow on minimal media containing glucose or glycerol as a sole carbon source. However, the strains were able to grow on media only when containing glycerol together with other substrate such as malate or succinate [41, 42]. These findings imply that the E. coli GAPDH deficient strain uses a distinct route for glycerol metabolism. Extensive metabolic flux analyses of the GAPDH deficient strain have never been performed. Therefore the details of the pathway are still unclear.
Improvement of 3-HP production by yqhD deletion
The titer of 1,3-PDO produced by TA3130_9 h (4.51 ± 1.2 mM) was significantly lower compared to that for TA2821_9 h (18.9 ± 3.1 mM) (Figs. 6d, 7d). The deletion of yqhD in the E. coli strain producing 3-HP led to reduced 1,3-PDO production. However small amounts of 1,3-PDO was still produced, suggesting that there are other enzymes that can convert 3-HPA to 1,3-PDO in E. coli [22, 33]. Additionally, the sum of the concentrations of 3-HP and 1,3-PDO produced by TA3130 (approximately 71.8 mM) was similar to that of TA2821_9 h (approximately 73.1 mM). These results reveal that deletion of yqhD did not affect the metabolic flux from glycerol to 3-HPA but decreased the metabolic flux from 3-HPA to 1,3-PDO.
Measurement of gapA activity and intracellular metabolites during fermentation
GAPDH activities during fermentation
0.249 ± 0.0037
0.117 ± 0.012
0.655 ± 0.013
0.576 ± 0.029
0.676 ± 0.018
0.0483 ± 0.0031
Recently, Jung et al. demonstrated that deletion of glpR encoding a regulation factor repressing the genes for glycerol utilization increased the glycerol utilization rate as well as the 3-HP titer . Chu et al. and Honjo et al. improved the conversion rate of 3-HPA to 3-HP using an aldehyde dehydrogenase mutant or via the introduction of an additional metabolic pathway [24, 44]. The conditional repression of gapA combined with these findings provides further improvement of 3-HP productivity. Such resultant strains will be applied to various cultivation conditions including fed-batch culture that could gave knowledge for the eventual commercialization of 3-HP production by E. coli with MTS from glycerol.
Here, we performed conditional repression of genes related to glycerol metabolism (glpK, tipA and gapA) for increasing 3-HP production from glycerol by E. coli. The conditional repression of glpK and tpiA did not improve 3-HP production, indicating that the conditional repression of every node on the routes of carbon utilization does not completely contribute to enhanced productivity of the final products. In contrast, the conditional repression of gapA with MTS, accompanied by deletion of yqhD, significantly increased 3-HP production from glycerol. The 3-HP titer and yield achieved by the resultant strain TA3130 were 67.3 ± 2.1 mM and 51.5 ± 3.2 %, respectively, which were 80 and 94 % greater, respectively, than those for TA2463, the wild type strain. To our knowledge, this is the first report demonstrating the effectiveness of conditional repression by using the MTS on 3-HP production from glycerol. Taking into account our previous report, which showed increased isopropanol production from glucose by conditional repression of gltA, this strategy can be applied to various kinds of bio-production from various substrates.
Chemicals and reagents
Chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA), Wako Pure Chemical Industries, Ltd. (Osaka, Japan), and MP Biomedicals (Solon, OH, USA), and all restriction enzymes were purchased from New England Biolabs (Ipswich, MA, USA), unless otherwise noted.
Media and growth conditions
For plasmid preparation, E. coli strains were cultured in 3 mL Luria–Bertani (LB) medium in test tubes incubated at 37 °C in a rotary shaker (250 rpm). The media and growth conditions for flask cultures were adopted from a previous study, with slight modifications . E. coli strains were grown in M9 minimal media containing 200 mM glycerol, 0.05 % (w/v) yeast extract, and 10 ppm thiamin hydrochloride. Preculture was performed at 37 °C in a rotary shaker (250 rpm), and the main culture was incubated at 37 °C in a rotary shaker (150 rpm) in the dark. Overnight precultures were diluted to 300 mL in a baffled flask containing 25 mL fresh M9 media with initial optical density at 600 nm (OD600) of 0.05 as the main culture. IPTG (0.1 mM) and cyanocobalamin (2 μM) were added at the indicated time points (0, 3, 6, or 9 h). All media were supplemented with appropriate antibiotics [kanamycin (50 μg/mL), spectinomycin (100 μg/mL), or chloramphenicol (40 μg/mL)].
Plasmid and strain construction
The plasmids and strains used in this study are listed in Table 1. All plasmids were prepared in E. coli XL1-blue (Agilent Technologies, Santa Clara, CA, USA). PCR analyses were performed using KOD plus NEO (Toyobo Co., Ltd., Osaka, Japan). To insert a DNA fragment containing the tryptophan terminator Ttrp flanked by an IPTG inducible promoter PllacO1 into the pTA867 vector  at the BamHI site, a fragment was generated by primer extension using the following primers: T1856 (5′ GCCAT CGGAT CCAGC CCGCC TAATG AGCGG GCTTT TTTTT TCTAG AAATT GTGAG CGGAT AACAA TTGAC ATTG 3′) and T2008 (5′ GCCAT CGGAT CCGGT CAGTG CGTCC TGCTG ATGTG CTCAG TATCT TGTTA TCCGC TCACA ATGTC AATTG TTATC CGCTC ACA 3′). The resultant fragment was digested with BamHI and cloned into the pTA867 vector, resulting in a plasmid designated as pTA1196. The kanamycin-resistance gene of the pTA695 plasmid  was replaced with spectinomycin. A fragment encoding the spectinomycin-resistance gene was digested with AvrII and SacI from PZS4Int-laci/tetR  and was ligated into the pTA695 vector, which did not contain an antimicrobial-resistance gene, resulting in a plasmid that was designated as pTA1065. glpK was amplified from the E. coli BW25113 genome by PCR using the following phosphorylated primers T1569: 5′ ATGAC TGAAA AAAAA TATAT CGTTG CGC 3′) and T1702 (5′ TTAAG CTGCT AAAGC GTAGT TTTCG TCGTT TGCTG CTTCG TCGTG TTCTT CCCAC GC 3′); the LAA tag was added at the C-terminus for rapid degradation [26, 27]. The resultant fragment was ligated with a fragment generated from pTA1065 by PCR performed using the primers T2083 (5′ CATGG TACGC GTGCT AGAGG CATC 3′) and T2084 (5′ GGATC CTTTC TCCTC TTTAA TGAAT TCGG 3′), thus creating a pTA1277 plasmid. gapA was amplified from the BW25113 genome using the primers T1551 (5′ GCCAT CGGAT CCATG ACTAT CAAAG TAGGT ATCAA CGGTT TTG 3′) and T1704 (5′ GCCAT CACGC GTTTA AGCTG CTAAA GCGTA GTTTT CGTCG TTTGC TGCTT TGGAG ATGTG AGCGA TCAGG TC 3′) for PCR; an LAA tag at the C-terminus. The resultant fragment was digested with BamHI and MluI, and cloned into pTA695, resulting in a plasmid designated as pTA958. The kanamycin-resistance gene of pTA958 was replaced with spectinomycin, similar to the pTA1065 plasmid, resulting in a plasmid designated as pTA1335. To exchange the origin of pTA1065, the plasmid was digested with AvrII and SpeI, and a pTA1065 fragment without origin was gel purified. The resultant fragment was ligated with a pSC101* origin generated from pZS*24MCS  by digestion with AvrII and SpeI, resulting in a plasmid designated as pTA1296. tpiA was amplified from the BW25113 genome by PCR using the primers T2302 (5′ AAAAA GGATC CATGC GACAT CCTTT AGTGA TGGG 3′) and T2304 (5′ AAAAA GGATC CTTAA GCTGC TAAAG CGTAG TTTTC GTCGT TTGCT GCAGC CTGTT TAGCC GCTTC TG 3′); an LAA tag was added at the C-terminus. The resultant fragment was digested with BamHI and introduced into the BamHI site of pTA1296, creating a plasmid designated as pTA1383. PllacO1-tpiA.LAA was amplified by PCR performed using the primers T2404 (5′ AAAAA AGCTT AGCCC GCCTA ATGAG CG 3′) and T2405 (5′ AAAAC CTAGG TCTAG GGCGG CGGA 3′) and digested with AvrII and HindIII. The fragment was cloned into pTA1065 at the AvrII-HindIII site, resulting in a plasmid designated as pTA1393.
All E. coli strains used for 3-HP production were based on BW25113. The gapA gene was deleted by Wanner method (, Nakahigashi unpublished data). The genes glpK, tpiA, and yqhD were inactivated by P1 transduction based on TA1015 or TA2814. TA2793 was constructed by P1 transduction using JW0336 based on TA2732 in which gapA was supplemented with pTA1335 . After P1 transduction, the residual kanamycin marker was removed using pCP20 in all strains .
Glyceraldehyde-3-phosphate dehydrogenase activity
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity was measured as previously reported, with slight modifications [30, 31]. E. coli strains, TA2463 and TA3130 were cultured in M9 medium as described in section media and growth. Cells were harvested by centrifugation, washed, and resuspended in wash buffer. Crude extracts were prepared by sonication on ice, and supernatants were collected after centrifugation for experiments. The protein concentration of the crude extracts was determined by measuring the absorbance at 280 nm using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, USA). The activity was measured by monitoring the increase in absorbance at 340 nm at 25 °C. Crude extracts were added to a reaction mixture containing 200 mM tricine buffer (pH 8.5), 30 mM 2-mercaptoethanol, 10 mM NAD, and 10 mM glyceraldehyde-3-phosphate. Measurement began when 100 mM inorganic phosphate was added. The unit of enzyme activity is defined as the formation of 1 μmol of NADPH per minute.
Measurement of intracellular metabolites
TA2463 and TA3130 were cultured in M9 medium as described in section media and growth conditions. IPTG (0.1 mM) was added after 9 h of culture for TA3130. Intracellular metabolite extraction and subsequent LC–MS/MS analysis were performed as previously described .
KT designed the experiments, carried out the cells and plasmids construction, fermentations, samples analysis, and intracellular metabolites analysis and wrote the manuscripts. HH participated in the cells and plasmids construction. TH supervised the study. All authors read and approved the final manuscript.
We thank K. Nakahigashi (Keio University, Tsuruoka, Japan) for providing the E. coli ΔgapA, Kanr plasmid. This research was supported by a JSPS Grant-in-Aid for Scientific Research on Innovative Areas (Grant number: 23119002).
Compliance with ethical guidelines
Competing interests The authors declare that they have no competing interests.
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