Engineering E. coli–E. coli cocultures for production of muconic acid from glycerol
© Zhang et al. 2015
Received: 22 July 2015
Accepted: 20 August 2015
Published: 15 September 2015
cis, cis-Muconic acid is an important chemical that can be biosynthesized from simple substrates in engineered microorganisms. Recently, it has been shown that engineering microbial cocultures is an emerging and promising approach for biochemical production. In this study, we aim to explore the potential of the E. coli–E. coli coculture system to use a single renewable carbon source, glycerol, for the production of value-added product cis, cis-muconic acid.
Two coculture engineering strategies were investigated. In the first strategy, an E. coli strain containing the complete biosynthesis pathway was co-cultivated with another E. coli strain containing only a heterologous intermediate-to-product biosynthetic pathway. In the second strategy, the upstream and downstream pathways were accommodated in two separate E. coli strains, each of which was dedicated to one portion of the biosynthesis process. Compared with the monoculture approach, both coculture engineering strategies improved the production significantly. Using a batch bioreactor, the engineered coculture achieved a 2 g/L muconic acid production with a yield of 0.1 g/g.
Our results demonstrate that coculture engineering is a viable option for producing muconic acid from glycerol. Moreover, microbial coculture systems are shown to have the potential for converting single carbon source to value-added products.
cis, cis-muconic acid (MA) is a diunsaturated linear dicarboxylic acid with recognized industrial applications. One of the primary uses of MA is to make adipic acid through hydrogenation [1–4]. As a precursor for the synthesis of several commodity chemicals, such as nylon and polyurethane, adipic acid has a long history of commercial applications in the plastics industry. In addition, MA can be utilized as the starting material for making terephthalic acid, which is one of the two constituent monomers of the high-demand plastic polymer polyethylene terephthalate (PET) . Adipic acid and terephthalic acid also have wide applications in the cosmetic, pharmaceutical and food industries . However, current industrial production of adipic acid and terephthalic acid relies exclusively on utilization of petroleum and coal feedstocks through chemical conversion processes, which has encountered major challenges due to the increasing sustainability, environmental and economic concerns.
Development of new production methods utilizing renewable resources, such as biomass feedstocks, offers more sustainable ways for producing valuable bulk compounds . This has fueled extensive research interest in the engineering of microbes to achieve MA biosynthesis from simple renewable carbon substrates . The use of microorganisms to convert aromatics to MA has been demonstrated before; however, the starting materials used in these reports, such as toluene and benzoic acid, are derived from petroleum or coal raw materials [7–13]. The first complete MA biosynthesis from glucose, a renewable sugar, was established in engineered E. coli by the Frost group, which utilized the metabolite 3-dehydroshikimic acid as key biosynthesis precursor [3, 14]. Further optimization of this biosynthetic pathway resulted in 18 g/L MA production from glucose using a fed-batch bioreactor . The same pathway was also employed to biosynthesize MA in the yeast S. cerevisiae, although the reported titers and yields of these studies were significantly lower due to the challenge of achieving sufficient functional expression of the heterologous pathway enzymes [15, 16]. In addition, Lin et al.  established a new MA biosynthetic route via salicyclic acid and achieved a MA production of 1.5 g/L. The same group also demonstrated a biosynthetic pathway in E. coli leading from simple carbon sources to MA via anthranilate .
Recently, our group developed a novel metabolic engineering approach based on a coculture concept  and successfully utilized it for achieving high-yield MA biosynthesis from sugar mixtures that can be derived from lignocellulose . In the present study, we further explore the potential of the microbial coculture approach by using a single carbon source, glycerol, to biosynthesize MA. Although E. coli–E. coli cocultures have been utilized to produce small molecules, such as lactic acid, previous studies focused on utilizing different carbon sources [21, 22]. Other studies engineered E. coli cocultures for the production of more complex molecules. For example, Saini et al.  employed an E. coli–E. coli coculture for biosynthesis of n-butanol from sole carbon source glucose. The novelty of the present study is the use of cocultures to balance metabolic pathways harboring a very slow step that leads to secretion of pathway intermediates and, as a result, low product yields. By using the coculture consisting of two different strains, the low activity of an enzyme can be overcome by increasing the relative amount of the strain harboring the slow step.
On the other hand, we aimed to produce value-added MA from the renewable and inexpensive glycerol substrate. As a major byproduct of the biodiesel industry (roughly 10 % w/w), crude glycerol is produced in surplus amounts relatively to the global market demand. Moreover, disposal of glycerol can cause environmental issues and thus needs to meet regulatory requirements that increase the operational cost of biodiesel production. Nevertheless, glycerol can be efficiently utilized by a variety of microbes for growth, and the related biodegradation pathways have been well studied. It is therefore of great research and industrial significance to develop new bioprocesses that convert glycerol to valuable compounds [24–26]. This study reports the conversion of the substrate glycerol to cis, cis-muconic acid using engineered E. coli–E. coli cocultures.
Results and discussion
MA synthesis from glycerol using an E. coli monoculture
In an effort to further improve DHS conversion, the downstream pathway enzyme AroZ, responsible for the DHS-to-PCA conversion, was fused to the ShiA permease so that imported DHS could be immediately processed by AroZ to produce the intermediate PCA before leaking out of the cell again. However, E. coli P5fs constructed using this design did not show any significant improvement of MA production (Fig. 2). In another construct, the AroZ enzyme was engineered to be localized in the periplasmic space between the E. coli inner and outer membranes through the use of a phage gIII capsid protein signal sequence [27, 28]. In this construct, imported DHS would not need to travel across both cell membranes to gain access to the cytosolic AroZ enzyme for downstream conversion, which could potentially alleviate any DHS cross-membrane transportation issues and improve downstream conversion efficiency. However, this strategy turned out to be unsuccessful and actually resulted in decreased MA production (Fig. 2). The reason could be that the periplasmic expression of AroZ generated a physiological impact on cell’s fitness and decreased the cell’s production performance.
MA production by a coculture of E. coli P5.2 and BLS
The lack of MA production improvement by the above metabolic engineering efforts suggested that alternative methods should be deployed in order to reduce DHS accumulation. To this end, we employed an E. coli–E. coli coculture engineering approach to improve MA biosynthesis from glycerol. In fact, a previous report demonstrated a coculture system for converting sugar mixtures to the MA product . Here, we further explored the potential of coculture engineering to utilize a single carbon source, glycerol.
Production of MA using P5s:BLS coculture
Product and intermediate concentrations (mg/L)
473 ± 31
114 ± 20
130 ± 26
562 ± 33
P5s:BLS inoculation ratio
416 ± 33
160 ± 48
92 ± 17
223 ± 33
562 ± 52
114 ± 22
95 ± 18
194 ± 15
639 ± 83
230 ± 38
69 ± 12
198 ± 14
418 ± 80
152 ± 39
78 ± 30
164 ± 31
26 ± 2
8 ± 5
0.2 ± 0.2
1.1 ± 0.7
Utilization of E. coli chaperones in the P5s:BLH coculture system
Production of MA using P5s:BLH coculture
Product and intermediate concentrations (mg/L)
473 ± 31
114 ± 20
130 ± 26
562 ± 33
P5s:BLH inoculation ratio
591 ± 57
182 ± 50
143 ± 3
238 ± 32
691 ± 112
189 ± 76
114 ± 25
255 ± 42
488 ± 55
77 ± 26
47 ± 3
144 ± 24
480 ± 3
79 ± 10
109 ± 26
123 ± 15
128 ± 21
190 ± 56
33 ± 6.0
3.2 ± 2.5
Despite different engineering strategies to improve DHS conversion in the monoculture strategy, such as utilization of the ShiA transporter, fusion expression of ShiA and AroZ, and periplasmic expression of AroZ, DHS accumulation was not completely eliminated in engineered E. coli. In contrast, when a second E. coli strain containing the DHS-to-MA pathway was introduced into the production process, more DHS conversion capacity was provided and DHS was better converted to the final MA product. In both cases of P5s:BLS and P5s:BLH cocultures, the benefit of increasing DHS-to-MA conversion outweighed the disadvantage of introduced growth competition between the two constituent strains. As a result, significant MA production improvement was observed.
Splitting the MA biosynthesis pathway between two strains cultivated in coculture
We next split the entire MA biosynthetic pathway into two modules, each of which was accommodated in a separate E. coli strain. The upstream strain E. coli P5.2 contained only the shikimate pathway ending with the synthesis of DHS, whereas the downstream strain E. coli BS was equipped with enzymes AroZ, AroY, CatA and ShiA under the control of the inducible T7 promoter to assimilate and convert DHS to MA. The overall design of this new coculture strategy is shown in Fig. 3b.
Consistent with previous observations, MA production depended on the inoculation ratio of P5.2 and BS (Fig. 3c). The highest MA production of 758 mg/L was achieved at the inoculation ratio of 1:1, indicating that the upstream and downstream pathways were best balanced for the complete glycerol-to-MA biosynthesis process under this condition. Although the DHS concentration gradually decreased as more BS cells were supplemented in the cocultures, DHS accumulation was still observed even at the highest inoculation ratio. This finding suggested that inducible expression of the enzymes responsible for DHS to MA conversion was not the best strategy for eliminating DHS accumulation. In fact, it was observed before that use of a milder constitutive expression strategy helped improve MA biosynthesis . Therefore, we constructed a new downstream strain E. coli BC that over-expressed the downstream pathway enzymes and the ShiA permease under the control of a constitutive pyruvate decarboxylase promoter isolated from Zymomonas mobilis. It should be noted that the upstream strain P5.2 also used constitutive promoters for DHS biosynthesis and thus the new P5.2:BC coculture system did not need any inducer for MA biosynthesis.
As shown in Fig. 3d, MA production with the new coculture system of P5.2:BC was improved at all inoculation ratios. At the optimal inoculation ratio of 1:1, MA concentrations reached 1016 mg/L. Moreover, DHS accumulation was also further reduced in this new coculture system. Notably, constitutive expression of the entire biosynthetic pathway in a single strain system, E. coli P5cS, resulted in poor growth and reduced MA production (56 mg/L), due to overwhelming metabolic burden imposed by consolidated over-expression of all pathway enzymes. These findings together suggest that lessening the metabolic stress on the microbial host improved its fitness for functional expression of the heterologous downstream enzymes; and the production improvement shown here highlights the importance and usefulness of the coculture strategy to address this issue.
The MA and intermediate production profiles are shown in Fig. 5b. The concentrations of intermediates CA, PCA and DHS were repressed throughout the cultivation process, indicating that these intermediates were well converted to downstream products in the downstream strain BC. The MA concentration increased rapidly within the first 36 h, and then plateaued at 2 g/L due to depletion of the glycerol substrate. The highest production yield of 0.1 g/g is similar to the results achieved in the previous section. The development of the production curves in Fig. 5b demonstrates that the intermediate-to-MA conversion capacity was reduced after 36 h, which is consistent with the decline of BC sub-population caused by the glycerol depletion.
The theoretical maximum yield for the pathway leading from glycerol to MA is calculated as 0.66 g/g, using the equation for pathway balance 7 Glycerol + 4 ATP + 10 NAD = 3 MA + 3 CO2 + 4 ADP + 10 NADH. Although carbon loss for biomass formation and respiration is not considered, the achieved yield is significantly lower than the theoretical maximum, indicating that there is room for further engineering efforts to improve MA production yield.
We report a coculture engineering approach to convert renewable glycerol substrate to an important value-added product. Through step-wise engineering and optimization efforts, we significantly improved the product concentration and yield. We also demonstrate that, despite growth competition between the two strains, the E. coli–E. coli coculture can be used in the context of sole carbon source for complex biosynthesis pathway engineering. The results of this study indicate that the combination of pathway modularization and microbial co-cultivation has strong potential for future metabolic engineering studies.
Plasmid and strain construction
Gene cloning and bacterial transformations were carried out according to the standard protocols . PCR primers were purchased from Lifetechnologies (Grand Island, NY, USA). Phusion DNA Polymerase for PCR amplification endonucleases, and the Gibson assembly kit used in this study were purchased from New England Biolabs (Ipswich, MA, USA). Chaperone expression plasmid pG-KJE was purchased from Clontech Laboratories (Mountain View, CA, USA).
The plasmids and E. coli strains utilized in this study
T7 promoter, AmpR
T7 promoter, KanR
A gTME plasmid carrying a mutated alpha subunit of RNA polymerase for enhancing the shikimate pathway
pET21c carrying the catA and auxiliary ACIAD1443 genes from Acinetobacter calcoaceticus
pACYCDuet-1 carrying the catA gene and ACIAD1443 genes
pET28a carrying the aroY gene from Klebsiella pneumoniae
pET28a carrying the aroZ gene from Klebsiella pneumoniae
pET28a carrying the aroY and aroZ genes from Klebsiella pneumonia and the E. coli shiA gene
pET28a carrying the aroY and fused shiA-aroZ genes
pY carrying aroZ that has a phage gIII leading sequence to localize AroZ enzyme into periplasm
A plasmid carrying E. coli chaperone dnaK-dnaJ-grpE and groES-groEL genes, CmR (only groES-groEL genes were induced for expression)
A low copy number plasmid expressing weak green fluorecence protein, KanR
pACYCDuet-1 carrying the shiA gene that is under the control of a constitutive lacuv5 promoter
pCDFDuet-1 carrying the shiA gene that is under the control of a constitutive lacuv5 promoter
pUC57(Kan) carrying the aroY, aroZ, catA and ACIAD1443 genes that are under the control of a constitutive Zymomonas mobilis pyruvate decarboxylase promoter
E. coli K12 ΔpheA ΔtyrR lacZ::PLtetO-1-tyrA fbr aroG fbr tyrR::PLtetO-1-tyrA fbr aroG fbr hisH(L82R) ΔaroE ΔydiB
P5 harboring pHACM-rpoA14, pAO and pYZ
P5 harboring pHACM-rpoA14, pAO and pYZS
P5 harboring pHACM-rpoA14, pAO and pYfsZ
P5 harboring pHACM-rpoA14, pAO and pYpZ
P5 harboring pHACM-rpoA14, pCYZAO and pCS2
P5 harboring pII and pHACM-rpoA14
E. coli BL21(DE3) harboring pHACM-rpoA14, pAO and pYZS
E. coli BL21(DE3) harboring pG-KJE, pAO and pYZS
E. coli BL21(DE3) harboring pYZS and pAOM
E. coli BL21(DE3) harboring pCYZAO and pCS
The MA biosynthesis was conducted using a glycerol medium. 1 L glycerol medium contained 10 g glycerol, 2.0 g NH4Cl, 5.0 g (NH4)2SO4, 3.0 g KH2PO4, 7.3 g K2HPO4, 8.4 g MOPS, 0.5 g NaCl, 0.24 g MgSO4, 0.5 g yeast extract, 40 mg tyrosine, 40 mg phenylalanine, 40 mg tryptophan, 10 mg 4-hydroxybenzoic acid, 0.4 mg/L Na2EDTA, 0.03 mg H3BO3, 1 mg thiamine, 0.94 mg ZnCl2, 0.5 mg CoCl2, 0.38 mg CuCl2, 1.6 mg MnCl2, 3.77 mg CaCl2, and 3.6 mg FeCl2. The initial medium pH was adjusted to 6.6 before use. Appropriate antibiotics (100 mg/L ampicillin, 50 mg/L kanamycin, and 34 mg/L chloramphenicol) were also added to the medium when needed. Except for the bioreactor experiments, the MA production was performed in 14 mL culture tubes.
For MA monoculture production, 2 mL glycerol medium was inoculated with 2 % (v/v) overnight E. coli P5g or P5s LB culture and cultivated under 37 °C with 250 rpm shaking. Since the addition of IPTG at the time of inoculation resulted in poor cell growth, IPTG was supplemented to the culture 24 h after inoculation to induce the heterologous enzyme expression. MA production was analyzed 72 h after induction.
For MA production using E. coli–E. coli cocultures, 2 % (v/v) overnight LB cultures of the first strain (i.e. P5s or P5.2) and the second strain (i.e. BLS, BLH, BS or BC) were inoculated into 2 mL glycerol medium and the LB medium, respectively. After 24 h, the optical density (OD) at 600 nm of the two cultures was measured. The second strain was then harvested by centrifugation and appropriate amounts of the cell pellets were re-suspended in the first strain culture to achieve the desired inoculation ratios. To be consistent with the single strain cultivation strategy, 0.1 mM IPTG was supplemented at the time of the second strain addition when E. coli BLS, BLH and BS (containing the inducible pathway and transporter enzymes) were used. For P5s:BLH coculture, 5 ng/mL tetracycline was also supplemented at the time of BLH addition to induce the expression of E. coli chaperones groES/groEL carried by plasmid pG-KJE in BLH (the chaperone genes dnaK, dnaJ and grpE in pG-KJE were not induced for expression). The cocultures were then cultivated under 37 °C with 250 rpm shaking for another 72 h.
For bioreactor cultivation, 14 mL overnight LB culture of E. coli P5.2 and appropriate amount (based on their cell densities) of overnight LB culture of E. coli BC were inoculated into 0.7 L glycerol medium to achieve 1:1 inoculation ratio. The coculture cultivation was performed in a 1.3-L BioFlo110 modular fermentor system (New Brunswick Scientific) running at 1.5 L/min air flow rate, pH = 7.0 and 37 °C. The agitation rate was controlled to maintain a constant DO of 12 %.
Assaying the cell-to-cell ratio in the coculture system
The ratio of the two constituent cell types in the E. coli–E. coli coculture was determined using the method described before . Specifically, 20 μL culture sample taken from the coculture system was first diluted around 105 times using sterile water. 20 μL diluted culture was then spread onto a LB plate containing 40 mg/L X-gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) and 50 mg/L kanamycin. The plate was then incubated at 37 °C overnight. E. coli P5.2 with disrupted lacZ gene only formed white colonies on the X-gal plate, whereas the colonies of E. coli BC with intact chromosomal lacZ gene generated blue color on X-gal. The number of colonies of each color were counted to determine the ratio of the two cell types in the coculture system.
Substrate and metabolite quantification
Glycerol concentration was monitored by HPLC analysis. Coculture samples were centrifuged at 12,000g for 2 min to collect cell-free broth, which was then filtered through 0.2-μm-pore-size polytetrafluoroethylene membrane syringe filters (VWR International) before subjected to HPLC analysis. The HPLC system consisted of a Bio-rad HPX-87H column, a Waters 2695 separation module and a Waters 410 differential refractometer. Isocratic elution was conducted using 14 mM sulfuric acid as mobile phase at a flow rate of 0.7 mL/min.
The concentration of MA product and the pathway intermediates were determined by LC/MS/MS analysis. Cell-free broth was collected by 12,000g centrifugation for 5 min followed by filtration through 0.2-μm-pore-size polytetrafluoroethylene membrane syringe filters (VWR International). 10 μL 1 g/L p-coumaric acid internal standard was added into 1 mL of the filtered broth. 10 μL of the mixed solution was then injected into an Applied Biosystems API2000 LC/MS/MS running on a previously established method .
HZ designed and carried out the experiments, analyzed the data and drafted the manuscript. ZL carried out the experiments and analyzed the data. BP carried out the experiments and analyzed the data. GS designed experiments, analyzed the data and drafted the manuscript. All authors read and approved the final manuscript.
This work was partially supported by US Department of Energy Grant DE-SC0006698.
Compliance with ethical guidelines
Competing interests The authors declare that they have no competing interests.
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