Construction of engineered E. coli for ectoine production
For heterologous synthesis of ectoine in E. coli, the basic strains E. coli MG1655 and E. coli BL21 (DE3) were chosen as hosts in light of their clear genetic background. The constructed plasmids pTrc-ectABC and pET- ectABC were transferred into E. coli MG1655 and E. coli BL21 (DE3), to form the strain ET01 and ET02 respectively (Table 1). The extracellular ectoine titer reached 0.52 g/L after 48 h cultivation with ET01 (Table 2). Although the protein expression levels of ectABC in ET02 were more powerful than those of ectABC in ET01 (see Additional file 1: Figure. S1), and the biomass of ET02 was higher than that of ET01, the ectoine titer of the strain ET02 could not be detected (Table 2). The results clearly indicated that ET01 performed best and thus was chosen for further genetic manipulation.
Table 1
Strains and plasmids used in this study.
Strains/plasmid | Relevant characteristic | Source |
Strains | | |
E. coli MG1655 | Wild type | This lab |
E. coli BL21 (DE3) | Expression host | This lab |
Halomonas venusta | Wild type | This lab |
ET01 | MG1655 (pTrc-ectABC) | This study |
ET02 | BL21 (pET-ectABC) | This study |
ET03 | ET01 ΔlysA | This study |
ET04 | ET01 ΔthrA | This study |
ET05 | ET03 ΔthrA | This study |
ET06 | ET03 Δpta | This study |
ET07 | ET03 ΔldhA | This study |
ET08 | ET03 ΔpykF | This study |
Plasmid | | |
pTrc99a | trc promoter, cloning vector, Ampr | This lab |
pET-28a | T7 promoter, cloning vector, Kanr | This lab |
pKD46 | Temperature sensitive vector carrying Red recombinase, Ampr | This lab |
pKD3 | Template vector, Cmr | This lab |
pCP20 | Temperature sensitive vector carrying FLP recombinase, Ampr | This lab |
pTrc-ectABC | pTrc99a containing Halomonas venuas ectABC gene | This study |
pET-ectABC | pET28a containing Halomonas venuas ectABC gene | This study |
Table 2
Comparison of fermentation parameters of different strains in shake flask cultivations.
Strain | Ectoine titer (g/L) | Glucose consumption (g/L) | DCW (g/L) | Ectoine yield on glucose (g/g) |
ET01 | 0.52 ± 0.01 | 65 ± 1.37 | 16.2 ± 0.39 | 0.01 |
ET02 | 0 | 65 ± 1.72 | 18 ± 0.41 | 0 |
ET06 | 1.09 ± 0.21 | 65 ± 1.43 | 14.3 ± 0.29 | 0.02 |
ET07 | 0.6 ± 0.07 | 45 ± 0.91 | 14.3 ± 0.32 | 0.01 |
ET08 | 1.87 ± 0.18 | 63 ± 1.8 | 15.02 ± 0.48 | 0.03 |
Elimination of branch and by-product metabolic pathways
L-aspartate-β-semialdehyde is the important precursor of L-threonine, L-lysine and ectoine synthesis pathway in E. coli [12]. To reduce L-aspartate-β-semialdehyde shunting by the branch metabolism, the genes of lysA (encoding diaminopimelate decarboxylase) and thrA (encoding aspartate kinase/homoserine dehydrogenase) in E. coli ET01 were knocked out individually and together, generating the strains E. coli ET03, E. coli ET04 and E. coli ET05 respectively. As shown in Figure. 2, in comparison to that of E. coli ET01, the ectoine titer of E. coli ET03 (1.08 g/L) increased by 1.08 folds but biomass (13.9 g/L) decreased at the same glucose consumption (65 g/L). When thrA was deleted, ectoine titer of E. coli ET04 decreased to 0.34 g/L while glucose consumption and cell growth showed the similar trend, declining to 58 g/L and 8.2 g/L respectively. Similarly, as lysA and thrA were knocked out together, ectoine titer of E. coli ET05 decreased to a lower level (0.2 g/L) with 53 g/L glucose consumption and 7.05 g/L DCW. The result indicated that the deletion of thrA greatly inhibited the growth of E. coli ET01 and had negative impact on the ectoine synthesis. This might be due to the fact that thrA is involved in the synthesis of many important amino acids such as homoserine, methionine and threonine which are necessary for the cell growth. The deletion of thrA inhibited the synthesis of amino acids, and consequently the biomass decreased notably. Finally, E. coli ET03 was chosen for further study.
In addition to ectoine, some quantitative organic acids such as acetate, pyruvate and lactate were thought to be in the substrate. The productions of organic acids distribute the carbon flux and could significantly inhibit the cell growth by reducing the pH level, thereby affecting the titer. To further increase the ectoine production, E. coli ET06, E. coli ET07, E. coli ET08 were constructed by deletion of pta, ldhA, and pykF in E. coli ET03 respectively, which are the genes encoding key enzyme in acetate, lactate and pyruvate synthetic pathway respectively (Table 1). When pta was deleted, the ectoine production and biomass of E. coli ET06 were nearly equal to those of E. coli ET03, but the lag phase of E. coli ET06 was extended. However, the ectoine titer of E. coli ET08 increased by 73.1% (reached to 1.87 g/L) compared with that of E. coli ET03. Meanwhile the titer of E. coli ET07 was 0.6 g/L with the lowest glucose consumption (45 g/L). These results indicated that the deletion of pta and ldhA had a negative impact on the production and biomass, while knocking out pykF could increase the ectoine titer. This might be due to the positive role that the weakening of pyruvate pathway played in the accumulation of phosphoenolpyruvate, a prerequisite for ectoine synthesis. E. coli ET08 was chosen as the potential strain for further study on optimization of fermentation.
Optimization of nitrogen sources for ectoine production
Nitrogen is a constituent of cellular components such as proteins, nucleic acids and several cofactors [27]. It also regulates primary and secondary metabolism in different bacteria. To further improve the ectoine production, the effects of nitrogen sources combined with yeast extract and inorganic nitrogen sources on ectoine titer were investigated. As shown in Table 3, with the increase of yeast extract concentration in the medium, the ectoine production of E. coli ET08 presented an increasing trend except for the titer obtained by sodium nitrate addition. The noticeable ectoine yield (7.3 g/L) was achieved at the combination of 20 g/L yeast extract and 152 mM ammonium chloride as inorganic nitrogen source. And the ectoine yield obtained by ammonium chloride addition was 3.9 times higher than that of the control. At the same yeast extract concentration, the ectoine titer gained by ammonium chloride addition was significantly higher than that of the blank control and that obtained by sodium nitrate addition. The combination of yeast extract and nitrate had no positive effect on the production of ectoine. Both the biomass and sugar consumption capacity of E. coli ET08 were also reduced to a certain extent (Table 3). This implied that ammonium salt played a more significant role in promoting the production of ectoine during fermentation compared with nitrate salt. The former was more conducive to microbial absorption in comparison with the latter and acted as exogenous amino donor to provide NH4+ involved in the synthesis of glutamate directly (Figure. 1). Besides, glutamate, as a co-substrate, was involved in the catalytic reaction of the key enzyme EctB in the synthesis pathway of ectoine, and it provided an amidogen to L-aspartate-β-semialdehyde [12].
Table 3
Effect of the yeast extract mixed with inorganic nitrogen on ectoine fermentation.
Yeast extract (g/L) | 10 | 15 | 20 |
Inorganic nitrogen | CK | 152 mM NH4Cl | 152 mM NaNO3 | CK | 152 mM NH4Cl | 152 mM NaNO3 | CK | 152 mM NH4Cl | 152 mM NaNO3 |
Ectoine titer (g/L) | 0.37 ± 0.03 | 1.35 ± 0.07 | 0.47 ± 0.04 | 1.26 ± 0.11 | 4.19 ± 0.18 | 0.23 ± 0.05 | 1.87 ± 0.18 | 7.3 ± 0.18 | 0.21 ± 0.02 |
Glucose consumption (g/L) | 59 ± 1.23 | 59 ± 1.30 | 41 ± 0.47 | 64 ± 1.63 | 60.6 ± 0.95 | 46 ± 0.94 | 63 ± 1.8 | 61.5 ± 1.6 | 47 ± 1.27 |
DCW (g/L) | 10.97 ± 0.5 | 12.06 ± 0.36 | 9.27 ± 0.39 | 12.79 ± 0.43 | 13.06 ± 0.34 | 11.03 ± 0.47 | 15.02 ± 0.48 | 14.8 ± 0.34 | 11.60 ± 0.42 |
Optimization of exogenous amino donor
From the above results, we consider that ammonium chloride as an amino donor plays an important role in improving the ectoine production. Therefore, the types and concentrations of amino donors should be optimized. As shown in Figure. 3, the maximal ectoine output (10.2 g/L) was obtained by ammonium sulfate addition, higher than that obtained by ammonium chloride addition (7.3 g/L) and sodium glutamate addition (6.2 g/L). With the concentration of amino donor increasing, the titer presented a trend that changed from increase to decrease. And the maximum titer was obtained when the concentration of NH4+ (for ammonium salt)/NH3+ (for sodium glutamate) was 152 mM, no matter it is under the condition of ammonium sulfate (Figure. 3A), ammonium chloride (Figure. 3B) or sodium glutamate addition (Figure. 3C). The concentration of NH4+/NH3+ had little effect on the growth rate of bacteria when the concentration of NH4+/NH3+ was less than or equal to 152 mM. However, with the concentration of NH4+/NH3+ reaching to 228 mM, the growth of bacteria was significantly inhibited, which was viewed as an important reason for the decrease of total glucose consumption and ectoine production. Although all these three amino donors improved ectoine biosynthesis, ammonium sulfate was the best while sodium glutamate was the worst. This might be attributed to the higher transport efficiency of ammonium sulfate compared with sodium glutamate. Overall, ammonium sulfate (76 mM) was chosen to act as final exogenous amino donor after optimization.
Effect of NH4+ on the relevant metabolic pathways
Results above indicated the supplement of NH4+ was of great importance for improving ectoine production. We anticipated the participation of ammonium sulfate made a difference to the key genes in ammonium metabolic pathways and ectoine synthesis pathway. Thus, the transcription levels of relative genes were investigated under addition of 76 mM ammonium sulfate or not (control).
Ammonium is utilized as the main nitrogen source via two stages of uptake and assimilation. After ammonium is transported into the cells, assimilation proceeds via glutamate dehydrogenase (GDH) encoded by gdhA or glutamine synthetase/glutamate synthase (GS/GOGAT) encoded by glnA and gltB separately, depending on the ammonium availability in the medium [28, 29]. In the study, the transcription levels of gdhA and gltB increased by 3.36 and 2.92 times respectively, whereas the transcription levels of glnA decreased significantly compared with those of the control (Figure. 4). The up-regulated gene gltB promoted the synthesis of glutamate in strains over the process of fermentation. The high expression of gdhA could also enhance the accumulation of glutamate [30]. And the down-regulated gene glnA indicated the inhibition of glutamine synthesis but the promotion of glutamate accumulation. Furthermore, the glutamate as an essential substance could be directly involved in ectoine synthesis through transamination (Figure. 1). Therefore, the ammonium sulfate addition could act as boost in ectoine synthesis by improving glutamate synthesis.
Aspartokinase (AK), EctA, EctB as well as EctC played significant roles in the pathway of ectoine synthesis. As depicted in Figure. 4, the transcription levels of the aspartate kinase gene (lysC) was up regulated by 2.05 folds, which demonstrated that the ability of aspartate metabolism to produce downstream products was improved. Obvious increases in the transcription levels of ectABC acted as a main gene cluster in ectoine synthesis could also be seen. the transcription levels of ectB was up-regulated by 6.84 folds, higher than ectA (3.93 folds) and ectC (1.78 folds). This result indicated that EctB was the most important enzyme among the key enzyme of the ectoine synthesis, which was consistent with the results previously reported [23]. It was speculated that the ammonium sulfate addition enhanced the supplement of the precursor L-aspartate-β-semialdehyde and glutamate, providing affluent co-substrate for the key enzyme EctB. At the same time, the strong expression of ectABC, especially ectB, promoted the flow of substrate to the ectoine synthesis pathway, improving the ectoine production.
Fermentation performance in a 7.5L bioreactor
To assess the overall production performance of E. coli ET08 under the addition of exogenous ammonium sulfate (76 mM), ectoine fermentation in a 7.5 L bioreactor with feeding batch fermentation using two-stage feed was conducted. As shown in Figure. 5, the ectoine concentration reached the maximum (36.5 g/L) at 36 h. At the first phase of fermentation (0 h ~ 18 h), the bacteria grew fast, but just 9.4 g/L extracellular ectoine titer could be seen. At the second phase of fermentation (18 h ~ 36 h), the ectoine titer accumulated rapidly even though there was a slow growth in dry cell weight, reaching 27 g/L at about 30 h and remaining until the end of the fermentation. The specific ectoine production and productivity reached 1.4 g/g DCW and 1.01 g/L/h respectively. Plus, the yield of ectoine was 0.3 g/g glucose. To our best knowledge, our work shows the highest ectoine titer and yield from glucose synthesized by E. coli., and still has room for further improvement. Compared with previous studies (Table 4), whole-cell catalysis seems to have certain advantages in yield, but it requires the addition of extra glycerol and aspartate, which results in high cost of extra substrate, cell culture before catalytic reaction. Although C. glutamicum ectABCopt [23] achieved higher titer, the yield was only 0.19 g/g ,which was lower than our yield. Thus, the metabolic engineered strain E. coli. ET08 and the fermentation strategy of supplementing amino donor shows a promising value for industrial production of ectoine.
Table 4
Microbial production of ectoine using different fermentative strains or biocatalysts.
Strain | Titer (g/L) | Specific production (g/g DCW) | Yield (g/g) | Productivity (g/L/h) | Process strategy | Reference |
E. coli ET08 | 36.5 | 1.4 | 0.30 | 1.01 | Fed-batch | This work |
E. coli Ect05 | 25.1 | 0.8 | 0.11 | 0.84 | Fed-batch | [22] |
E. coli BW25113 (pBAD-ectABC) | 25.1 | 4.1 | - | 1.04 c | Whole-cell catalysis a | [21] |
E. coli ECT2 | 12.7 | - | 1.27 | 0.53 c | Whole-cell catalysis a | [34] |
C. glutamicum ectABCopt | 65.3 | - | 0.19 | 1.16 | Fed-batch | [23] |
Chromohalobacter salexigens DSM3043 | 32.9 | 0.5 | - | 1.35 | continuous reactors with cell b | [19] |
a Whole-cell catalysis using aspartate and glycerol as substrates at a high cell density. |
b A special fermentation process using two continuously operated bioreactors. |
c Achieved by calculating reported data. |