The comprehensive profile of fermentation products during in situ CO2 recycling by Rubisco-based engineered Escherichia coli
© The Author(s) 2016
Received: 21 January 2016
Accepted: 21 July 2016
Published: 2 August 2016
In our previous study, the feasibility of Rubisco-based engineered E. coli (that contains heterologous phosphoribulokinase (PrkA) and Rubisco) for in situ CO2 recycling during the fermentation of pentoses or hexoses was demonstrated. Nevertheless, it is perplexing to see that only roughly 70 % of the carbon fed to the bacterial culture could be accounted for in the standard metabolic products. This low carbon recovery during fermentation occurred even though CO2 emission was effectively reduced by Rubisco-based engineered pathway.
In this study, the heterologous expression of form I Rubisco was found to enhance the accumulation of pyruvate in Escherichia coli MZLF [E. coli BL21(DE3) Δzwf, Δldh, Δfrd]. This may be attributed to the enhanced glycolytic reaction supported by the increased biomass and the ethanol/acetate ratio. Besides, it was found that the transcription of arcA (encodes the redox-dependent transcriptional activators ArcA that positively regulates the transcription of pyruvate formate-lyase) was down-regulated in the presence of Rubisco. The enhanced accumulation of pyruvate also occurs when PrkA is co-expressed with Rubisco in E. coli MZLF. Furthermore, E. coli containing Rubisco-based engineered pathway has a distinct profile of the fermentation products, indicating CO2 was converted into fermentation products. By analyzing the ratio of total C-2 (2-carbon fermentation products) to total C-1 (1-carbon fermentation product) of MZLFB (MZLF containing Rubisco-based engineered pathway), it is estimated that 9 % of carbon is directed into Rubisco-based engineered pathway.
Here, we report for the first time the complete profile of fermentation products using E. coli MZLF and its derived strains. It has been shown that the expression of Rubisco alone in MZLF enhances the accumulation of pyruvate. By including the contribution of pyruvate accumulation, the perplexing problem of low carbon recovery during fermentation by E. coli containing Rubisco-based engineered pathway has been solved. 9 % of glucose consumption is directed from glycolysis to Rubisco-based engineered pathway in MZLFB. The principle characteristics of mixotroph MZLFB are the high bacterial growth and the low CO2 emission.
The Calvin–Benson–Bassham (CBB) cycle, also known as the reductive pentose phosphate cycle, is a metabolic pathway involving the conversion of three molecules of carbon dioxide into one molecule of glyceraldehyde-3-phosphate. The CBB cycle uses 11 enzymes to complete autotrophic CO2 fixation. Interestingly, most of enzymes are also involved in the central metabolism including glycolysis and the pentose phosphate pathway. Therefore, it is generally accepted that the functional CBB cycle that emerged billions of years ago did not evolved as a whole, but instead the individual enzymes have their own phylogenetic history that is independent of the biochemical distribution of the CBB cycle . Consequently, one way to genetically assess whether a microorganism utilizes the CBB cycle is to characterize the manifestation of two key genes that encodes phosphoribulokinase (PRK) and Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). Rubisco is the one enzyme that is specific to the CBB cycle. Rubisco catalyzes the carboxylation and the oxygenation of ribulose-1,5-bisphosphate with CO2 and O2, respectively. The products of carboxylation and oxygenation are 3-phosphoglycerate and 2-phosphoglycolate and the former will be sequentially converted into glyceraldehyde-3-phosphate by phosphoglycerate kinase and glyceraldehyde-3 phosphate dehydrogenase whereas the latter will enter photorespiration .
The CBB cycle has been used in the engineering perspective for converting CO2 into bio-based chemicals. For example, microalgae have been used to produce carbohydrates from CO2, where the carbohydrates produced were subsequently used for bioethanol production . A sequential fermentation process was proposed for a hydrogen production process with zero CO2 emission . Cyanobacteria have been engineered to produce various bio-based chemicals from autotrophic CO2 assimilation [5–8]. On the other hand, PRK and Rubisco without the function of the full CBB cycle were shown to improve oil production in developing embryos of Brassica napus L . This partial CBB cycle was also developed in Rubisco-dependent Escherichia coli (RDE) for the selection of Rubisco mutants that had better performance in terms of reaction rate and CO2/O2 selectivity [10–13]. The quantitative analysis of CO2 fixation in recombinant E. coli has been reported recently where 17 % of carbon was found to be directed to Rubisco-based engineered pathway in the presence of the carbonic anhydrase . In our previous study, the feasibility of Rubisco-based engineered E. coli for in situ CO2 recycling during the fermentation of pentoses was demonstrated . By enhancing the function of the homologous non-oxidative pentose phosphate pathway, Rubisco-based engineered E. coli can be further used to achieve a low CO2 emission during the fermentation of hexoses . While Rubisco-based engineered pathway (that contains PrkA and Rubisco) is arguably compatible to the central metabolism of E. coli , it is perplexing to see the enhanced biomass accumulation and other physiological responses when form I Rubisco is heterologously expressed in E. coli . It is also perplexing to see that only roughly 70 % of the carbon fed to the bacterial culture could be accounted for in the standard metabolic products. This low carbon recovery (the carbon recovery is defined as the mole of carbon of total fermentation products/the mole of carbon of consumed glucose) during fermentation occurred even though CO2 emission was effectively reduced by Rubisco-based engineered pathway [15, 16]. The low carbon recovery of 73 % can be seen for MZLFB + IP when pyruvate was not included for the calculation, see below. This unforeseen carbon distribution is also observed when Rubisco-based engineered pathway is heterologously expressed in Saccharomyces cerevisiae . In this study, a recombinant E. coli strain with the deactivations of ldh and frd genes (encoding the lactate dehydrogenase and the fumarate reductase, respectively) was constructed to study the partition of carbon flow among the wild-type fermentation routes and Rubisco-based engineered pathway was quantified by examining the profiles of the end-metabolites. During which, several interesting phenotypes due to the overexpression of form I Rubisco were presented. Finally, the feasibility of mixotrophic E. coli as a platform for bio-based chemical productions was discussed.
Results and discussion
The pyruvate accumulation was found to be enhanced in E. coli in the presence of Rubisco
List of bacterial strains and plasmids used in this study
E. coli BL21 (DE3)
F-, dcm, ompT, gal, lon, hsdSB (rB−, mB−), λ (DE3[lacI, lacUV5-T7 gene 1, ind1, sam7,nin5])
E. coli BL21 (DE3) harboring rbcLS-pET30a + (M259T)
E. coli BL21 (DE3) harboring both PBAD-his6-prkA-pACYC184 and rbcLS-pET30a + (M259T)
E. coli BL21 (DE3) Δzwf
MZLF harboring PBAD-his6-prkA-pACYC184
MZLF harboring rbcLS-pET30a + (M259T)
MZLF harboring both PBAD-his6-prkA-pACYC184 and rbcLS-pET30a + (M259T)
araC, bla, oriR101, repA101 (Ts), araC-P araB -γ-β-exo (encode λ Red recombinases), temperature-conditional replicon
FLP +, λ cI857+,λ PR Pepts, bla, catF
Recombinant plasmid carries prkA gene (derived from Synechococcus PCC7492) for the overexpresion of phosphoribulokinase (PrkA) under the control of PBAD
rbcLS-pET30a + (M259T)
Recombinant plasmid carries engineered rbcLS gene (originated from Synechococcus PCC6301) for the overexpresion of engineered Rubisco (M259T) under the control of PT7
rbcLS-pET30a + (M259T, K198G)
Recombinant plasmid carries engineered rbcLS gene (originated from Synechococcus PCC6301) for the overexpresion of engineered Rubisco (M259T and K198G) under the control of PT7
rbcLS-pET30a + (M259T, K198G, D200G, E201G)
Recombinant plasmid carries engineered rbcLS gene (originated from Synechococcus PCC6301) for the overexpresion of engineered Rubisco (M259T, K198G, D200G, and E201G) under the control of PT7
rbcLS-pET30a + (M259T, K198G, K172G)
Recombinant plasmid carries engineered rbcLS gene (originated from Synechococcus PCC6301) for the overexpresion of engineered Rubisco (M259T, K198G, and K172G) under the control of PT7
rbcLS-pET30a + (M259T, K198G, K331G)
Recombinant plasmid carries engineered rbcLS gene (originated from Synechococcus PCC6301) for the overexpresion of engineered Rubisco (M259T, K198G, and K331G) under the control of PT7
The distribution of C-2 fermentation products (i.e., ethanol and acetate) partly reflects the intracellular balances of ATP and the reducing equivalents. It has been demonstrated that the glycolytic flux is mainly controlled by the demand for ATP during the anaerobic growth of E. coli in the minimal medium [19, 20]. When the ATP demand, resulting from the anabolism, is strong and intense, the glycolic flux will accordingly increase to replenish ATP supply by the substrate-level phosphorylation. Moreover, additional ATP production can be achieved by the conversion of pyruvate derived from glycolysis. However, this route for the additional ATP production is restrained by surplus reducing equivalents generated from glycolysis where the conversion of pyruvate to ethanol is one of the main mechanisms to respire the reducing equivalents. The increase in the molar ratio of ethanol to acetate as shown in Fig. 1b infers that NADH may be produced in excess when Rubisco was overexpressed (see MZLF3 + IP). The introduction of PrkA in MZLFB (containing both Rubisco and PrkA), representing the extra demand for ATP, resulted in an additional increase of the ethanol:acetate ratio from 2.14 to 2.23 for MZLFB + IP. It is therefore suggested that the overproduction of NADH may result from an increase in glycolysis, which is because of the high demand for ATP in MZLF3 + IP and MZLFB + IP. These observations are consistent with the results when 30 mM of acetate were added, further increasing the molar ratio of ethanol to acetate from 2.23 to 3.28. The addition of acetate lowered the conversion of pyruvate to acetate by roughly 40 % (data not shown). The reduced acetate production resulted in reduced ATP production, thus further stimulating the glycolytic pathway to some extent. Note that the addition of 30 mM acetate during the cultivation of MZLF provided a pyruvate yield of 0.13 and an ethanol/acetate ratio of 1.25 (data not shown). This relatively same performance compared to MZLF as shown in Fig. 1 indicates that the enhanced pyruvate production observed for MZLFB + ace + IP (Fig. 1a) is because of the presence of Rubisco. And the enhanced pyruvate production can be attributed to the unbalanced ATP production. The enhanced glycolytic activity accompanied by enhanced bacterial growth is consistent with our previous study when Rubisco was aerobically overexpressed in E. coli BL21 (DE3) .
Pyruvate is not a typical fermentation product of anaerobic cultivation of E. coli. Instead, it is an important metabolic node that is associated with the metabolisms of lactate, acetate, ethanol, and succinate . It is also involved in the anaplerosis (through phosphoenolpyruvate-pyruvate-oxaloacetate) as well as glucose metabolism (through phosphotransferase system, PTS) . The enhanced glycolysis due to the overexpression of Rubisco should lead to vigorous function of PTS where PEP is converted to pyruvate. At the same time, the enhanced anaplerotic metabolism has been shown to occur when Rubisco is aerobically overexpressed in E. coli BL21 (DE3)  or anaerobically in E. coli MZ  and MZLFB (see results below). The enhanced anaplerosis competes with PTS for PEP. This may further intensify the activity of glycolysis and PTS and thus results in the enhanced pyruvate production.
Transcriptional levels of global regulators in Rubisco-based E. coli
log2Ratio in J3
log2Ratio in JB
Carbon dioxide is recycled and converted into fermentation products and biomass by Rubisco-based engineered pathway
Fermentation profiles of E. coli MZLF derived strains
E. coli strains
Molar yield (mmol/mmol)
Carbon recovery (%)
59 ± 7
0.16 ± 0.12
0.66 ± 0.09
0.84 ± 0.14
0.04 ± 0.02
1.54 ± 0.28
0.02 ± 0.005
0.37 ± 0.05
92 ± 9
MZLF3 + IP
55 ± 1
0.46 ± 0.02
0.41 ± 0.00
0.87 ± 0.01
0.03 ± 0.00
1.30 ± 0.02
0.001 ± 0.00
0.54 ± 0.02
98 ± 1
58 ± 5
0.14 ± 0.03
0.67 ± 0.07
0.82 ± 0.10
0.04 ± 0.01
1.56 ± 0.19
0.01 ± 0.003
0.40 ± 0.03
92 ± 5
MZLFB + IP
50 ± 2
0.46 ± 0.04
0.40 ± 0.03
0.87 ± 0.07
0.03 ± 0.01
1.24 ± 0.11
0.52 ± 0.04
96 ± 7
MZLFB + ace + IP
51 ± 1
0.61 ± 0.06
0.25 ± 0.05
0.82 ± 0.04
0.02 ± 0.0
1.13 ± 0.06
0.50 ± 0.03
94 ± 4
The feasibility of mixotrophic E. coli as a platform for bio-based chemical productions
The feasibility of mixotrophic E. coli can also be perceived by examining ethanol production and the associated C-1 accumulation. It can be seen in Fig. 5b that E. coli strain MZ produced 0.47 mol-ethanol/mol-glucose with a 0.85 mol-C-1/mol-glucose. By de-activating ldh and frd genes from MZ, E. coli strain MZLF produced 0.84 mol-ethanol/mol-glucose (79 % increase) while C-1 production was proportionally increased to 1.56 mol-C-1/mol-glucose. The introduction of Rubisco-based engineered pathway into MZLF enhanced the ethanol production by 4 % while decreasing total C-1 production by 21 %. This is in agreement with the theoretical calculation that 9 % of carbon flux was directed into Rubisco-based engineered pathway. Note that some recycled C-1 could arguably become biomass since the biomass yield increase from 0.37 for MZLF to 0.52 for MZLFB + IP.
Here, we report for the first time the complete profile of fermentation products using E. coli MZLF and its derived strains. The profiles reveal that the heterologous expression of form I Rubisco has a strong impact on the central metabolism of a non-phototrophic heterotroph E. coli, resulting in significant increases in the production of biomass and pyruvate. By examining the comprehensive profile of fermentation products, it is estimated that 9 % of glucose consumption is directed from glycolysis to Rubisco-based engineered pathway. The co-existence of glycolysis and Rubisco-based engineered pathway in E. coli MZLFB poses an example as the mixotroph where the principle characteristics of MZLFB are the high bacterial growth and the low CO2 emission.
Bacterial strains and plasmids
All strains and plasmids used in this study are listed in Table 1. The ldh gene knock-out mutant derived from E. coli strain MZ and the frd gene knock-out mutant derived from E. coli strain MZL were constructed through the one-step inactivation procedure [29, 30]. The sequences of primers used for the synthesis of linear DNA fragments for Red-mediated recombination can be found in Additional file 1. More information regarding the construction of mutant strains can be found in .
The recombinant plasmid rbcLS-pET30a + (M259T,K198G) was derived from rbcLS-pET30a + (M259T) by the Q5 Site-Directed Mutagenesis Kit (New England BioLabs® Inc., the USA), where primers rbcL-K198G-F and rbcL-K198G-R were used, see Additional file 1. rbcLS-pET30a + (M259T,K198G,D200G,E201G), rbcLS-pET30a + (M259T,K198G,K172G), and rbcLS-pET30a + (M259T,K198G,K331G) were generated subsequently from rbcLS-pET30a + (M259T,K198G). Four recombinant plasmids were subject to DNA sequencing (serviced by Biotechnology center at National Chung Hsing University, Taiwan). All primers used for the amplification of mutation were listed in (Additional file 1).
Culture media and growth conditions
Escherichia coli strains used for fermentation study were grown anaerobically at 200 rpm and 37 °C in fresh 25-ml M9 defined medium containing (per liter): 12.8 g Na2HPO4·7 H2O; 3 g KH2PO4; 0.5 g NaCl; 1.0 g NH4Cl; 0.24 g MgSO4; 0.011 g CaCl2; and 20 g glucose. An anaerobic culture environment was achieved in an sealed serum bottle as described previously . Initial OD600 was adjusted to 0.05. The pH was adjusted to 8 at the fermentation times of 0, 8, and 24 h. The respective concentrations of streptomycin, chloramphenicol and kanamycin used were 50, 34, and 50 μg/ml. The isopropyl-β-d-1-thiogalactopyranoside (IPTG) and acetate was added at 8 h when needed.
The cell density was measured at 600 nm using a UV–Vis spectrophotometer (GENESYS 10S, Thermo Scientific, USA). The gaseous CO2 concentration in the headspace of the cultures was measured by a diffusive infrared-based CO2 analyzer (Sentry ST303). The total CO2 concentration was calculated based on the gaseous CO2 concentration and the detailed calculation has been described in [15, 16].
Samples for quantification of residual glucose or extracellular metabolites were collected from the culture solutions followed by the centrifugation for 5 min at 17,000×g to remove cell pellets. The supernatant was filtered by a 0.2 μm PVDF filter before sample injection. The concentration of residual glucose was determined by either HPLC or DNS methods . Characterization and quantification of extra-cellular formate, acetate, ethanol, lactate, succinate, and pyruvate were performed by Thermo ScientificTM DionexTM Ulitmate 3000 LC Systems. The separation of the mixture was achieved with the HPLC column Thermo scientific HyperREZ XP Carbohydrate H+ (300 mm × 7.7 mm 8 μm) where the measurement was done with a refractive index (RI) detector. The mobile phase was 5 mM H2SO4. The temperature was maintained at 65 °C while the flow rate was maintained at 1.0 ml per minute. The sample injection was done by an autosampler whereas the injection volume is 20 μl.
The qualitative and quantitative analysis of pyruvate was further confirmed by the use of Pyruvate Colorimetric/Fluorometric Assay Kit (Biovision Inc., the USA). Basically, 50 µl proprietary Reaction Mix, containing 46 µl of Assay Buffer, 2 µl of Pyruvate Probe, and 2 µl Enzyme Mix, were mixed with 50 µl of sample. Incubate the mixtures in a light-free condition for 30 min at room temperature. The absorbance of samples at 570 nm was measured using a microplate reader.
Quantification of mRNA expression level
The RNA sequencing was done by Genomics BioSci & Tech Ltd and used to quantify the expression level of mRNA. Primary sequencing data of cDNA library were performed by Illumina HiSeq™ 2000 and the data mapping was done with SOAPaligner/SOAP2. E. coli strains J3 together with E. coli BL21 (DE3) as the control experiment were aerobic cultivated in LB medium supplemented with 20 g/l L-arabinose . Meanwhile, E. coli strains JB together with E. coli BL21 (DE3) as the control experiment were anaerobic cultivated in M9 medium supplemented with 20 g/l d-glucose as presented above. The samples for J3 transcriptome analysis were collected at 24 h while the samples for JB transcriptome analysis were collected at 24 h. Four samples for RNA-seq were shipped out with dry ice bath. Detailed protocol for sample preparation, RNA-seq, and data analysis can be found in Text S1 and .
- The CBB cycle:
The Calvin–Benson–Bassham cycle
phosphoribulokinase originated from Synechococcus PCC7492
Rubisco-dependent Escherichia coli
- C-1 compounds:
represent formate and CO2
- C-2 compounds:
represent ethanol and acetate
aerobic respiration control protein
sodium dodecyl sulfate–polyacrylamide gel electrophoresis
CHY constructed recombinant strains and plasmids, carried out the fermentation experiments, and analyzed fermentation and RNA-seq data. EJL carried out the fermentation experiments and analyzed fermentation and RNA-seq data. YLC constructed recombinant plasmids for expressing mutant Rubiscos and carried out fermentation experiments. FYOY carried out the fermentation experiments and analyzed RNA-seq data. SYL conceived and designed research, analyzed data, and wrote the manuscript. All authors read and approved the final manuscript.
This work was funded by the Ministry of Science and Technology Taiwan.
The authors declare that they have no competing interests.
Availability of data and materials
The datasets supporting the conclusions of this article are included within the article and its supplementary materials.
Consent for publication
Ethics approval and consent to participate
This work was funded by the Ministry of Science and Technology Taiwan, MOST-103-2221-E-005-072-MY3 and MOST-104-2621-M-005 -004 -MY3.
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