Synthesis of pCA from D-glucose using mono culture E. coli MCA
To evaluate the ability of single E. coli strain to synthesize p-coumaric acid, this strain was cultured in minimal M9 plus 15 g/L glucose. In another report, we tested the production of pCA from different carbon sources as glucose and glycerol (Thuan et al. 2021) (submitted). As a result, D-glucose was allowed to produce better titer of pCA (25.3 mg/L) after 48 h. Hence it was used for further study (Fig. 2).
Synthesis of eriodictyol from pCA using mono-culture E. coli
Different E. coli host strains were constructed with genes in the biosynthetic pathway of ED (4CL, CHS, CHI, and M13) to assess their ability for protein expression and functional activity. Mainly, E. coli BL21(DE3) containing those genes were designated as E. coli ED1. Dependence of biosynthetic dynamic of ED using E. coli ED1 on incubation time was shown in Fig. 3. ED concentration was gradually increased from 12-48 h of culture and reached the highest amount (21.3 mg /L) after 48 h. Hence, the host E. coli ED1 was further used for the study.
Production of eriodictyol in the mono-culture E. coli MED strain
E. coli MED strain contained all genes that were to synthesize ED from D-glucose (Table 1). This work aimed to demonstrate that whole genes in the biosynthetic pathway of eriodictyol working well in E. coli. Production of ED and its intermediates was shown in Fig. 4. Moreover, the highest titer of ED was reached 18.1 mg/L after 48 h.
Co-culture for the synthesis of ED
Design of synthetic co-culture system
E. coli - based co-culture included two modules. Module 1 (E. coli MCA, upstream) contained genes for the biosynthetic pathway of L-tyrosine from D-glucose. Notably, gene doses of ppsA and tktA were increased to enhance the intracellular accumulation of main precursors, PEP and E4P. Furthermore, the inactivation of pheA was to restrict the bioconversion of L-tyrosine to L-phenylanaline (Fordjour et al. 2019; Patnaik et al. 2008). And it resulted in the mutant E. coli /ΔpheA/ppsA/tktA. On the other hand, this strain was introduced TAL gen for conversion of tyrosine to pCA. Module 2 (E. coli ED1) contained genes (4CL, CHS, CHI) for the synthesis of NRN from pCA and flavonoid-3’-hydroxylase type M13 to convert NRN to ED (Fig. 1).
Effect of initial inoculum ratio
Up and downstream strains were mixed at different ratio (100:1; 50:1; 10:1; 1:1; 1:10; 1:50 and 1:100) and cultured under the same condition (minimal M9 medium + 15 g/L glucose and 33 oC) with supplementation of D-glucose. The initial inoculum ratio affected the formation of intermediates (pCA, NRN) and ED, as shown in Fig. 5.
Effect of temperature
Various temperature ranges were used to set up culture conditions, including 25, 30, 33, and 37 oC. Production titer of ED was gradually increased at 25 oC and achieved 38.4 mg/L at 33 oC as the best result before decreasing (Fig. 6).
Effect of initial net inoculum
To investigate the impact of increasing initial net cell density of each module on the titer of ED production, the initial cell density of each up and downstream module strain was enhanced from 5x106 to 5x107 cells per mL and cultured at a temperature of 33oC. Firstly, this work was tested on a test tube scale. As shown in Table 2, the highest concentration of ED was reached 44.5 mg/L at 33 oC after 48 h. Moreover, comparatively, it resulted in an increased titer 1.15-fold (44.5/38.4).
Table 2
Effect of initial net inoculum on production of eriodictyol.
Temperature (oC)
|
pCA (mg/L)
|
NRN (mg/L)
|
ED (mg/L)
|
25
|
13.8
|
22.7
|
37.6
|
28
|
15.2
|
24.8
|
39.8
|
33
|
16.5
|
26.9
|
44.5
|
37
|
13.4
|
26.1
|
42.1
|
Scale up the co-culture for production of eriodictyol using shake flask
Finally, to evaluate the scalability of this synthetic consortium for the production of ED and its intermediates, shake flask culture was utilized under mentioned above conditions, including the ratio of MCA : ED1 = 1 : 1, 33 oC, and initial net inoculum density of 5x107 cells per mL. The titer of ED was measured and achieved of 51.5 mg/L at 48 h as the highest amount (Fig. 7).