iGEM 2023 — Team jiangnan-china

NOTEBOOK

NOTEBOOK

↑

WEEK

01

11.13

Instructor’s speech
The brief introduction of the team members.
Classification into five groups, including wet lab, dry lab, human practice, network, and design.

Safety training
Our advisor trained us on experimental safety, including proper use of the instrument, proper wear in the lab,
and other considerations.

![](https://static.igem.wiki/teams/4759/wiki/note01.jpg)

strategy development:

Get access to necessary articles to have a full understanding of the project;
Summarize the difficulty of hydroxylating steroid substance via chemical synthesis;

WEEK

02

11.21

* Experiment preparation:
  (1) Configure the TB medium

  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
  | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
  | Integrant | Dosage || Tryptone | 12 g/L |
  | Glycerinum | 5 g/L |
  | Yeast extract | 24 g/L |
  | KH2PO4 | 17 mmol/L |
  | K2HPO4 | 72 mmol/L |
  | 121℃ sterilization for 15 min | |

  (2) Configure the LB medium

  |  |  |  |  |  |  |  |  |  |  |
  | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
  | Integrant | Dosage/th>|  |  | | --- | --- | | Tryptone | 10 g/L | | Yeast extract | 5 g/L | | NaCl | 10 g/L | | 121℃ sterilization for 15 min | | |

  11.25
* (1) Clean reagent bottles
* (2) Insert pipette tips

![](https://static.igem.wiki/teams/4759/wiki/note02.jpg)

strategy development:

Review of literature related to hydroxylation of steroids;
Turn to biosynthesis for help and discover an appropriate enzyme that is important to hydroxylate the site we
want.
Decide to use CYP107D1 (Olep) to hydroxylate steroids.

WEEK

03-04

11.27

Construction of the strain *E. coli* O1.

12.1

Full wavelength scanning of Olep

12.4

SDS-PAGE analysis of crude enzymes, intracellular P450 enzymes, intracellular and extracellular P450 enzymes

12.8

Explore TLC methods for detecting DCA and 6β-OH DCA
HPLC test product content

![](https://static.igem.wiki/teams/4759/wiki/contribution/notebook-3-new.png)

TLC results for DCA and 6β-OH DCA

WEEK

05-09

Increase the soluble expression of Olep

12.12-12.21: Screening of
different plasmid
vectors

12.12

* Construct plasmids:
* pETDuet-camA-camB-oleP;
* pACYCDuet-camA-camB-oleP;
* pRSFDuet-camA-camB-oleP.

12.14

* Plasmid extraction
* The constructed plasmid was sent to the company for sequencing.

![](https://static.igem.wiki/teams/4759/wiki/note04.jpg)

12.18

* Whole-cell-catalyzed reactions(based on final concentration): Strain cell OD600=30, deoxycholic
  acid 1
  mg/mL, NADPH 1 mM, glucose dehydrogenase 1 U/mL.

12.20

* TLC test product content
* HPLC test product content

![](https://static.igem.wiki/teams/4759/wiki/note05.jpg)

12.22-27: Screening of host cells

12.22

* Transfection plasmid pRSFDuet-camA-camB-oleP into different host cells

12.26

* TLC methods detect DCA and 6β-OH DCA

12.28 – 1.7：Screening for
pro-soluble labels

12.29

* Construct plasmids: PCR
* SUMO-linker-Olep
* MBP-linker-Olep
* TF-linker-Olep
* Trx-linker-Olep
* Nus-linker-Olep
* GST-linker-Olep

12.31

* The constructed plasmid was sent to the company for sequencing.

1.2

* Some strains didn't grow very well, so the agar streak culture was re-performed.
  For those strains that grow well, we selected some strains with complete shapes on each plate to be sent for
  sequencing.

1.4

* The strains were still not growing very well. We suspect this may be because the receptive cells have been
  preserved for too long. So we reprepared the receptive cells.

1.8

* The growth of the strain was significantly improved compared with that before

1.11

* Whole-cell-catalyzed reactions

1.13-1.18: Optimization of culture conditions

1.15

* Transfer to shaker and culture, then induce.
  When inducing, we culture the cells respectively at 16 °C, 20 °C, 25 °C, and 30 °C.
  We control the inducer(IPTG) concentration at 0.1mmol/L, 0.25mmol/L, 0.5 mmol/L, 0.75 mmol/L, and 1.0
  mmol/L,
  respectively

1.17

* TLC test product content

![](https://static.igem.wiki/teams/4759/wiki/contribution/notebook-6-new.png)

WEEK

11

Spring Festival (Chinese New Year)!

WEEK

12-16

Increase intracellular heme supply

1.30-2.5：Measurement of heme binding efficiency of MBP and TF

1.30

* Agar streak: The previously preserved MBP-oleP and TF-oleP recombinant strains were removed and marked on
  LB
  medium for activation
  Cultured for 12h, then inoculated into liquid LB medium

2.2

* Whole-cell-catalyzed reactions

2.4

* Measured the heme concentration by UHPLC-QTOF-MS

2.6-2.12：Exogenous addition of heme and its precursor

2.8

* During induction, added 0.025 mL of inducer isopropyl-β-d-thiogalactoside (1 M IPTG, final concentration
  0.5
  mM). Then added 5-amino-ketovaleric acid (ALA)＋FeSO4
  ，5-amino-ketovaleric acid (ALA)＋FeCl3, heme, respectively.
  Then the culture was continued at 25℃ for 24-32 h.

2.11

* TLC detected product content;
* HPLC detected product content;

2.13-2.25: Enhancing heme biosynthetic pathway

2.14

* Construct plasmids: PCR
* pCDFDuet-hemA-hemL;
* pETDuet-hemB-hemC-hemD-hemH;

2.20

* Transfer to shaker and culture, then induce.

2.23

* Whole-cell-catalyzed reactions

2.24

* TLC test product generation

![](https://static.igem.wiki/teams/4759/wiki/noteg.png)

WEEK

17-25

Screening and modification of redox partners

Week 17 - Week 19: Redox partner Sensor Construction

3.15

* Construct plasmids: PCR
* pRSFDuet-BM3-GFP-1-10-GFP-11-olep;
* pRSFDuet-camA-camB-GFP-1-10-GFP-11-olep;
* pRSFDuet-FdR0978- Fdx1499-GFP-1-10-GFP-11-olep;
* pRSFDuet-petH-petF-GFP-1-10-GFP-11-olep.

3.18

* Plasmid extraction, send them to the company for sequencing

3.22

* After the fermentation was completed, add 200 uL of the bacterium to a 96-well plate to determine the
  biomass
  (wavelength 600 nm) and fluorescence value (excitation wavelength 488 nm, emission wavelength 520 nm) using
  an
  enzyme labeler.

![](https://static.igem.wiki/teams/4759/wiki/note08.jpg)

Week 20 - Week 22: Verifying the reliability of the redox partner sensor

3.25

* Construct plasmids: PCR
* pRSFDuet-BM3-olep;
* pRSFDuet-camA-camB-olep;
* pRSFDuet-FdR0978-Fdx1499-olep;
* pRSFDuet-petH-petF-olep.

3.26

* The target gene fragment is attached to the plasmid vector and then transferred into the host cell

3.29

* One plasmid (pRSFDuet-FdR0978-Fdx1499-olep) failed to build, rebuild

4.3

* Transfer to shaker and culture, then induce.

4.7

* HPLC test product content

Week 23 - Week 25: Redox partner fusion assortment

4.11

* Construct plasmids: PCR
* recombinant strains R6-R9

4.16

* Whole-cell-catalyzed reactions

WEEK

26-30

Construct the optimal whole-cell catalytic system

WEEK

31-42

Targeted mutate key sites on ferredoxin petF

6.20

* Construct plasmids: PCR

![](https://static.igem.wiki/teams/4759/wiki/note09.jpg)

Bands were detected by DNA gel electrophoresis

![](https://static.igem.wiki/teams/4759/wiki/contribution/notebook-10-new.png)

6.22

* The target gene fragment is attached to the plasmid vector and then transferred into the host cell

6.25

* The sequencing results of some plasmids were incorrect, so we tried different methods to attach DNA
  fragments to plasmids.

7.1

* The sequencing results of some plasmids were incorrect. This time we redesigned the primers and did PCR
  again.

7.6

* The sequencing results were correct. Agar streak, cultured 12 h

7.8

* Bacteria with complete shape were selected for sequencing.

7.9

* The sequencing results of some plasmids were incorrect. So we selected several strains from the original
  culture medium and sent them to the company for sequencing again.

7.10

* The sequencing results of some plasmids were still incorrect. So we repeated the agar streak.

7.11

* The chromogenic agent used for TLC is used up. Reconfigure.

| Integrant | Dosage |
| --- | --- |
| H2O | 225 mL |
| Ammonium molybdate tetrahydrate | 12.5 g |
| Cerous molybdate | 2.5 g |
| H2SO4 | 25 mL |

7.13

* The sequencing results of several strains were incorrect. However, the sequence comparison results showed
  that only a few nucleotides were inconsistent, so we selected several strains from the original plate and
  sent them with the previous strains for sequencing.

7.15

* It turned out that the previous sequencing results were wrong. All strains had been constructed, and the
  agar streak began.

7.17

* Transfer to shaker and culture, then induce.

7.20

* After the fermentation was completed, 200uL of the bacterium was added to a 96-well plate to determine the
  biomass (wavelength 600 nm) and fluorescence value (excitation wavelength 488 nm, emission wavelength 520
  nm) using an enzyme labeler.

![](https://static.igem.wiki/teams/4759/wiki/note11.jpg)

7.30

* We counted all the fluorescence intensities, and selected the recombinant strains with strong fluorescence
  intensity. The primers were redesigned for catalysis.

8.3

* Construct plasmids: PCR

8.5

* The target gene fragment is attached to the plasmid vector and then transferred into the host cell. This
  time we took the previous lesson and used a variety of enzymes to link.

8.7

* The sequencing results were incorrect, so we attached DNA fragments to plasmids again.

8.10

* The sequencing results were correct. Agar streak, cultured 12h.

8.12

* We selected some strains with complete shapes on each plate to be sent for sequencing.

8.14

* The sequencing results of several strains were incorrect, so we selected several strains from the original
  plate and sent them with the previous strains for sequencing. At the same time, we also attached DNA
  fragments to plasmids again.

8.17

* The sequencing results of the reconstructed plasmids were satisfactory. Agar streak, cultured 12h.

8.20

* The results of strain sequencing were correct, all strains had been constructed, and the agar streak
  began.

![](https://static.igem.wiki/teams/4759/wiki/note12.jpg)

8.21

* Transfer to shaker culture.

![](https://static.igem.wiki/teams/4759/wiki/note13.jpg)

9 mutants + wild type + negative control were fermented in 50 ml/250 ml system

8.23

* Whole-cell-catalyzed reactions.

![](https://static.igem.wiki/teams/4759/wiki/note14.jpg)

8.24

* TLC test product content
  200 uL methanol redissolved sample was centrifuged at 14000 rpm for 20 min,
  for the liquid phase and TLC

![](https://static.igem.wiki/teams/4759/wiki/note15.jpg)

8.26

* HPLC test product content

ENGINEERING

ENGINEERING

↑

* Outline
* Enhancing the heme supply in *E. coli* to increase
  conversion
  rate
  + Cycle1
  + Cycle2
  + Cycle3
* Screening and modification of redox partners
  + Cycle1
  + Cycle2
  + Cycle3
  + Cycle4
  + Cycle5

![](https://static.igem.wiki/teams/4759/wiki/robot/head.png)
![](https://static.igem.wiki/teams/4759/wiki/robot/gear-left.png)
![](https://static.igem.wiki/teams/4759/wiki/robot/gear-right.png)
![](https://static.igem.wiki/teams/4759/wiki/robot/body.png)
![](https://static.igem.wiki/teams/4759/wiki/robot/hand-left.png)
![](https://static.igem.wiki/teams/4759/wiki/robot/hand-right.png)
![](https://static.igem.wiki/teams/4759/wiki/robot/foot-left.png)
![](https://static.igem.wiki/teams/4759/wiki/robot/foot-right.png)

Outline

"Engineering success can be achieved by making an effort to follow the engineering design cycle: Design →
Build → Test → Learn → Design..."

![](https://static.igem.wiki/teams/4759/wiki/engineering/group-368.png)

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.

Enhancing the heme supply in *E. coli* to increase conversion
rate

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.

Measurement of heme binding efficiency of MBP and TF

Cycle1

![](https://static.igem.wiki/teams/4759/wiki/engineering/frame.png)

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.

![](https://static.igem.wiki/teams/4759/wiki/engineering/frame-1.png)

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.

![](https://static.igem.wiki/teams/4759/wiki/engineering/frame-2.png)

Build

P450 enzyme CYP107D1 (Olep), derived from *Streptomyces antibioticus*, was synthesized after codon
optimization ([BBa\_K4759001](http://parts.igem.org/Part%3ABBa_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](https://example.com/your-link); [BBa\_K4759206](https://example.com/your-link).

![](https://static.igem.wiki/teams/4759/wiki/engineering/frame-3.png)

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).

![](https://static.igem.wiki/teams/4759/wiki/engineering/frame-4.png)

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.

![](https://static.igem.wiki/teams/4759/wiki/es1-1-3-1.png)

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.

Supplementing heme or the essential precursor of heme

Cycle2

![](https://static.igem.wiki/teams/4759/wiki/engineering/frame.png)

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*.

![](https://static.igem.wiki/teams/4759/wiki/engineering/frame-1.png)

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.

![](https://static.igem.wiki/teams/4759/wiki/engineering/frame-2.png)

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.

![](https://static.igem.wiki/teams/4759/wiki/engineering/frame-3.png)

Test

Samples were handled in the same way as the previous iteration.

The heme concentration was measured by UHPLC-QTOF-MS (Agilent 1290-6495C).

![](https://static.igem.wiki/teams/4759/wiki/engineering/frame-4.png)

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.

![](https://static.igem.wiki/teams/4759/wiki/es1-2-3-2.png)

Fig. 1-2: The effect of supplements on the Olep catalysis

Endogenous synthesis of the heme

Cycle3

![](https://static.igem.wiki/teams/4759/wiki/engineering/frame.png)

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.

![](https://static.igem.wiki/teams/4759/wiki/es1-3-3-3.png)

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

![](https://static.igem.wiki/teams/4759/wiki/engineering/frame-1.png)

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*.

![](https://static.igem.wiki/teams/4759/wiki/engineering/frame-2.png)

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](http://parts.igem.org/Part%3ABBa_K4759020)) and *hemL* ([BBa\_K4759025](http://parts.igem.org/Part%3ABBa_K4759025)) were expressed in [BBa\_K4759210](http://parts.igem.org/Part%3ABBa_K4759210), *hemH* ([BBa\_K4759024](http://parts.igem.org/Part%3ABBa_K4759024)) was expressed in [BBa\_K4759211](http://parts.igem.org/Part%3ABBa_K4759211). *hemB* ([BBa\_K4759021](http://parts.igem.org/Part%3ABBa_K4759021)) /*hemC* ([BBa\_K4759022](http://parts.igem.org/Part%3ABBa_K4759022)) /*hemD* ([BBa\_I716155](http://parts.igem.org/Part%3ABBa_I716155)) were expressed in [BBa\_K4759270](http://parts.igem.org/Part%3ABBa_K4759270).

![](https://static.igem.wiki/teams/4759/wiki/engineering/frame-3.png)

Test

Samples were handled in the same way as the previous iteration.

Heme concentration was measured by UHPLC-QTOF-MS (Agilent 1290-6495C).

![](https://static.igem.wiki/teams/4759/wiki/engineering/frame-4.png)

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%.

![](https://static.igem.wiki/teams/4759/wiki/es1-4-3-5.png)

Fig. 1-4: The effect of enhancing heme biosynthesis on the Olep catalysis

![](https://static.igem.wiki/teams/4759/wiki/es1-5-3-4.png)

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

Screening and modification of redox partners

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.

Constructing a sfGFP sensor to screen redox partners

Cycle1

![](https://static.igem.wiki/teams/4759/wiki/engineering/frame.png)

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.

![](https://static.igem.wiki/teams/4759/wiki/engineering/frame-1.png)

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.

![](https://static.igem.wiki/teams/4759/wiki/es2-1-4-1.png)

Fig. 2-1: The scheme of constructing sfGFP sensor

![](https://static.igem.wiki/teams/4759/wiki/es2-2-4-2.png)

Fig. 2-2: The self-assembly of Olep and Fdx based on the three-dimensional
structure of sfGFP (PDB: 5BT0)

![](https://static.igem.wiki/teams/4759/wiki/es2-3-4-3.png)

Fig. 2-3: BL21 morphology diagram seen under excitation light 488nm and
emitted light 520nm, green is green fluorescence of sfGFP

![](https://static.igem.wiki/teams/4759/wiki/engineering/frame-2.png)

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.

The following table is to show
parts used in this section.

|  |  |  |
| --- | --- | --- |
| [BBa\_K4759212](http://parts.igem.org/Part%3ABBa_K4759212) | composite | T7-RBS4-GFP11-linker-Olep |
| [BBa\_K4759214](http://parts.igem.org/Part%3ABBa_K4759214) | composite | T7-RBS1-petH-RBS2-petF-linker-GFP1-10 |
| [BBa\_K4759215](http://parts.igem.org/Part%3ABBa_K4759215) | composite | T7-RBS1-camA-RBS2-camB-linker-GFP1-10 |
| [BBa\_K4759217](http://parts.igem.org/Part%3ABBa_K4759217) | composite | T7-RBS1-Fdr-0978-RBS2-Fdx-1499-linker-GFP1-10 |
| [BBa\_K4759219](http://parts.igem.org/Part%3ABBa_K4759219) | composite | T7-RBS1-BM3-linker-GFP1-10 |

![](https://static.igem.wiki/teams/4759/wiki/es-2-4.png)

Fig. 2-4: Screening proper redox partners for Olep from different sources

![](https://static.igem.wiki/teams/4759/wiki/engineering/frame-3.png)

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.

![](https://static.igem.wiki/teams/4759/wiki/engineering/frame-4.png)

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.

![](https://static.igem.wiki/teams/4759/wiki/es2-5.png)

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.

Testing the catalytic capacity of different redox partners

Cycle2

![](https://static.igem.wiki/teams/4759/wiki/engineering/frame-1.png)

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.

![](https://static.igem.wiki/teams/4759/wiki/engineering/frame-2.png)

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.

The following table is to show
parts used in this section.

|  |  |  |
| --- | --- | --- |
| [BBa\_K4759203](http://parts.igem.org/Part%3ABBa_K4759203) | composite | T7-RBS4-Olep |
| [BBa\_K4759218](http://parts.igem.org/Part%3ABBa_K4759218) | composite | T7-RBS1-BM3 |
| [BBa\_K4759201](http://parts.igem.org/Part%3ABBa_K4759201) | composite | T7-RBS1-camA-RBS2-camB |
| [BBa\_K4759216](http://parts.igem.org/Part%3ABBa_K4759216) | composite | T7-RBS1-Fdr-0978-RBS2-Fdx-1499 |
| [BBa\_K4759218](http://parts.igem.org/Part%3ABBa_K4759218) | composite | T7-RBS1-BM3 |
| [BBa\_K4759213](http://parts.igem.org/Part%3ABBa_K4759213) | composite | T7-RBS1-petH-RBS2-petF |

![](https://static.igem.wiki/teams/4759/wiki/engineering/frame-3.png)

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%.

![](https://static.igem.wiki/teams/4759/wiki/engineering/frame-4.png)

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.

![](https://static.igem.wiki/teams/4759/wiki/es2-6.png)

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

The fusion expression and different expressional ratio between Olep and
PetH/PetF

Cycle3

![](https://static.igem.wiki/teams/4759/wiki/engineering/frame-1.png)

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.

![](https://static.igem.wiki/teams/4759/wiki/engineering/frame-2.png)

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).

The following table is to show parts used in this
section.

|  |  |  |
| --- | --- | --- |
| [BBa\_K4759220](http://parts.igem.org/Part%3ABBa_K4759220) | composite | T7-RBS1-petH-linker-petF-linker-Olep |
| [BBa\_K4759221](http://parts.igem.org/Part%3ABBa_K4759221) | composite | T7-RBS1-petH-linker-petF |

![](https://static.igem.wiki/teams/4759/wiki/es2-7-4-6.png)

Fig. 2-7: The fusion expression and different expressional ratio between
Olep and PetH/PetF

![](https://static.igem.wiki/teams/4759/wiki/engineering/frame-3.png)

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%.

![](https://static.igem.wiki/teams/4759/wiki/engineering/frame-4.png)

Learn

The transformation rate of recombinant strains R6 to R9 were lower than that of the recombinant strain
R5.

![](https://static.igem.wiki/teams/4759/wiki/es2-8-4-7.png)

Fig2-8: The conversion rate of DCA for R5-R9 strains. The blue-filled
triangle represents the biomass (OD600)

Semi-rational design of redox partner PetH/PetF

Cycle4

![](https://static.igem.wiki/teams/4759/wiki/engineering/frame-1.png)

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.

![](https://static.igem.wiki/teams/4759/wiki/engineering/frame-2.png)

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](http://parts.igem.org/Part%3ABBa_K4759053) to [BBa\_K4759075](http://parts.igem.org/Part%3ABBa_K4759075), and constructed modeling screening for
redox partners can be search from [BBa\_K4759076](http://parts.igem.org/Part%3ABBa_K4759076) to [BBa\_K4759099](http://parts.igem.org/Part%3ABBa_K4759099))

![](https://static.igem.wiki/teams/4759/wiki/engineering/frame-3.png)

Test

Measurement methods were the same as in Cycle 2.

The fluorescence intensity (fluorescence value/biomass) of the strains were calculated.

![](https://static.igem.wiki/teams/4759/wiki/engineering/frame-4.png)

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.

![](https://static.igem.wiki/teams/4759/wiki/es2-9.png)

Fig2-9: Fluorescence intensity of wild type with 23 mutants

Testing the catalytic capacity of different mutants

Cycle5

![](https://static.igem.wiki/teams/4759/wiki/engineering/frame-1.png)

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.

![](https://static.igem.wiki/teams/4759/wiki/engineering/frame-2.png)

Build

Nine sets of mutants were subcloned to high-copy plasmid pRSFDuet to obtain recombinant plasmids,
respectively.

(From [BBa\_K4759223](http://parts.igem.org/Part%3ABBa_K4759223) to [BBa\_K4759245](http://parts.igem.org/Part%3ABBa_K4759245))

And they were transformed to C41 (DE3) to obtain mutant strains.

![](https://static.igem.wiki/teams/4759/wiki/engineering/frame-3.png)

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%.

![](https://static.igem.wiki/teams/4759/wiki/es2-10.png)

Fig. 2-10: 9 mutants + wild-type + negative control, 50 ml/250 ml system
fermentation

![](https://static.igem.wiki/teams/4759/wiki/engineering/frame-4.png)

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!

![](https://static.igem.wiki/teams/4759/wiki/es2-11.png)

Fig. 2-11: Conversion of the 9 mutants with the highest fluorescence
intensity with wild type


RESULTS

RESULTS

↑

* The characteristics of
  expressed Olep in E. coli.
  + The expression of Olep
  + The soluble expression of Olep
  + Identification of whole-cell
    biocatalytic products
* The suitable strategy to
  express enough Olep for the hydroxylation of DCA in *E. coli*
  + Selection of suitable plasmid
  + Selection of proper host
  + The application of protein tags
  + The co-expression of molecular
    chaperones
  + Optimization of induction conditions
  + Conclusion
* Enhancing the heme supply
  in E. coli to increase the conversion rate
  + Discovery of the problem
  + Supplementing heme or the essential
    precursor of heme
  + Enhancing heme biosynthetic
    pathway
* Screening and
  modification of the optimal redox partners for OleP by sfGFP and semi-rational design
  + Building a redox partner library
  + Constructing a sfGFP sensor to screen
    redox partner
  + Testing the catalytic capacity of
    different redox partners
  + The fusion expression and different
    expressional ratio between OleP and PetH/PetF.
  + Semi-rational design of redox
    partner PetH/PetF
* The optimal whole-cell
  catalytic system for Olep to perform most efficient conversion
  + The catalytic form
  + The biomass of resting whole-cell
  + The concentration of substrate DCA
  + Catalytic time
  + The catalytic time
* Constructing an
  intracellular NADPH regeneration system

![](https://static.igem.wiki/teams/4759/wiki/robot/head.png)
![](https://static.igem.wiki/teams/4759/wiki/robot/gear-left.png)
![](https://static.igem.wiki/teams/4759/wiki/robot/gear-right.png)
![](https://static.igem.wiki/teams/4759/wiki/robot/body.png)
![](https://static.igem.wiki/teams/4759/wiki/robot/hand-left.png)
![](https://static.igem.wiki/teams/4759/wiki/robot/hand-right.png)
![](https://static.igem.wiki/teams/4759/wiki/robot/foot-left.png)
![](https://static.igem.wiki/teams/4759/wiki/robot/foot-right.png)

01

The characteristics of expressed Olep in *E. coli*.

1.1 The expression of Olep

① plasmid construction

P450 enzyme CYP107D1 (Olep), derived from *Streptomyces antibioticus*, was synthesized after codon
optimization [(BBa\_K4759001)](http://parts.igem.org/Part%3ABBa_K4759001) and
subcloned to the plasmid pET-28a to obtain the recombinant plasmid pET-28a-oleP[(BBa\_K4759202)](http://parts.igem.org/Part%3ABBa_K4759202). Since the CYP107 family
was a typical three-component P450 enzyme that
required a pair of redox partners to transfer electrons from the electron donor NAD(P)H to the P450 enzyme
activity center, ferredoxin reductase CamA and ferredoxin CamB were selected as redox partners. The
codon-optimized *camA* gene and *camB* gene were synthesized and subcloned to the plasmid
pACYCDuet to obtain
the recombinant plasmid pACYCDuet-camA/camB[(BBa\_K4759201)](http://parts.igem.org/Part%3ABBa_K4759201).

② Expression of Olep in a recombinant strain

The above two recombinant plasmids were then transformed into the *E. coli* expression host C43
(DE3) to
obtain one recombinant strain, named *E. coli* O1.

③ Enzyme purification and CO spectral assay

The purified Olep was obtained by Ni-NTA affinity chromatography. The
expressed Olep presented a characteristic peak at 450 nm after the reaction with sodium dithionite and
carbon monoxide. The results showed that Olep was an active P450 enzyme.

![](https://static.igem.wiki/teams/4759/wiki/results/1-1.png)

Fig. 1-1: Full wavelength scan of purified Olep in three states.
The blue, orange, and red circles mark the characteristic absorbance of the purified Olep, Olep-CO complex
and Olep-CO-sodium dithionite complex, respectively.

1.2 The soluble expression of Olep

The recombinant strain *E. coli* O1 was cultivated using TB culture and grown at 37 °C until an
OD600 value
of 0.6-0.8 was reached. 0.5 mM IPTG (isopropyl-β-D-thiogalactopyranoside) was added and the culture was
incubated for another 20 h at 25 °C.

After the fermentation was completed, the fermentation broth was centrifuged at 4°C, 8000 rpm for 10 min.
The bacteria were collected and washed with potassium phosphate buffer at pH 8.0. Then the bacteria were
resuspended with potassium phosphate buffer (containing 500 mM sodium chloride, and 20 mM imidazole) at pH
8.0 and performed cell disruption with an ultrasonic disruptor on ice. The procedure of the ultrasonic
crusher was as follows: POWER, 38%; 2 s on /3 s off; 5 min. The cell disruption solution was centrifuged
at 4°C, 8000 rpm for 10 min, and the supernatant was collected as Olep crude enzyme. Olep pure enzyme was
obtained by Ni-NTA affinity chromatography with a pH 8.0 potassium phosphate buffer (containing 500 mM
sodium chloride and 200 mM imidazole). The purified Olep was desalted with Amicon Ultra 30 K
ultrafiltration centrifuge tubes.
SDS-PAGE showed that the proportion of soluble Olep only can reach 32.4%.

![](https://static.igem.wiki/teams/4759/wiki/results/1-2.png)

Fig. 1-2: SDS-PAGE analysis of Olep. Lane 1, soluble expression of
Olep; Lane 2, inclusion body of Olep; Lane 3, purified soluble Olep. M, marker

1.3 Identification of whole-cell biocatalytic products

① Whole-cell biocatalysis

The washed bacteria were resuspended with potassium phosphate at pH 8.0 (glycerol containing 10% v/v) to be
the whole-cell catalyst.
The whole cell catalyzed reaction system was (in terms of final concentration): strain cell
OD600=30,
deoxycholic acid 1 mg/mL, NADPH 1 mM, glucose dehydrogenase 1 U/mL.

② The analysis of the hydroxylated product of DCA by TLC

Thin layer chromatography (TLC) is a type of chromatography used to separate mixtures by covering a glass
sheet, plastic sheet, or aluminum foil with a thin layer of adsorbent.
In our project, we explored TLC methods for detecting DCA and 6β-OH DCA. Among them, the formula of the
organic phase was a ratio of dichloromethane to methanol of 10:1. The color developer was CAM stain (225 mL
of water + 12.5 g ammonium molybdate tetrahydrate + 2.5 g of cerium molybdate, 25 mL of concentrated
sulfuric acid (slowly added in ice bath)).

![](https://static.igem.wiki/teams/4759/wiki/f1-3.png)

Fig. 1-3: TLC results for DCA and 6β-OH DCA
From left to right, DCA, 6β-OH DCA, and the mixture

③ The analysis of the hydroxylated product of DCA by HPLC

High-performance liquid chromatography (HPLC) is mainly used for the analysis of high boiling point
non-volatile, heat instability, and large molecular weight of organic compounds instrument equipment.
With HPLC testing, we found that the recombinant strain *E. coli* O1 (containing recombinant plasmids
pET28a-oleP and pACYCDuet-camA-camB) yields 0.048 mg/mL 6β-OH deoxycholic acid (conversion rate
4.8%).
Therefore, it was necessary to improve the soluble expression of Olep in *E. coli* to
improve the
efficiency
of obtaining 6β-OH deoxycholic acid.

![](https://static.igem.wiki/teams/4759/wiki/results/1-4.png)

Fig. 1-4: HPLC results of catalytic products

02

The suitable strategy to express enough Olep for the hydroxylation of DCA in *E. coli*

2.1 Selection of suitable plasmid

First, we selected high-copy plasmid pRSFDuet, medium-high copy plasmid pETDuet and pET28a, and low-copy
plasmid pACYCDuet to express Olep in *E. coli*. The results showed that the recombinant strain
constructed
with high-copy plasmid pRSFDuet grew better and its conversion rate increased to 29.4%.
In WT strain, Olep was expressed in [(BBa\_K4759202)](http://parts.igem.org/Part%3ABBa_K4759202)and CamA/CamB was expressed in [(BBa\_K4759201)](http://parts.igem.org/Part%3ABBa_K4759201); In the
plasmid pETDuet, Olep was expressed in [(BBa\_K4759203)](http://parts.igem.org/Part%3ABBa_K4759203) and CamA/CamB was expressed in [(BBa\_K4759201)](http://parts.igem.org/Part%3ABBa_K4759201); In the
plasmid pACYCDuet and pRSFDuet, Olep was expressed in [(BBa\_K4759203)](http://parts.igem.org/Part%3ABBa_K4759203) and CamA/CamB was expressed in
[(BBa\_K4759201)](http://parts.igem.org/Part%3ABBa_K4759201).

![](https://static.igem.wiki/teams/4759/wiki/results/2-1.png)

Fig. 2-1: Selection of suitable plasmid. WT represents the *E.
coli*O1
strain. The blue-filled triangle represents the biomass (OD600). The red hollow triangle
represents the
conversion rate (%). Values and triangles represent the means and standard deviations of biological
triplicates.

2.2 Selection of proper host

Subsequently, we selected *Escherichia coli* BL21 (DE3), C41(DE3), and C43(DE3) to express Olep.
The
results showed that C41(DE3) was the suitable expression host and the conversion rate of 6β-OH DCA was
increased to 31.7%.

![](https://static.igem.wiki/teams/4759/wiki/results/2-2.png)

Fig. 2-2: Selection of proper host

2.3 The application of protein tags

Next, we fused six protein tags (SUMO, GST, MBP, TF, Trx, Nus) at the N-terminal of the Olep. Protein
tags MBP and TF can significantly improve the solubility of Olep, but the catalytic efficiency of Olep was
reduced.

The expression framework of elements is shown in the following parts:[BBa\_K4759204](http://parts.igem.org/Part%3ABBa_K4759205)、
[BBa\_K4759205](http://parts.igem.org/Part%3ABBa_K4759205)、[BBa\_K4759206](http://parts.igem.org/Part%3ABBa_K4759206)、[BBa\_K4759207](http://parts.igem.org/Part%3ABBa_K4759207)、[BBa\_K4759208](http://parts.igem.org/Part%3ABBa_K4759208)、[BBa\_K4759209](http://parts.igem.org/Part%3ABBa_K4759209)

![](https://static.igem.wiki/teams/4759/wiki/results/2-3.png)

Fig. 2-3: The application of protein tags

2.4 The co-expression of molecular chaperones

Then, we co-expressed commonly used commercial chaperone plasmids (pGro7, pKJE7, pGKJE8, PTf16, and
pTf2) and plasmid pRSFDuet-camA-camB-oleP. However, these chaperones did not significantly increase the
expression of Olep, nor did they improve the catalytic efficiency of substrates.

![](https://static.igem.wiki/teams/4759/wiki/results/2-4.png)

Fig. 2-4: The co-expression of molecular chaperones

2.5 Optimization of induction conditions

Furthermore, the conditions of Olep expression were optimized. The results suggested that the lower
temperature (25°C) and moderate concentrations of IPTG (0.5 mM) were crucial for Olep expression (67.1%
solubility) and the highest conversion rate of 6β-OH DCA reached 34.8% at the optimal conditions.

![](https://static.igem.wiki/teams/4759/wiki/results/2-5.png)

Fig. 2-5: (A) The optimal induction temperature for Olep expression
(°C).
(B) The proper concentration of IPTG for Olep expression (mM).

2.6 Conclusions

By screening different copy number plasmids, *E. coli* expression host, protein tags, molecular
chaperones,
and optimizing of induction conditions, the recombinant strain *E. coli* O2 was constructed. The
expression
system of *E. coli* O2 was high-copy recombinant plasmid pRSFDuet-camA-camB-oleP, expressing host *E.
coli* C41(DE3), induction temperature 25°C, inducer IPTG concentration 0.5 mM.
The soluble expression of Olep reached 67.1%, and under the optimized conditions, 0.348 mg/mL
6β-OH
deoxycholic acid (conversion rate 34.8%) could be produced using a final concentration of 1
mg/mL of
deoxycholic acid.
Compared with *E. coli* O1, the soluble expression of Olep was increased by 51.7%, and the
catalytic efficiency was increased by 6.25 times.

03

Enhancing the heme supply in *E. coli* to increase the conversion rate

3.1 Discovery of the problem

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
.

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 were collected by centrifugation at 14,000 rpm for
20 min, and it was used to detect intracellular heme.

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
.
There were 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*.

![](https://static.igem.wiki/teams/4759/wiki/results/3-1.png)

Fig. 3-1: 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.

3.2 Supplementing heme or the essential precursor of heme

First, three different combinations of exogenous additions (direct addition of heme, addition of
precursor ALA+FeSO4, addition of precursor ALA+FeCl3) were tested. 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.

![](https://static.igem.wiki/teams/4759/wiki/results/3-2.png)

Fig. 3-2: The effect of supplements on the Olep catalysis

3.3 Enhancing heme biosynthetic pathway

We divided the synthesis pathway of heme into two parts, one was the synthesis pathway of heme premise
ALA
(upstream), and the other part was 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*. So we constructed
plasmids containing *hemA* and *hemL*
, and overexpressed them by transforming recombinant plasmids to *E. coli*.

For the downstream pathway, that was, the synthesis pathway of heme, it involved 7 genes, of which 4
genes
(*hemB*, *hemD*, *hemC*, *hemH*) were more critical, according to the literature.
Therefore,
we also
constructed
and overexpressed plasmids containing *hemB*, *hemD*, *hemC*, and *hemH*.

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. Therefore, we also
constructed
and overexpressed plasmids containing *hemB*, *hemD*, *hemC*, and *hemH*.

![](https://static.igem.wiki/teams/4759/wiki/results/3-3.png)

Fig. 3-3: 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

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](http://parts.igem.org/Part%3ABBa_K4759020)）and *hemL*（[BBa\_K4759025](http://parts.igem.org/Part%3ABBa_K4759025)）were
expressed in [BBa\_K4759210](http://parts.igem.org/Part%3ABBa_K4759210), *hemH*（[BBa\_K4759024](http://parts.igem.org/Part%3ABBa_K4759024)）was
expressed
in [BBa\_K4759211](http://parts.igem.org/Part%3ABBa_K4759211).
*hemB*（[BBa\_K4759021](http://parts.igem.org/Part%3ABBa_K4759021)）/*hemC*（[BBa\_K4759022](http://parts.igem.org/Part%3ABBa_K4759022)）/*hemD*（[BBa\_I716155](http://parts.igem.org/Part%3ABBa_I716155)）were
expressed in [BBa\_K4759270](http://parts.igem.org/Part%3ABBa_K4759270)

![](https://static.igem.wiki/teams/4759/wiki/results/3-4.png)

Fig. 3-4: 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 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%.

![](https://static.igem.wiki/teams/4759/wiki/results/3-5.png)

Fig. 3-5: The effect of enhancing heme biosynthesis on the Olep
catalysis

04

Screening and modification of the optimal redox partners for OleP by sfGFP and semi-rational design

4.1 Building a redox partner library

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.

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.

4.2 Constructing a sfGFP sensor to screen redox partners

Generally, the method of determining whether the redox partners were 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.
We used the fluorescent protein sfGFP and successfully constructed a sensor to detect redox partners.

We divided sfGFP into N-terminal and C-terminal, and although these two parts were cut off, there was an
interaction force between them. 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.

The following table is to show parts used in this section.

|  |  |  |
| --- | --- | --- |
| [BBa\_K4759212](http://parts.igem.org/Part%3ABBa_K4759212) | composite | T7-RBS4-GFP11-linker-Olep |
| [BBa\_K4759214](http://parts.igem.org/Part%3ABBa_K4759214) | composite | T7-RBS1-petH-RBS2-petF-linker-GFP1-10 |
| [BBa\_K4759215](http://parts.igem.org/Part%3ABBa_K4759215) | composite | T7-RBS1-camA-RBS2-camB-linker-GFP1-10 |
| [BBa\_K4759217](http://parts.igem.org/Part%3ABBa_K4759217) | composite | T7-RBS1-Fdr-0978-RBS2-Fdx-1499-linker-GFP1-10 |
| [BBa\_K4759219](http://parts.igem.org/Part%3ABBa_K4759219) | composite | T7-RBS1-BM3-linker-GFP1-10 |

![](https://static.igem.wiki/teams/4759/wiki/results/4-1.png)

Fig. 4-1: The scheme of constructing sfGFP sensor

![](https://static.igem.wiki/teams/4759/wiki/results/4-2.png)

Fig. 4-2: The self-assembly of Olep and Fdx based on the
three-dimensional structure of sfGFP (PDB: 5BT0)

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
strain.
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).

![](https://static.igem.wiki/teams/4759/wiki/results/4-3.png)

Fig. 4-3: BL21 morphology diagram seen under excitation light 488nm
and emitted light 520nm, green is green fluorescence of sfGFP

4.3 Testing the catalytic capacity of different redox partners

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. The recombinant strains R2 to R5 were subjected to shaker
fermentation experiments.

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.

The following table is to show parts used in this section.

|  |  |  |
| --- | --- | --- |
| [BBa\_K4759203](http://parts.igem.org/Part%3ABBa_K4759203) | composite | T7-RBS4-Olep |
| [BBa\_K4759218](http://parts.igem.org/Part%3ABBa_K4759218) | composite | T7-RBS1-BM3 |
| [BBa\_K4759201](http://parts.igem.org/Part%3ABBa_K4759201) | composite | T7-RBS1-camA-RBS2-camB |
| [BBa\_K4759216](http://parts.igem.org/Part%3ABBa_K4759216) | composite | T7-RBS1-Fdr-0978-RBS2-Fdx-1499 |
| [BBa\_K4759218](http://parts.igem.org/Part%3ABBa_K4759218) | composite | T7-RBS1-BM3 |
| [BBa\_K4759213](http://parts.igem.org/Part%3ABBa_K4759213) | composite | T7-RBS1-petH-RBS2-petF |

![](https://static.igem.wiki/teams/4759/wiki/results/4-4.png)

Fig. 4-4: (A) Screening proper redox partners for Olep from
different
sources. 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.
(B) 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 blue-filled triangle
represents the fluorescent intensity/OD600. The red hollow triangle represents the conversion
rate (%).
Values and triangles represent the means and standard deviations of biological triplicates

![](https://static.igem.wiki/teams/4759/wiki/results/4-5.png)

Fig. 4-5: The structures and interactions between Olep and Fdxs are
presented. The key interacting residues in Olep-Fdx complexes are depicted as sticks and highlighted in
yellow. Heme and substrates are displayed as sticks, colored in red and wheat, respectively. The Fe2S2
cluster is visualized as spheres. The distances (Å) between the iron–sulfur cluster and heme-iron are
measured and indicated by dashed red lines. The interaction areas of Olep-Fdx are calculated by NovoPro
(https://www.novopro.cn/). The numbers of hydrogen bonds and salt bridges are predicted by PDBePISA
(https://www.ebi.ac.uk/pdbe/)

4.4 The fusion expression and different expressional ratio between Olep
and PetH/PetF.

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.
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).

![](https://static.igem.wiki/teams/4759/wiki/results/4-6.png)

Fig. 4-6: The fusion expression and different expressional ratio between Olep and PetH/PetF

The following table is to show parts used in this section.

|  |  |  |
| --- | --- | --- |
| [BBa\_K4759220](http://parts.igem.org/Part%3ABBa_K4759220) | composite | T7-RBS1-petH-linker-petF-linker-Olep |
| [BBa\_K4759221](http://parts.igem.org/Part%3ABBa_K4759221) | composite | T7-RBS1-petH-linker-petF |

Recombinant strains R6 to R9 were subjected to shake flask fermentation. HPLC assay for product
generation.
The transformation rate of recombinant strains R6 to R9 were lower than that of the
recombinant strain R5.

![](https://static.igem.wiki/teams/4759/wiki/results/4-7.png)

Fig. 4-7: The conversion rate of DCA for R5-R9 strains. The blue-filled triangle represents the biomass
(OD600).

4.5 Semi-rational design of redox partner PetH/PetF

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.

(Site-directed Mutagenesis parts can be search from [BBa\_K4759053](http://parts.igem.org/Part%3ABBa_K4759053) to [BBa\_K4759075](http://parts.igem.org/Part%3ABBa_K4759075), and constructed
modeling
screening for redox partners can be search from [BBa\_K4759076](http://parts.igem.org/Part%3ABBa_K4759076)  to [BBa\_K4759099](http://parts.igem.org/Part%3ABBa_K4759099) )

![](https://static.igem.wiki/teams/4759/wiki/results/4-8.png)

Fig. 4-8: Fermentation of 23 mutants and control groups

We conducted control tests with the positive control group, negative control group, and wild-type
strains, and finally selected 9 mutants with the highest fluorescence intensity for
subsequent catalytic
verification by detecting their green fluorescence intensity.

![](https://static.igem.wiki/teams/4759/wiki/results/4-9.png)

Fig. 4-9: 9 mutants + wild-type + negative control, 50 ml/250 ml
system fermentation

By verifying the catalytic ability, we found that the
substrate conversion of D68P was higher than that
of the wild type in the nine strains with high fluorescence intensity, reaching 89.2%.

![](https://static.igem.wiki/teams/4759/wiki/results/4-10.png)

Fig. 4-10: A:Fluorescence intensity of wild type with 23 mutants

 B: Conversion of the 9 mutants with the highest fluorescence intensity with wild type

05

The optimal whole-cell catalytic system for Olep to perform most efficient conversion

The above three strategies were combined to obtain a highly effective recombinant strain *E. coli* C1
(containing recombinant plasmids pRSFDuet-petH-petF-oleP and pCDFDuet-hemA-hemL). The conversion rate of the
C1 strain without the addition of ALA and FeCl3 during induction reached 89.7%. Next, the C1 strain
was
optimized for whole-cell catalytic conditions.

5.1 The catalytic form

First, we explored catalytic forms. We measured the substrate conversion rate of Whole-Cell,
Permeabilized Cell, Crude enzyme, and fermentation, respectively, and found
that the substrate conversion
rate was the highest under whole-cell catalytic conditions.

![](https://static.igem.wiki/teams/4759/wiki/results/5-1.png)

Fig. 5-1: Effect of catalytic form.

The red hollow triangle represents the conversion rate (%). Values and triangles represent the means and
standard deviations of biological triplicates.

5.2 The biomass of resting whole-cell

In the next step, we explored the optimal biomass by changing the size of the cellular biomass added to
the whole cell catalytic system. When the substrate concentration at the time of reaction was set to 1
mg/mL and the catalysis time was set to 12 h, the effect of biomass OD600 (10, 15, 20, 25, 30,
35) on
whole cell catalysis was investigated. The results showed that
the conversion rate was the highest when
OD600 was selected as 30
.

![](https://static.igem.wiki/teams/4759/wiki/results/5-2.png)

Fig. 5-2: Effect of different biomass of resting whole-cell
(OD600)

5.3 The concentration of substrate DCA

Next, when the biomass OD600 was set to 30 and the catalysis time was set to 12 h, the effect
of
substrate concentrations (0.5 mg/mL, 1.0 mg/mL, 1.5 mg/mL, 2.0 mg/mL, 2.5 mg/mL, 3.0 mg/mL, 3.5 mg/mL) on
whole-cell catalysis was investigated and we found that
the conversion efficiency was the highest when the
substrate concentration was 0.5 mg/mL
.

![](https://static.igem.wiki/teams/4759/wiki/results/5-3.png)

Fig. 5-3: Effect of concentration of DCA (mg/mL)

5.4 The catalytic time

Subsequently, when the biomass OD600 was set to 30 and the substrate concentration was set to
2.0 mg/mL,
the effect of catalysis time (0 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 14 h, 16 h, 18 h, 20 h, 22 h, 24 h) on
whole cell catalysis was investigated and we found that the conversion rate of 20-24 h reached the highest
and remained unchanged by controlling different catalytic time. Considering the time was too long, we
chose the catalytic time of 12h, and the conversion rate could reach more than 90%.

![](https://static.igem.wiki/teams/4759/wiki/results/5-4.png)

Fig. 5-4: Effect of catalytic time (h)

5.5 Constructing an intracellular NADPH regeneration system

P450 enzyme catalysis required redox partners to transfer NADPH electrons to heme to catalyze the
substrate. The content of NADPH in *E. coli* cells was low, which can only meet its own growth needs.
By
constructing a cofactor circulatory system, the effect of NADPH on whole-cell activity was investigated.
However, the cost of exogenous NADPH addition was higher and the catalytic efficiency was low. NAD+ kinase
*(NadK)* and membrane-bound hydrogenase *(PntAB)* can be used to enhance the circulation of
NADPH.
Thus, 3
recombinant strains are constructed. The recombinant plasmid pACYCDuet-nadK is expressed in *E. coli*C1 (C2
strain). The recombinant plasmid pACYCDuet-pntAB ([BBa\_K4759269](http://parts.igem.org/Part%3ABBa_K4759269)) was expressed in *E. coli* C1 (C3
strain).
The recombinant plasmid pACYCDuet-pntAB-nadK([BBa\_K4759271](http://parts.igem.org/Part%3ABBa_K4759271)) was expressed in *E. coli* C1 (C4
strain).
Without the addition of NADPH, the conversion rate of whole-cell catalysts prepared by the C4 strain was
as high as 99.1%
.

![](https://static.igem.wiki/teams/4759/wiki/results/5-5.png)

Fig. 5-5: Effect of NADPH addition and intracellular NADPH
regeneration system

06

Applying the constructed whole-cell catalytic system to produce other bile acid

The recombinant strain *E. coli* C4 was fermented using TB mediums under the same fermentation
conditions as in
the first part, and a whole-cell catalyst was prepared according to the method used in the first
part. Whole-cell catalysis was performed with a final concentration of 0.5 mg/mL of lithocholic acid as the
substrate.
The results are shown in Figure 6. Compared with *E. coli* O1,
the conversion rate of murine deoxycholic acid
catalyzed by the C4 strain to its 6-position β hydroxylation product increased from 1.6% to 42.7%.

![](https://static.igem.wiki/teams/4759/wiki/results/6-1.png)

Fig. 6-1: LCMS analysis of the hydroxylation of LCA by Olep. (A)
Biocatalytic reaction of LCA. a control reaction catalyzed by *E. coli* C41 (DE3) strain harboring
pRSFDuet-1
empty plasmid; b the hydroxylation of LCA catalyzed by *E. coli* O1 strain harboring pET28a-oleP and
pACYCDuet-camA-camB plasmids; c the hydroxylation of LCA catalyzed by *E. coli* C4 strain harboring
pRSFDuet-petH-petF-oleP, pCDFDuet-hemA-hemL, and pACYCDuet-pntAB-nadK plasmids. The black and red arrows point
to the substrate (LCA) and the hydroxylated product (MDCA), respectively.
 (B) MS analysis of substrate
LCA.
(C) MS analysis of hydroxylated product MDCA