Modification of targets related to the Entner–Doudoroff/pentose phosphate pathway route for methyl-d-erythritol 4-phosphate-dependent carotenoid biosynthesis in Escherichia coli
- Chun Li†1,
- Lan-Qing Ying†1,
- Sha-Sha Zhang1, 2,
- Nan Chen1, 2,
- Wei-Feng Liu1Email author and
- Yong Tao1Email author
© Li et al. 2015
Received: 13 February 2015
Accepted: 20 July 2015
Published: 12 August 2015
In engineered strains of Escherichia coli, bioconversion efficiency is determined by not only metabolic flux but also the turnover efficiency of relevant pathways. Methyl-d-erythritol 4-phosphate (MEP)-dependent carotenoid biosynthesis in E. coli requires efficient turnover of precursors and balanced flux among precursors, cofactors, and cellular energy. However, the imbalanced supply of glyceraldehyde 3-phosphate (G3P) and pyruvate precursors remains the major metabolic bottleneck. To address this problem, we manipulated various genetic targets related to the Entner–Doudoroff (ED)/pentose phosphate (PP) pathways. Systematic target modification was conducted to improve G3P and pyruvate use and rebalance the precursor and redox fluxes.
Carotenoid production was improved to different degrees by modifying various targets in the Embden–Meyerhof–Parnas (EMP) and ED pathways, which directed metabolic flux from the EMP pathway towards the ED pathway. The improvements in yield were much greater when the MEP pathway was enhanced. The coordinated modification of ED and MEP pathway targets using gene expression enhancement and protein coupling strategies in the pgi deletion background further improved carotenoid synthesis. The fine-tuning of flux at the branch point between the ED and PP pathways was important for carotenoid biosynthesis. Deletion of pfkAB instead of pgi reduced the carotenoid yield. This suggested that anaplerotic flux of G3P and pyruvate might be necessary for carotenoid biosynthesis. Improved carotenoid yields were accompanied by increased biomass and decreased acetate overflow. Therefore, efficient use of G3P and pyruvate precursors resulted in a balance among carotenoid biosynthesis, cell growth, and by-product metabolism.
An efficient and balanced MEP-dependent carotenoid bioconversion strategy involving both the ED and PP pathways was implemented by the coordinated modification of diverse central metabolic pathway targets. In this strategy, enhancement of the ED pathway for efficient G3P and pyruvate turnover was crucial for carotenoid production. The anaplerotic role of the PP pathway was important to supply precursors for the ED pathway. A balanced metabolic flux distribution among precursor supply, NADPH generation, and by-product pathways was established.
Bioconversion by engineered microbes in cell factories is a promising alternative strategy to produce valuable chemicals, many of which are expensive to produce in their native hosts or by chemical processes [1, 2]. To construct an engineered strain with optimal phenotypes, the first steps are to enhance native pathways or introduce superior heterogeneous pathways for biosynthesis of target chemicals. Another strategy that can be more important and laborious is to seek and integrate separate genetic modification targets that improve productivity, even though the mechanisms of these targets are sometimes poorly understood.
The heterologous production of carotenoids in Escherichia coli is a well-studied example of an engineered microbial cell factory in which there has been coordinated modification of multiple targets [3–5]. Carotenoids are isoprenoid pigments naturally produced by plants, algae, and photosynthetic bacteria. They have attracted much attention for industrial use because of their diverse physiological effects and their potential applications as antioxidants and nutraceuticals [6–8]. Carotenoids are structurally derived from two universal isoprene units; isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). There are two independent pathways for the biosynthesis of IPP and DMAPP: the mevalonate pathway from the precursor acetyl-CoA in eukaryotic and archaea cells, and the methyl-d-erythritol 4-phosphate pathway (MEP) pathway from the precursors pyruvate and glyceraldehyde-3-phosphate (G3P) in prokaryotic cells and plastids in plants. Bioconversion of carotenoids by engineered strain has been developed based on either of these two pathways [9, 10].
In E. coli, MEP pathway-dependent carotenoid biosynthesis was initially engineered by introducing the carotenoid synthesis gene cluster crtEBI and by enhancing the expressions of genes in the MEP pathway, such as dxs and idi . The modification of chromosomal targets has been shown to further improve carotenoid production [3, 11, 12]. PykFA was the first gene target identified to improve carotenoid production. Deletion of pykFA reduced the flux from G3P to pyruvate, suggesting that the balance between G3P and pyruvate was important for carotenoid synthesis . Additional rate-limiting targets for carotenoid biosynthesis were further explored using either of two major methods: systematic approaches using genome-scale modeling; and combinatorial approaches based on phenotypic diversification and screening. The targets included the global regulators Hnr and ClpXP; the central metabolism enzymes YtjC and AceE, which may directly affect precursor levels by catalyzing G3P- and pyruvate-related metabolic reactions; and GdhA and FdhF, which might indirectly contribute to cofactors or flux balance [5, 11, 12, 14]. Thus, as suggested, improving and balancing the precursor supply remains an obstacle for MEP pathway-based carotenoid production.
The redistribution of metabolic flux at the pathway level is an alternative strategy to improve chemical biosynthesis and precursor supply . This strategy is always more complex, because desirable flux distribution is not usually achieved by modifying single genes. The MEP pathway begins with the condensation of G3P and pyruvate in equal amounts. These precursors are primarily supplied by the Embden–Meyerhof–Parnas pathway (EMP pathway). The Entner–Doudoroff pathway (ED pathway), as a variant glycolysis pathway, produces equal amounts of G3P and pyruvate. This superior stoichiometric feature makes the ED pathway a preferable route for precursor supply. The strategy to redistribute central metabolic pathways has been used to improve MEP-dependent carotenoid production by engineered microbes [16–18]. However, the exact flux profile is not well understood. Systematic modification and analysis of gene targets in the EMP, ED, and pentose phosphate (PP) pathways may provide new insights on the flux profile.
In addition to the precursor supply, cofactor generation might be another key factor for the MEP pathway and carotenoid biosynthesis. Cofactors such as NAPDH and ATP are necessary for MEP reactions. Furthermore, the bioconversion efficiency could be affected by the accumulation of by-products such as acetate. Thus, it is necessary to elucidate flux distribution among precursor supply, cofactor generation, and by-product pathways. To this end, we implemented an engineering strategy that combined systematic genetic target modification and central metabolic flux redistribution. Multiple gene targets were either knocked out or overexpressed to redistribute the fluxes of the EMP, ED, and PP pathways. The effects of these modifications on the supply of G3P and pyruvate precursors, redox generation, and the metabolism of by-products were analyzed. These results will provide guidance for further MEP-pathway engineering studies.
Analysis of metabolic engineering targets within EMP, PP, and ED pathways
Analysis of targets responsible for directing flux from EMP pathway to ED/PP pathways
Initially, we constructed two reporter vectors for high carotenoid production, pLY036 and pLY10RK; their products were neurosporene and lycopene, respectively. The crtEBI cassette alone was insufficient for high carotenoid production (data not shown). Therefore, an additional E. coli idi gene was inserted downstream of crtEBI. The yields of neurosporene and lycopene from pLY036 and pLY10RK in wild E. coli BW25113 were 2.05 and 1.64 mg/g dry cell weight (DCW), respectively.
Efficient substrate use of G3P and pyruvate by overexpression of dxs in ED/PP pathways
To further verify the role of the ED pathway, we attempted to improve the precursor supply by coordinated improvements to Eda and Dxs. First, we overexpressed Eda and Dxs simultaneously in the Δpgi strain. Eda and Dxs were expressed in the pBAD and pSB1s plasmids (P-AE036 and P-SX036, and co-transformation of the two plasmids for P-AE-SX036), respectively. As shown in Fig. 4b, the production of neurosporene was increased 25% by Eda enhancement, as compared with those of P-SX036 and P-AE-SX036. Second, a protein scaffold was used to spatially recruit Eda and Dxs through the interaction between tagged ligands. We designed a protein scaffold platform based on two proteins from Clostridium sp.; cellulosome cohesin and dockerin. Eda and Dxs were each fused with different dockerins and were co-expressed with scaffold protein (SS) containing a cohesin domain in the Δpgi strain. The function of SS was confirmed in a His-tag pull down assay (Additional file 3: Figure S2). Eda and Dxs co-localized with each other via the interaction between dockerins and SS. To balance the expression level with the scaffold, Eda and Dxs were co-transcribed under the control of the araBAD promoter using the low-copy pSB1s plasmid in the Δpgi strain (P-A-SEX036), compatible with SS expression using the pBAD plasmid. As shown in Fig. 4c, neurosporene production was increased by approximately 30% in the scaffold platform strain (P-AS-SEX036). These results confirmed the significant role of the ED pathway in carotenoid production.
Fine-tuning gnd expression to balance PP and ED pathway flux
Different flux distributions between the PP and ED pathways were achieved by coordinated engineering of gnd and eda expression. In this experiment, gnd was overexpressed at two different levels in either the wild-type or the Δgnd background: high-level expression was obtained using the araBAD promoter; low-level expression was obtained by introducing rare codons (Gly-Pro) at the N-terminal of Gnd peptides under the control of the araBAD promoter (Additional file 4: Figure S3). gnd was co-expressed with eda. The results indicated that moderate gnd expression combined with eda was most favorable for neurosporene synthesis in the wild-type background (Fig. 5b).
Anaplerotic role of PP pathway is important for carotenoid biosynthesis
Enhanced carotenoid biosynthesis is accompanied with improved biomass and decreased acetate overflow
Batch fermentation for neurosporene production in a microbioreactor
Biomass (OD600 value)
Neurosporene production (mg neurosporene/g DCW)
Titer (mg neurosporene/L)
Acetate accumulation and glucose consumption of strains
Acetate production (mg acetate/mL)
Residual glucose (mg glucose/mL)
Combined modification of targets in ED/PP pathways for efficient lycopene production
Discussion and conclusions
An ideal microbial cell factory for bioconversion should maximize the target metabolic flux with an exact stoichiometric ratio of precursors and cofactors. However, this is hard to achieve because of the complexity of metabolic flux and its regulation. For MEP-dependent carotenoid biosynthesis, imbalanced precursor supply is one of the major metabolic bottlenecks, especially considering the limit of precursor (G3P) availability. Disruption of the EMP pathway target pykFA has been shown enhance lycopene production by retarding the catabolism of G3P to pyruvate . In another study, the unexpected accumulation of indole may indicate that the pool of the G3P precursor was insufficient, because indole may be a by-product of an anaplerotic supply of G3P through the tryptophan biosynthesis pathway during MEP-dependent taxol production . Research on non-EMP pathway precursor supply routes has mainly focused on the deletion of pgi [16, 18]. However, a parallel study has shown that lycopene yield was improved in a reverse redistribution pathway route with knocked-out zwf . This indicated that detailed studies of both flux and targets were necessary. Herein, we conducted a systematic analysis and modification of genetic targets within central metabolic pathways for the redistribution of metabolic flux for MEP-dependent carotenoid biosynthesis.
The bioconversion efficiency of a given chemical is determined by both the stoichiometric yield of the pathway and the kinetic efficiency of pathway reactions. The metabolic route established by deleting pgi not only provided a balanced precursor ratio for efficient turnover in the MEP pathway, but also created a well-distributed metabolic flux between carotenoid biosynthesis and other pathways. Acetate overflow is always a major metabolic burden in E. coli K-12 strains because of the imbalance of central metabolic flux in high-glucose culture conditions. Surprisingly, the improvement of carotenoid biosynthesis in the redistribution route led to both greater cell growth and less acetate accumulation. This indicated that MEP-dependent carotenoid biosynthesis might relieve the metabolic burden by balancing the level of redox or metabolites, e.g., pyruvate. Indeed, redistribution of central metabolic flux always has complex effects. A global flux response could occur when MEP precursors are used efficiently or when redox conditions are balanced, resulting in flexible adaptation of flux distribution. Additional factors in central metabolic pathways, such as the level of the regulator intermediate fructose 6-bisphosphate, might be affected in the redistribution route, resulting in various responses and effects on the carotenoid biosynthesis pathway. Further research should focus on the effects of global regulation of central metabolic networks and their flux in carotenoid biosynthesis.
Bacterial strains and plasmids
Strains and plasmids used in this study
Escherichia coli strains
Wild type K-12 strain
For genetic manipulation
PT5-dxs K-12 strain, for construction of strains that harboring PT5-dxs phenotype
PT5-idi K-12 strain, for construction of strains that harboring PT5-idi phenotype
galR::P119-glk K-12 strain, for construction of strains that harboring galR::P119-glk phenotype
BW25113, pLY036 plasmid
Δpgi, pLY036 plasmid
This study, KEIO
ΔpfkA, pLY036 plasmid
This study, KEIO
ΔpfkB, pLY036 plasmid
This study, KEIO
ΔpfkAΔpfkB, pLY036 plasmid
ΔfbaB, pLY036 plasmid
This study, KEIO
BW25113, pBAD-zwf and pLY036 plasmids
BW25113, pBAD-eda and pLY036 plasmids
BW25113, pBAD-edd and pLY036 plasmids
PT5-dxs, pLY036 plasmid
ΔpgiPT5-dxs, pLY036 plasmid
BW25113, pSB1s-dxs and pLY036 plasmids
Δpgi, pSB1s-dxs and pLY036 plasmids
This study, KEIO
PT5-dxs, pSB1s-dxs and pLY036 plasmids
Δpgi PT5-dxs, pSB1s-dxs and pLY036 plasmids
Δpgi, pBAD-eda and pLY036 plasmids
This study, KEIO
Δpgi, pBAD, pSB1s-eda-dxs and pLY036 plasmids
This study, KEIO
Δpgi, pBAD-eda, pSB1s- dxs and pLY036 plasmids
This study, KEIO
Δpgi, pBAD-SS, pSB1s-eda-dxs and pLY036 plasmids
This study, KEIO
ΔpgiΔedd, pSB1s-dxs and pLY036 plasmids
ΔpgiΔgnd, pSB1s-dxs and pLY036 plasmids
Δpgi, pBAD-eda-gnd and pLY036 plasmids
This study, KEIO
Δpgi, pBAD-eda-GCgnd and pLY036 plasmids
This study, KEIO
ΔpgiΔgnd, pLY036 plasmid
ΔpgiΔgnd, pBAD-eda and pLY036 plasmids
ΔpgiΔgnd, pBAD-eda-gnd and pLY036 plasmids
ΔpgiΔgnd, pBAD-eda-GCgnd and pLY036 plasmids
BW25113, pSB1s-dxs-idi-ispDF and pLY036 plasmids
Δpgi, pSB1s-dxs-idi-ispDF and pLY036 plasmids
This study, KEIO
ΔpfkA, pSB1s-dxs-idi-ispDF and pLY036 plasmids
ΔpfkAΔpfkB, pSB1s-dxs-idi-ispDF and pLY036 plasmids
ΔpgiΔpfkAΔpfkB, pSB1s-dxs-idi-ispDF and pLY036 plasmids
ΔpgiΔfbaB, pSB1s-dxs-idi-ispDF and pLY036 plasmids
ΔpgiΔytjC, pSB1s-dxs-idi-ispDF and pLY036 plasmids
Δpgi PT5-dxs PT5-idi, pLY036 plasmids
Δpgi PT5-dxs PT5-idi, pSB1s-dx -idi-ispDF and pLY036 plasmids
BW25113, pLY10RK plasmid
PT5-dxs PT5-idi, pSB1s-dx -idi-ispDF and pLY10RK plasmids
Δpgi PT5-dxs PT5-idi, pSB1s-dx -idi-ispDF and pLY10RK plasmids
This study, KEIO
Δpgi, PT5-dxsPT5-idi, ΔptsGgalR::P119-glk, pBAD-eda-GCgnd, pSB1s-dxs-idi-ispDF and pLY10RK plasmids
This study, KEIO
Other bacterial strains
Anabaena sp. PCC7120
crtE, crtB for pLY036
Rhodobacter sphaeroides 2.4.1
crtI for pLY036
crtB. crtI for pLY10RK
crtE for pLY10RK
For lambda-Red mediated recombination
For lambda-Red mediated recombination, derived from pKD4 by inserting multiple cloning site closed to FRT
For lambda-Red mediated recombination
For construction of TX and TI, derived from pUKM by inserting T5 promoter at multiple cloning site
ColE1 origin, araBAD promoter, AmpR
pSC101 origin, araBAD promoter, StrR
pSC101 origin, constitutive P119 promoter (derived from iGEM part BBa_J23119), StrR
pSC101 origin, constitutive P119 promoter, StrR,E. coli glk, for construction of pUKM-glk
For construction of GRK, derived from pUKM by inserting P119-glk fragment at multiple cloning site
p15A origin, araBAD promoter, CmR, Anabaena sp crtEB, R. sphaeroides crtI, E. coli idi
RSF1030 origin, araBAD promoter, KanR, P. agglomerans crtE, P. ananatis crtBI, E. coli idi
pSC101 origin, araBAD promoter, StrR,E. coli dxs
pSC101 origin, araBAD promoter, StrR, dockerin-fused E. coli eda, dockerin-fused E. coli dxs
pSC101 origin, araBAD promoter, StrR, E. coli dxs,idi,ispDF
ColE1 origin, araBAD promoter, AmpR, E. coli zwf
ColE1 origin, araBAD promoter, AmpR, E. coli edd
ColE1 origin, araBAD promoter, AmpR, E. coli eda
ColE1 origin, araBAD promoter, AmpR, E. coli eda,gnd
ColE1 origin, araBAD promoter, AmpR, E. coli eda,GCtag-gnd
ColE1 origin, araBAD promoter, AmpR, clostridium sp cellulose scaffold protein
Construction of plasmids and genomic integration
Plasmids were constructed using standard molecular biological protocols . For chromosomal promoter replacement and gene integration, heterogeneous gene fragments were inserted at the MCS site downstream of the FRT-kanamycin resistance cassette in pUKM. The gene-FRT-kan-FRT fragment was then amplified by PCR. Lambda-Red-mediated recombination was performed as described elsewhere . For chromosomal phenotype integration, P1 virus-mediated transfection was performed as described elsewhere . The primers used in this study are listed in Additional file 5: Table S2.
LB medium was used for all molecular construction experiments and strain cultures. Auto-inducing medium  was used to induce protein expression. Bioconversion medium was prepared by adding 4% (w/v) glucose to M9 medium or LB medium.
All strains were stored at −80°C until use. Strains were pre-cultured in LB medium supplemented with appropriate antibiotics at 37°C overnight and then transferred into auto-inducing medium to induce expression of recombinant proteins. For neurosporene and lycopene production, cells were harvested by centrifugation after protein induction. Cells were then transferred to bioconversion medium at a starting biomass of 4 OD/L. For bioconversion experiments, cells were cultured in a flask at 37°C with shaking at 220 rpm for indicated times, and then harvested to quantify products and metabolites.
Measurement of carotenoid production and production of other metabolites
Cells were harvested by centrifugation at 12,000g for 5 min. The cell pellet was washed and then extracted in 1 mL acetone at 4°C for 1 h. The mixture was centrifuged at 12,000g, and the absorbance of the supernatant was measured at 470 nm. Neurosporene was quantified as described elsewhere . Lycopene was quantified using a standard curve of pure standard lycopene (Sigma). The biomass was determined by measuring cell density (OD600). The acetate concentration was determined by HPLC using a C18 column (Agilent), and the glucose concentration was measured using a glucose monitor (SDBI).
Batch fermentation in a microbioreactor
For batch fermentation of carotenoids, the strains were pre-cultured in auto-inducing medium to induce the synthesis of recombinant proteins. Then, cells were transferred into bioconversion medium in a bioreactor (BioLector), with a beginning cell density of 4 OD. The cells were cultured at 37°C with shaking at 800 rpm.
YT participated in the conception and design of the study and revised the manuscript. W-FL participated in data collection and analysis, and drafted the manuscript. CL and L-QY participated in data collection and analysis. S–SZ and NC provided original data for some sections. All authors read and approved the final manuscript.
The authors gratefully acknowledge funding from the following organizations: the National Basic Research Program of China (973 Program) (Grant No. 2012CB721105); the Key Research Program of the Chinese Academy of Sciences (Grant No. KSZD-EW-Z-016-1); and the Key Research Program of the Chinese Academy of Sciences (Grant No. KGZD-EW-606-2).
Compliance with ethical guidelines
Competing interests The authors declare that they have no competing interests.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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