"Engineering success can be achieved by making an effort to follow the engineering design cycle: Design → Build → Test → Learn → Design..."
The “design-build-test-learn” (DBTL) cycle, which is the core principle of synthetic biology engineering design, can effectively and efficiently screen and optimize the functions of the targeted biosynthetic devices and systems. To successfully improve the catalytic activity of Olep, we have carried out several engineering cycles.
P450 enzyme has heme as its active center and needs to bind to heme to be active. Since Escherichia coli (E. coli) has a low intracellular heme supply, we hoped to increase the P450 enzyme’s activity by increasing the supply of heme.
However, this idea was not something we had listed in the framework from the beginning but rather came from an attempt to explore ways to increase the soluble heterologous expression of Olep.
Research
In the part of the soluble expression of Olep, it was found that when the N-terminal of Olep was fused with the solubilizing label MBP or TF, although the soluble expression of Olep increased, the catalytic efficiency decreased.
By reading the literature and consulting with our teacher, we realized that this might be related to the change in intracellular heme content, so we decided to measure the intracellular heme content of MBP-Olep and TF-Olep.
Design
To perform this assay, we repeated the experiments done in enhancing the soluble expression of Olep by fusing the pro-solubilization tags MBP and TF to the N-terminal of the oleP expressing gene, respectively, and constructing recombinant plasmids.
Build
P450 enzyme CYP107D1 (Olep), derived from Streptomyces antibioticus, was synthesized after codon optimization (BBa_K4759001). The oleP expression gene, codon-optimized ferredoxin reductase gene camA, and ferredoxin gene camB were then subcloned to the plasmid pRSFDuet to obtain the recombinant plasmid pRSFDuet-camA-camB-olep.
In the plasmid pRSFDuet, Olep was expressed in BBa_K4759203 and CamA/CamB was expressed in BBa_K4759201.
The expression host was E.coli C41 (DE3), induction temperature was 25°C, and the inducer IPTG concentration was 0.5 mM (E. coli O2).
The MBP and TF sequences were fused at the N-terminal of the oleP expression genes on the recombinant plasmid. We successfully constructed the above sequences and established the element BBa_K4759205; BBa_K4759206.
Test
After the end of induction, 1 mL of fermentation broth was collected and the cells were harvested by centrifugation at 14,000 rpm for 10 min at 4°C. 0.2 mL of the supernatant was taken and mixed with 0.2 mL of ammonia and 1.6 mL of acetonitrile. Then centrifuged it at 14,000 rpm for 20 min, collected the supernatant, which was used to detect extracellular heme. Resuspended the pellet with 0.2 mL of ultrapure water, 0.2 mL of ammonia, and 1.6 mL of acetonitrile. The samples were then placed on ice and the cell suspension was sonicated with an ultrasonic disruptor for 10 min (power 30 W, 3 mm conical microtip probe with a pulse period of 3 s on/3 s off). The supernatant was collected by centrifugation at 14,000 rpm for 20 min, and it was used to detect intracellular heme.
Heme concentration was measured by UHPLC-QTOF-MS (Agilent 1290-6495C).
Learn
When measuring the heme-binding rate, it was found that the heme binding rate of MBP-Olep and TF-Olep was only 21.1% and 16.8%, which was lower than that of the heme binding rate of the analytic label Olep (39.4%). Therefore, insufficient heme supply may be a key factor limiting the catalytic efficiency of Olep.
Fig. 1-1: The heme-binding ratio of wild-type Olep, MBP-Olep and TF-Olep The blue-filled triangle represents the heme-binding ratio (%). The red hollow triangle represents the conversion rate (%). Values and triangles represent the means and standard deviations of biological triplicates.
Research
In the previous DBTL, we verified that the addition of MBP and TF led to a decrease in catalytic efficiency associated with an insufficient heme supply.
In E. coli, the precursor of heme: 5-aminolevulinic acid (ALA), synthesized heme through a series of pathways. There are currently two main ways to enhance the heme supply of the P450 enzyme. One strategy was to exogenously add the precursor 5-aminolevulinic acid (ALA) of heme during P450 enzyme expression, and the other strategy was to enhance the synthetic pathway of heme in E. coli.
Design
In this iteration, to compare the effect of the exogenous addition of ALA and heme, we decided to exogenously add ALA, heme, and ALA＋heme, respectfully.
Build
ALA and heme were obtained from Sigma-Aldrich, and three experimental groups with three different conditions were set up, each with three sets of parallel samples.
Group 1: addition of precursor ALA+FeSO4, Group 2: addition of precursor ALA+FeCl3, and Group 3: direct addition of heme. The control groups were also prepared.
Test
Samples were handled in the same way as the previous iteration.
The heme concentration was measured by UHPLC-QTOF-MS (Agilent 1290-6495C).
Learn
The results showed that the addition of 0.64 mM ALA and 0.3 mM FeCl3 during the induction of Olep increased the heme binding rate to 67.7% and the conversion rate of deoxycholic acid to 40.7%. Through the above experiments, we verified that the increase in heme content had a positive effect on P450 enzyme activity, but the exogenous addition of ALA increased the cost of whole-cell catalysis by 60%, so we tried to increase intracellular heme content by strengthening the heme synthesis pathway of E. coli itself.
Fig. 1-2: The effect of supplements on the Olep catalysis
Research
Based on the literature, we divided the synthesis pathway of heme into two parts, one is the synthesis pathway of heme precursor ALA (upstream), and the other part is the synthesis pathway of ALA to heme (downstream).
For the upstream pathway, there were two pathways from glutamate to ALA, one was the C4 pathway and the other was the C5 pathway. The C4 pathway had been enhanced by the existing team, and the experimental results showed that the effect was not as good as the C5 pathway, so we chose the C5 pathway for modification. The C5 pathway had two key genes, one was hemA and the other was hemL.
The downstream pathway, that was, the synthesis pathway of heme, involved 7 genes, of which 4 genes (hemB, hemD, hemC, hemH) were more critical, according to the literature.
Fig. 1-3: Heme biosynthetic pathways in E. coli. The purple arrow represents the C5 pathway and the pink arrow represents the downstream biosynthetic pathway of heme. The pCDFDuet-hemA-hemL plasmid was constructed to enhance the C5 pathway; the pETDuet-hemBDC-hemH plasmid was constructed to enhance the downstream biosynthetic pathway
Design
We planned to set up three sets of experiments: 1: overexpression of hemA and hemL; 2: overexpression of hemB, hemD, hemC, and hemH; 3: overexpression of hemA and hemL together with hemB, hemD, hemC, and hemH.
Build
We constructed plasmids containing hemA and hemL, and overexpressed them by transforming recombinant plasmids to E. coli. We also constructed and overexpressed plasmids containing hemB, hemD, hemC, and hemH.
Therefore, 3 recombinant strains were constructed, E. coli AL strain(The recombinant plasmid pCDFDuet-hemA-hemL was expressed in E. coli O2), E. coli BCDH strain (The recombinant plasmid pETDuet-hemB-hemC-hemD-hemH was expressed in E. coli O2), E. coli AL-BCDH strain(The recombinant plasmids pCDFDuet-hemA-hemL and pETDuet-hemB-hemC-hemD-hemH were expressed in E. coli O2).
hemA (BBa_K4759020) and hemL (BBa_K4759025) were expressed in BBa_K4759210, hemH (BBa_K4759024) was expressed in BBa_K4759211. hemB (BBa_K4759021) /hemC (BBa_K4759022) /hemD (BBa_I716155) were expressed in BBa_K4759270.
Test
Samples were handled in the same way as the previous iteration.
Heme concentration was measured by UHPLC-QTOF-MS (Agilent 1290-6495C).
Learn
The experimental results showed that, without the addition of ALA and FeCl3, the heme binding rate of Olep increased to 53.9%, and the conversion rate of deoxycholic acid increased to 41.4%.
Fig. 1-4: The effect of enhancing heme biosynthesis on the Olep catalysis
Fig. 1-5: The color of engineered strains and pure enzyme. 1: E. coli O1 strain; 2: E. coli O2 strain; 3: E. coli O2 strain cultivated with ALA and FeCl3; 4: E. coli AL strain; 5: Olep enzyme purified from E. coli AL strain. The lower values represent the content of intracellular heme in different engineered strains
The P450 enzyme requires electrons (NADH, NADPH) to function. Electrons are first transferred to FDR (ferredoxin reductase), to FDX (ferro-reducing protein), and then to P450 enzyme under the action of redox partners, and finally to the heme center, after which the enzyme can react with the substrate and undergo hydroxylation.
Redox partners play an important role in the electron transfer of P450 enzymes, and different redox partners have different electron transfer roles.
Therefore, we hoped to find a pair of redox partners that were well adapted to Olep to improve its catalytic activity.
Research
Through extensive reading of the literature, we summarized 11 pairs of redox partners with good results. After that, we screened four pairs of redox partners with good effects by molecular docking and mathematical modeling and then verified them experimentally.
Generally, the method of determining whether the redox partners was suitable required tedious steps such as the construction of plasmids, heterologous expression, construction of catalytic systems, and detection of conversion rate after catalysis. Therefore, we wanted to find a convenient way to do a quick screening.
Design
To rapidly screen the redox partners that were most suitable for Olep, a novel redox patner sensor was constructed based on the bimolecular fluorescence complementation technique.
We divided sfGFP into N-terminal and C-terminal, and although these two parts were cut off, there was an interaction force between them.
Fig. 2-1: The scheme of constructing sfGFP sensor
Fig. 2-2: The self-assembly of Olep and Fdx based on the three-dimensional structure of sfGFP (PDB: 5BT0)
Fig. 2-3: BL21 morphology diagram seen under excitation light 488nm and emitted light 520nm, green is green fluorescence of sfGFP
Build
Thus, four iron redox proteins were fused to the N-terminal of sfGFP-1-10 and Olep to the C-terminal of sfGFP-11, respectively, to obtain the recombinant plasmid pRSFDuet-BM3-GFP-1-10-GFP-11-oleP, pRSFDuet-camA-camB-GFP-1-10-GFP-11-oleP, pRSFDuet-FdR_0978-Fdx_1499-GFP-1-10-GFP-11-oleP, and pRSFDuet-petH-petF-GFP-1-10-GFP-11-oleP.
The above four recombinant plasmids were converted to BL21(DE3) to obtain recombinant strains G2 to G5, respectively.
| BBa_K4759212 | composite | T7-RBS4-GFP11-linker-Olep |
| BBa_K4759214 | composite | T7-RBS1-petH-RBS2-petF-linker-GFP1-10 |
| BBa_K4759215 | composite | T7-RBS1-camA-RBS2-camB-linker-GFP1-10 |
| BBa_K4759217 | composite | T7-RBS1-Fdr-0978-RBS2-Fdx-1499-linker-GFP1-10 |
| BBa_K4759219 | composite | T7-RBS1-BM3-linker-GFP1-10 |
Fig. 2-4: Screening proper redox partners for Olep from different sources
Test
The recombinant strains G2 to G5 were subjected to shaker fermentation experiments. After the fermentation was completed, 200 uL bacteria were added to the 96-well plate with a microplate reader to determine biomass (wavelength 600 nm) and fluorescence value (excitation wavelength 488 nm, emission wavelength 520 nm). Then we calculated the fluorescence intensity (fluorescence value/biomass) of the strains.
Learn
The fluorescence intensity of the recombinant strain G5 (containing recombinant plasmid pRSFDuet-petH-petF-GFP-1-10-GFP-11-olep) was the highest (1.2×106) and 6 times higher than that of the control strain G2 (containing recombinant plasmid pRSFDuet-camA-camB-GFP-1-10-GFP-11-olep).
We confirmed the feasibility of the redox partner sensor, however, their reliability needed to be further explored.
Fig. 2-5: The G1 strain that contains the empty pRSFDuet-1 plasmid was used as a control. The fluorescent intensities were calculated and the color of cells and fluorescent images were presented for G2-G5 strains that express different redox partners-sfGFP-1-10 and sfGFP-11-Olep, respectively. The blue-filled triangle represents the fluorescent intensity/OD600.
Design
In order to verify the reliability of the sensor, the catalysis experiment was necessary.
In the previous iteration, the sfGFP gene was only used to detect fluorescence. Therefore, the sfGFP gene in the plasmid used for catalysis should be removed so as not to affect the experimental results.
Build
We selected four conventional redox partners (BM3, CamA/CamB, SelFdR0978/SelFdx1499, PetH/PetF) in combination with the P450 enzyme. Four groups of redox partners were constructed on the high-copy plasmid pRSFDuet to obtain recombinant plasmids: pRSFDuet-BM3-olep, pRSFDuet-camA-camB-olep, pRSFDuet-FdR0978-Fdx1499-olep, and pRSFDuet-petH-petF-olep. Then they were transformed to C41 (DE3) to obtain the recombinant strain R2 to R5.
| BBa_K4759203 | composite | T7-RBS4-Olep |
| BBa_K4759218 | composite | T7-RBS1-BM3 |
| BBa_K4759201 | composite | T7-RBS1-camA-RBS2-camB |
| BBa_K4759216 | composite | T7-RBS1-Fdr-0978-RBS2-Fdx-1499 |
| BBa_K4759218 | composite | T7-RBS1-BM3 |
| BBa_K4759213 | composite | T7-RBS1-petH-RBS2-petF |
Test
The recombinant strains R2 to R5 were subjected to shaker fermentation experiments.
HPLC assay for product generation.
Calculation of conversion rate = Concentration of product (mg/mL)/Concentration of initially added substrate (mg/mL) × 100%.
Learn
The recombinant strain R5 (containing recombinant plasmid pRSFDuet-petH-petF-olep) had the highest conversion rate, which significantly increased from 41.4% to 85.6%.
Therefore, the redox partners PetH/PetF derived from Synechocystis was successfully screened as the most suitable redox partners for the P450 enzyme Olep, and the construction of the sfGFP sensor was verified, which could efficiently and accurately screen the redox partner adapted by the P450 enzyme.
Fig. 2-6: The conversion rates were calculated for R2-R5 strains that express different redox partners and Olep, respectively. The R1 strain that contains the empty pRSFDuet-1 plasmid was used as a control. The red hollow triangle represents the conversion rate (%). Values and triangles represent the means and standard deviations of biological triplicates
Design
In the previous cycle, we explored to obtain the optimal redox partners PetF and PetH. The fusion combination strategy of redox partners can help to improve the catalytic activity of Olep. To further screen the optimal redox partner, we decided to make a fusion combination of PetF\PetH.
Build
We adopted the following strategies and all recombinant plasmid were transformed to C41 (DE3),
respectively:
recombinant strains R6 (PetH, PetF, and Olep were fused by two linkers);
recombinant strains R7 (petH and petF were fused by a linker);
recombinant strains R8 (PetH and PetF was constructed on pACYCDuet, while oleP expression gene was
constructed on pRSFDuet);
recombinant strains R9 (petH and petF were fused by a linker, and were constructed on pACYCDuet, while the
oleP expression gene was constructed on pRSFDuet).
| BBa_K4759220 | composite | T7-RBS1-petH-linker-petF-linker-Olep |
| BBa_K4759221 | composite | T7-RBS1-petH-linker-petF |
Fig. 2-7: The fusion expression and different expressional ratio between Olep and PetH/PetF
Test
Recombinant strains R6 to R9 were subjected to shake flask fermentation.
HPLC assay for product generation.
Calculation of conversion rate = Concentration of product (mg/mL)/Concentration of initially added substrate (mg/mL) × 100%.
Learn
The transformation rate of recombinant strains R6 to R9 were lower than that of the recombinant strain R5.
Fig2-8: The conversion rate of DCA for R5-R9 strains. The blue-filled triangle represents the biomass (OD600)
Design
After obtaining the best redox partners PetH/PetF, we performed alanine scanning on petF to speculate which sites had a greater impact on its electron transport capacity. Finally, we found that after mutations in seven of them, the electron transport effect would change greatly, so we mutated the amino acids of these sites into other 19 amino acids by modeling, and selected 23 of them to get better results.
Build
We fused the mutants to the N-terminal of sfGFP-1-10 and Olep to the C-terminal of sfGFP-11 to obtain the recombinant plasmids.
The mutant strains D21Y, D21F, D21W, D58E, D58W, D58Y, D58F, D61I, D61T, D61V, D61R, Q62W, Q62E, Q62Y, Q62F, F64P, F64I, F64D, D67W, D67Y, D67F, D68P, D68I were obtained by transforming the recombinant plasmids into BL21 (DE3), respectively. BL21(DE3), which was transformed into the empty plasmid, was used as the control group.
(Site-directed Mutagenesis parts can be search from BBa_K4759053 to BBa_K4759075, and constructed modeling screening for redox partners can be search from BBa_K4759076 to BBa_K4759099)
Test
Measurement methods were the same as in Cycle 2.
The fluorescence intensity (fluorescence value/biomass) of the strains were calculated.
Learn
Among all the 23 mutant strains, strain D58Y, D58F, D61I, D61T, Q62E, F64P, F64I, F64D, D68P exhibited higher fluorescence intensity compared to the unmodified strain.
Fig2-9: Fluorescence intensity of wild type with 23 mutants
Design
We designed a screen from these mutants with high fluorescence values. The sfGFP gene in the plasmid used for catalysis was removed so as not to affect the experimental results.
Build
Nine sets of mutants were subcloned to high-copy plasmid pRSFDuet to obtain recombinant plasmids, respectively.
(From BBa_K4759223 to BBa_K4759245)
And they were transformed to C41 (DE3) to obtain mutant strains.
Test
Mutant strains were subjected to shake flask fermentation.
HPLC assay for product generation.
Calculation of conversion rate = Concentration of product (mg/mL)/Concentration of initially added substrate (mg/mL) × 100%.
Fig. 2-10: 9 mutants + wild-type + negative control, 50 ml/250 ml system fermentation
Learn
Mutant strain D68P showed the highest conversion rate, which was increased from 85.6% to 89.2%.
Through a sequence of experiments, we successfully screened the most suitable redox partners and improved the catalytic efficiency of Olep!
Fig. 2-11: Conversion of the 9 mutants with the highest fluorescence intensity with wild type
