iGEM 2023 — Team zjfh-nanjing


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# Engineering Success

### Overview

We constructed xylose reductase-expressing strains with different enzyme sources to catalyze xylose for
xylitol
production in E. coli BL21. The strategy of promoter engineering was utilized to improve the efficiency of
conversion.
We overexpressed the erythritol synthesis pathway enzyme in Yarrowia lipolytica. Based on a high-throughput
screening
strategy, we constructed fluorescent reporter system for characterizing erythritol production and improving
erythritol
production in Y. lipolytica.

### Cycle 1: Construction of xylose reductase expression plasmid

##### Design

Through literature reading, we selected xylose reductase from Kluyveromyces sp. IIPE453 (KsXR, [BBa\_K4941010](http://parts.igem.org/Part%3ABBa_K4941010),
GenBank:
KJ563917.1), xylose reductase from Pichia stipites (PsXR, [BBa\_K4941009](http://parts.igem.org/Part%3ABBa_K4941009), GenBank: X59465.1), and xylose
reductase from
Debaryomyces nepalensis NCYC 3413 (DnXR, [BBa\_K4941008](http://parts.igem.org/Part%3ABBa_K4941008), GenBank: KT239024.1).

##### Build

In order to verify the catalytic performance of three different sources of xylose reductase in E. coli BL21,
we need to
construct three xylose reductase expression plasmids, including pET28a-KsXR-kana ([BBa\_K4941034](http://parts.igem.org/Part%3ABBa_K4941034)),
pET28a-PsXR-kana
([BBa\_K4941033](http://parts.igem.org/Part%3ABBa_K4941033)), pET28a-DnXR-kana ([BBa\_K4941035](http://parts.igem.org/Part%3ABBa_K4941035)). Here we take the process
of constructing pET28a-PsXR as an
example: i)
Firstly, the DNA fragment of PsXR ([BBa\_K4941009](http://parts.igem.org/Part%3ABBa_K4941009)) was amplified by PCR with primers PsXR\_F
and PsXR\_R;
ii) Next, the
DNA fragment of the vector plasmid pET28a ([BBa\_K4941040](http://parts.igem.org/Part%3ABBa_K4941040)) was amplified by PCR to obtain linearized
pET28a;
iii) Then,
the linearized pET28a and PCR-amplified PsXR fragment were assembled by Gibson to obtain pET28a-PsXR; iv)
The
Gibson-assembled reaction mixture was transformed into Escherichia coli DH5α; and v) Positive transformants
were
sequenced by Sangon Biotech (Shanghai, China). In order to be able to screen transformants using the
resistance
screening markers, we replaced the kan resistance marker in the pET28a-PsXR plasmid using Gibson assembly
(replaced with
the Cm resistance marker).

![](https://static.igem.wiki/teams/4941/wiki/pages/engineering-success/image1.png)

Figure 1. a. Fragments of xylose reductase-encoding genes from three different sources; b. Gibson
assembly; c. Positive
transformants of pET28a- PsXR, pET28a-DnXR, and pET28a-PsXR; d. Sequence comparison results of constructed
plasmids.

##### Test

The correctly sequenced xylose reductase expression plasmids pET28a-KsXR-kana ([BBa\_K4941034](http://parts.igem.org/Part%3ABBa_K4941034)),
pET28a-PsXR-kana
([BBa\_K4941033](http://parts.igem.org/Part%3ABBa_K4941033)), pET28a-DnXR-kana ([BBa\_K4941035](http://parts.igem.org/Part%3ABBa_K4941035))), and T7 RNAP expression
plasmids ([BBa\_K4941063](http://parts.igem.org/Part%3ABBa_K4941063)) were
transferred into
E. coli BL21. Here is an example of the transformation of the T7RNAP plasmid ([BBa\_K4941063](http://parts.igem.org/Part%3ABBa_K4941063)) and
pet28a-PsXR-kana
plasmid:

1. Firstly, in order to screen the transformants using resistance screening markers, we
   replaced the kan
   resistance
   marker in the pET28a-PsXR-kana plasmid using Gibson assembly (replacing it with the Cm resistance marker)
   to obtain
   the strain pET28a-PsXR ([BBa\_K4941100](http://parts.igem.org/Part%3ABBa_K4941100)) (using CM as the screening marker);
2. Next, the T7 RNAP expression plasmid PlacUV5 ([BBa\_K4941063](http://parts.igem.org/Part%3ABBa_K4941063)) was transformed into BL21 and
   plated on LB
   plates
   containing kan resistance using the chemical transformation method;
3. The grown positive transformants were selected and inoculated into 5 mL LB tubes containing
   kan
   resistance for
   incubation, and the pET28a-PsXR plasmid was then transformed into these transformants using the chemical
   transformation method. The transformed cells were plated on LB plates containing both kan and Cm
   resistance;
4. Positive transformants were selected for colony PCR confirmation and prepared for
   subsequent
   fermentation tests.

The transformants were selected and transferred into the liquid medium and cultured in a shaker at 37 ℃ and
220 rpm for
12 hours. The seed solution was then transferred into 30 ml TB medium and cultured at 37 ℃ and 220 rpm for 3
hours.
After that, the expression of xylose reductase was induced by adding 30 ul of IPTG inducer and the culture
was continued
at 28 ℃ and 160 rpm for 16 hours. Subsequently, xylose was added to a concentration of 5 g/L, and the
reaction was
carried out at 30 ℃ and 220 rpm for 36 hours. Samples were taken every 12 hours for HPLC detection.

The results showed that BL21-pET28a-PsXR catalyzed the production of 4.6 g/L xylitol from the substrate 5
g/L xylose
after 36 hours. It was demonstrated that the xylose reductase derived from Pichia stipitis has the ability
to catalyze
xylose to produce xylitol in the E. coli BL21 protein expression system, and xylitol production through
fermentation in
E. coli is achieved

![](https://static.igem.wiki/teams/4941/wiki/pages/engineering-success/image2.png)

Figure 2. a. Xylitol yields of xylitol-expressing strains expressing three xylose reductase enzymes; b.
Standard curve
of xylitol in HPLC assay.

##### Learn

The key issue in this work is to obtain the xylose reductase with the strongest catalytic capacity in
expression system
by assaying the activity of xylose reductase from different sources.

### Cycle 2: Promoter engineering to optimize xylose reductase expression

##### Design

With our newly constructed T7RNAP expression library based on the lacUV5 promoter mutant, we optimized the
expression
using the pET expression system against xylose reductase (PsXR, GenBank: X59465.1) of Pichia stipitis
origin. This was
done to find the most suitable expression intensity in order to further improve the conversion to xylose and
increase
xylitol production.

##### Build

In order to verify the effect of three catalytic properties of the pET expression system on PsXR by
modulating the
intensity of T7RNAP expression, and thus affecting the catalytic properties of PsXR, we simultaneously
transformed
T7RNAP expression plasmids and pet28a-PsXR containing different lacUV5 promoters into E. coli BL21 hosts.
Here is an
example of the transformation process for the T7RNAP plasmid driven by PlacUV5MB7 ([BBa\_K4941056](http://parts.igem.org/Part%3ABBa_K4941056)) and the
pET28a-PsXR
plasmid:

1. Firstly, plasmid PlacUV5MB7-T7RNAP-T7t ([BBa\_K4941083](http://parts.igem.org/Part%3ABBa_K4941083)) was transformed into BL21 using the
   chemical
   transformation
   method and plated onto LB plates containing kanamycin resistance;
2. Next, the positively grown transformants were selected and transferred into Kanamycin-resistant 5 mL LB
   tubes for
   3-4 hours to allow the receptor cells to grow. The pET28a-PsXR plasmid was then transformed into the above
   transformants using the chemical transformation method and plated onto LB plates containing both Kanamycin
   and
   Chloramphenicol resistances;
3. Positive transformants were selected for colony PCR test confirmation and prepared for subsequent
   fermentation
   testing.

##### Test

Three transformants containing different lacUV5 promoters were selected and transfected into 96-well culture
plates
containing LB liquid medium. The plates were then cultured in a shaker at 37 °C and 220 rpm for 12 hours.
The seed
solution was further transferred into 48-well culture plates containing 2 ml of fermentation medium and
cultured at 37
°C and 220 rpm for 3 hours. Afterwards, 30 μl of IPTG was added as an inducer, and the culture was continued
at 28 °C
and 160 rpm for 16 hours to allow for the expression of xylose reductase.For xylose reductase expression,
the plates
were incubated at 16°C and 160 rpm for 16 hours. Subsequently, xylose was added to a final concentration of
8 g/L, and
the reaction was carried out at 30°C and 220 rpm for 36 hours. HPLC detection was performed to analyze the
reaction.

The results showed that MB7-PsXR successfully catalyzed the production of 6.8 g/L of xylitol from the
substrate of 8 g/L
xylose after 36 hours. This outcome demonstrates the feasibility of the strategy employed, which involved
optimizing the
pET expression system through the construction of a promoter library for T7RNAP expression.

![](https://static.igem.wiki/teams/4941/wiki/pages/engineering-success/image3.png)

Figure 3. Xylitol production under different promoter variants.

##### Learn

The key objective of this study was to optimize the expression intensity of the pET expression system,
specifically the
activity of xylose reductase. This was achieved by utilizing a T7RNAP expression library based on a mutated
lacUV5
promoter. The aim was to identify the combination that exhibited the optimal expression intensity during
fermentation.

### Cycle 3: Construction of expression plasmids for erythrosine 4-phosphate phosphatase and erythrosine reductase

##### Design

A natural pathway for erythritol synthesis exists in Y. lipolytica, with the genes Yida and ER playing
important roles.
Yida encodes the enzyme 4-phosphate erythritol phosphatase, which converts erythrose 4-phosphate to
erythrose. ER, on
the other hand, encodes erythritol reductase, which converts erythrose to erythritol. However, the yield of
erythritol
was too low to be detected using HPLC conditions. It was hypothesized that the expression levels of Yida and
ER might be
insufficient, leading to undetectable erythritol production. To address this issue, we attempted to
overexpress LhYida
([BBa\_K4941041](http://parts.igem.org/Part%3ABBa_K4941041)), which is an erythritol
4-phosphate phosphatase derived from Lactobacillus helveticus, as
well as ylER
([BBa\_K4941019](http://parts.igem.org/Part%3ABBa_K4941019)) in Y. lipolytica. The
goal was to enhance the expression of erythrose 4-phosphate (E4P) and
facilitate
the de novo synthesis of erythritol, thereby increasing its yield.

##### Build

To enable the de novo synthesis of erythritol in Y. lipolytica, we selected the Yida and ER genes from Y.
lipolytica and
constructed the LhYida-ylER expression plasmid pYLXP’-LhYida-ylER ([BBa\_K4941105](http://parts.igem.org/Part%3ABBa_K4941105)). We also constructed single
gene
expression plasmids, namely pYLXP’-LhYida ([BBa\_K4941041](http://parts.igem.org/Part%3ABBa_K4941041)), pYLXP’-ylER ([BBa\_K4941037](http://parts.igem.org/Part%3ABBa_K4941037)), and pYLXP’-ura
([BBa\_K4941030](http://parts.igem.org/Part%3ABBa_K4941030)).
Let's take pYLXP’-LhYida as an example:

1. The target fragments LhYida ([BBa\_K4941020](http://parts.igem.org/Part%3ABBa_K4941020)), pTEF ([BBa\_K4941012](http://parts.igem.org/Part%3ABBa_K4941012)), and XPR2 ([BBa\_K4941014](http://parts.igem.org/Part%3ABBa_K4941014)) were obtained
   using PCR
   and designed with 4bp sticky ends for plasmid assembly;
2. Plasmid pYLXP' ([BBa\_K4941039](http://parts.igem.org/Part%3ABBa_K4941039)) was
   digested with nuclease BsaI to obtain linearized pYLXP'
   ([BBa\_K4941039](http://parts.igem.org/Part%3ABBa_K4941039));
3. The resulting reaction mixture from the golden gate assembly was transformed into E. coli DH5α.iv) The
   colonies
   were verified by PCR and sent for sequencing by Sangon Biotech (Shanghai, China) to confirm the correct
   sequence.

After obtaining the single gene expression plasmids, we proceeded to construct the pYLXP’-LhYida-ylER
expression
plasmid:

1. Firstly, plasmids were digested with AvrII and NotI to obtain PTEF1-LhYida-XPR1 ([BBa\_K4941044](http://parts.igem.org/Part%3ABBa_K4941044)) and
   PTEF-URA-XPR2
   ([BBa\_K4941026](http://parts.igem.org/Part%3ABBa_K4941026)) fragments for plasmid
   assembly;
2. Plasmid pYLXP-ylER ([BBa\_K4941037](http://parts.igem.org/Part%3ABBa_K4941037))
   was digested with NheI and NotI to obtain linearized pYLXP-ylER0.
3. The linearized pYLXP-ylER and the two standardized fragments were ligated using T4 ligation;
4. The resulting T4 assembly reaction mixture was transformed into Escherichia coli DH5α.v) Colonies were
   verified by
   PCR and sent for sequencing by Sangon Biotech (Shanghai, China) to confirm successful construction and
   sequence accuracy.

![](https://static.igem.wiki/teams/4941/wiki/pages/engineering-success/image4.jpg)

Figure 4. a. T4 DNA Ligase; b. expression plasmid pylxp-LhYida-ylER ([BBa\_K4941105](http://parts.igem.org/Part%3ABBa_K4941105)); c. Validation results
of enzymatic
digestion of the expression plasmid.

##### Test

Next, we proceeded to integrate the LhYida-ylER expression fragment ([BBa\_K4941106](http://parts.igem.org/Part%3ABBa_K4941106)) into the genome of
*Y.
lipolytica* po1g in order to achieve de novo synthesis of erythritol. For successful integration, we
employed
the lithium acetate transformation method, following a standard protocol previously reported [1, 2]. Here is
a brief
overview of the protocol:

1. During the exponential growth phase (16-24 hours), 1 mL of the *Y. lipolytica* culture was
obtained from 2
mL of YPD medium (containing yeast extract 10 g/L, peptone 20 g/L, and dextrose 20 g/L) in a 14 mL shaker
tube;

2. The bacterial cells were then washed twice with 100 mM phosphate buffer (pH 7.0);

3. The cells were resuspended in 105 µL of transformation solution, consisting of 90 µL of 50% PEG4000
solution, 5 µL
of lithium acetate (2M), 5 µL of boiled single-stranded DNA (denatured salmon spermatozoa), and 5 µL of DNA
products
(including 200-500 ng of plasmid, linear plasmid, or DNA fragment);

4. The mixture was thoroughly mixed and heated, followed by incubation for 1 hour at 37°C in a water bath;

5. After incubation, the mixture was coated onto selected solid plates. It is important to note that the
transformation mixtures needed to be oscillated every 15 minutes for 15 seconds during the incubation at
37°C;

6. The selected marker for integration in this study was Uracil. As a result, we successfully integrated
the
LhYida-ylER fragments into the genome of *Y. lipolytica* po1g, obtaining the engineered strain
po1g-LhYida-ylER
(po1g-1).

Subsequently, we selected positive transformants for fermentation experiments. The strain was transferred
to YNB-URA
medium and cultured for 48 hours to obtain the seed liquid. Samples were taken after transferring 500 µL of
seed
liquid to 30 mL of fermentation medium, which was then cultured at 30°C and 220 rpm on a shaker for 120
hours. The
content of erythritol was detected using HPLC. The results revealed that the engineered strain po1g-1
overexpressing
LhYida and ylER achieved a yield of 2.1 g/L of erythritol, thus successfully realizing de novo synthesis of
erythritol
in *Y. lipolytica*.

![](https://static.igem.wiki/teams/4941/wiki/pages/engineering-success/image5.png)

Figure 5. a. Integration of LhYida-ylER into the genome for erythritol production; b. Erythritol
production of
po1g-LhYida-ylER (po1g-1).

##### Learn

Although the erythritol synthesis pathway exists in the wild-type *Y. lipolytica*, the expression of
the
pathway enzymes is insufficient, resulting in a low yield of erythritol that cannot be detected by HPLC.
Therefore, in
this project, we successfully achieved the de novo synthesis of erythritol in *Y. lipolytica* by
overexpressing
the endogenous LhYida and ylER, which enhanced the expression of E4P for erythritol production.

### Cycle 4: Construction of the fluorescent reporter system for characterizing erythritol production

##### Design

The fluorescent reporter system has been widely employed to assess protein expression levels [1] [2].
Inducible
promoters generate varying intensities depending on the concentration of the inducer, thereby modifying the
expression
level of the protein [3]. Jean Marc Nicaud [4] combined an erythritol-inducible promoter with a luciferase
reporter
system that correlates fluorescence intensity with erythritol concentration. Consequently, erythritol
concentration
was determined using a more straightforward and intuitive method. Building upon this, we utilized an
erythritol-inducible promoter to express the luciferase-encoding gene. Our aim was to construct an
erythritol-luciferase reporter system in *Y. lipolytica* and assess erythritol yield through
fluorescence
intensity.

##### Build

To construct the fluorescent reporter system for characterizing erythritol production, we created the
expression
plasmid pylxp-pEYK1-Nluc ([BBa\_K4941036](http://parts.igem.org/Part%3ABBa_K4941036)) which contains the Nluc ([BBa\_K4941022](http://parts.igem.org/Part%3ABBa_K4941022)) gene driven by an
erythritol-inducible
promoter (pEYK1, [BBa\_K4941036](http://parts.igem.org/Part%3ABBa_K4941036)). The
construction process involved the following steps:

1. The target fragments Nluc ([BBa\_K4941022](http://parts.igem.org/Part%3ABBa_K4941022)), pEYK1 ([BBa\_K4941023](http://parts.igem.org/Part%3ABBa_K4941023)), and XPR2 ([BBa\_K4941014](http://parts.igem.org/Part%3ABBa_K4941014)) were obtained
using the
PCR method, with 4bp sticky ends added to both ends for plasmid assembly；

2. Plasmid pYLXP' ([BBa\_K4941039](http://parts.igem.org/Part%3ABBa_K4941039)) was
then digested with nuclease BsaI, resulting in linearized pYLXP'
([BBa\_K4941039](http://parts.igem.org/Part%3ABBa_K4941039))；

3. The linearized pYLXP' and the standardized processed fragments were then ligated together using Golden
Gate
assembly；

4. The resulting reaction mixture from the Golden Gate assembly was transformed into Escherichia coli DH5α；

5. Corrected colonies were verified using PCR and subsequently sequenced by Sangon Biotech (Shanghai,
China).

![](https://static.igem.wiki/teams/4941/wiki/pages/engineering-success/image6.png)

Figure 6. a. The Plasmid of pylxp-pEYK1-Nluc, b. PCR results of the target fragment Nluc, c. Plot of
plasmid sequencing
results

##### Test

Next, we proceeded to transform the pylxp-pEYK1-Nluc ([BBa\_K4941036](http://parts.igem.org/Part%3ABBa_K4941036)) plasmid into the *Y. lipolytica*po1g
strain. For the transformation, we utilized the lithium acetate method, as previously described in studies
by Islam,
Zhisheng et al. (2015) and Xu, Wu et al. (2022). In our project, we employed leucine as the selection
marker. The
transformation was successful, resulting in the creation of the engineered strain po1g pylxp-pEYK1-Nluc
(po1g-2).

Subsequently, we proceeded to select positive transformants for fermentation experiments. The strain was
transferred
to YNB-leu medium and cultured for 48 hours to obtain the seed liquid. Then, 500 μl of the seed liquid was
transferred
to 30 ml of fermentation medium with varying concentrations of erythritol (0 g/L, 1 g/L, 2 g/L, 3 g/L, 4
g/L, 5 g/L, 6
g/L). The samples were taken after 48 hours of culturing in a shaking bed at 30°C and 220 rpm. The
luciferase
substrate was added to initiate the reaction, and a kinetic assay was performed to compare the fluorescence
intensity
of the fermentation broths with different erythritol concentrations.

The results demonstrated a notable variation in the fluorescence intensity of the fermentation broth upon
the
addition of different concentrations of erythritol, as depicted in Figure 7. The fluorescence intensity
provided a
visual representation of the varying erythritol concentrations within the fermentation broth.

![](https://static.igem.wiki/teams/4941/wiki/pages/engineering-success/image7.png)

Figure 7. Construction of the fluorescent reporter system for characterizing erythritol production

##### Learn

In this project, we successfully constructed a fluorescent reporter system to characterize erythritol
production by
correlating the concentration of erythritol in the fermentation broth with the fluorescent intensity. This
approach
allowed us to identify and obtain engineered strains with higher erythritol production through the detection
of fluorescent intensity.

### Cycle 5: Screening of Erythrose-4P phosphatase

##### Design

The successful construction of the fluorescent reporter system played a crucial role in our ability to
effectively
screen erythritol-producing strains. In order to enhance the expression of Erythrose-4P phosphatase (Yida)
for
increased erythritol production, we selected this enzyme from various sources. These selected enzymes were
then
integrated into *Y. lipolytica* for erythritol production. We utilized the expression of
pYLXP-pEYK1-Nluc
([BBa\_K4941036](http://parts.igem.org/Part%3ABBa_K4941036)) to evaluate the
erythritol production of each engineered strain based on fluorescence
intensity.

##### Build

We selected *EcYida* ([BBa\_K4297032](http://parts.igem.org/Part%3ABBa_K4297032)) from *E. coli* [1] and
*StYida* ([BBa\_K4297028](http://parts.igem.org/Part%3ABBa_K4297028)) from
*Streptococcus
thermophilus* ([BBa\_K4297032](http://parts.igem.org/Part%3ABBa_K4297032)).
Through PCR, we obtained the target fragments EcYida ([BBa\_K4941018](http://parts.igem.org/Part%3ABBa_K4941018)) and
StYida ([BBa\_K4297028](http://parts.igem.org/Part%3ABBa_K4297028)). Reference to
the construction of pylxp-LhYida-ylER ([BBa\_K4941105](http://parts.igem.org/Part%3ABBa_K4941105)), we obtained two
expression
plasmids, pylxp-EcYida-ylER ([BBa\_K4941104](http://parts.igem.org/Part%3ABBa_K4941104)) and pylxp-StYida-ylER ([BBa\_K4941107](http://parts.igem.org/Part%3ABBa_K4941107)).

![](https://static.igem.wiki/teams/4941/wiki/pages/engineering-success/image8.jpg)

Figure 8. a. pylxp-EcYida-ylER and sequencing results, b. pylxp-StYida-ylER and sequencing results

##### Test

Next, we proceeded to transform the expression fragments EcYida-ylER ([BBa\_K4941103](http://parts.igem.org/Part%3ABBa_K4941103)) and StYida-ylER
([BBa\_K4941108](http://parts.igem.org/Part%3ABBa_K4941108))
into the genome of *Y. lipolytica* po1g, enabling integration of their expression [1, 2]. In our
project, the
selection marker used was Uracil. Consequently, we successfully integrated the EcYida-ylER and StYida-ylER
expression
fragments into the genome of *Y. lipolytica* po1g, resulting in the engineered strains
po1g-EcYida-ylER plate
(po1g-3) and po1g-StYida-ylER plate (po1g-4).

Subsequently, we utilized the lithium acetate transformation method to introduce pylxp-pEYK1-Nluc into the
mixed
bacteria of po1g-1, po1g-3, and po1g-4 separately. The strains were then transferred to YNB-URA medium and
cultured
for 48 hours to obtain the fermentation broth. The fermentation broth was cultivated at 30°C and 220rpm. In
order to
initiate the reaction, the luciferase substrate was added, and a kinetic assay was performed to compare the
fluorescence intensity of the fermentation broths of the three engineered strains. The strain with the
highest yield
was selected for further analysis. The chosen engineered strains were subsequently transferred into YNB-leu
fermentation broth and allowed to ferment for 120 hours. The fermentation broth was then collected for HPLC
detetion.
According to the results, strain po1g-3, expressing EcYida-ylER, exhibited the highest erythritol yield, as
depicted in
Figure 9. The HPLC assay confirmed that the erythritol yield reached 5.9 g/L.

![](https://static.igem.wiki/teams/4941/wiki/pages/engineering-success/image9.png)

Figure 9. a. Screening of Erythrose-4P phosphatase, b. Fluorescence results of high throughput screening,
c. Erythritol
yields of control po1g-3 after 120h.

##### Learn

In this project, we employed the EcYida fragment from *E. coli* and the ylER fragment from *Y.
lipolytica*. The expression construct EcYida-ylER was integrated into the genome of *Y.
lipolytica* po1g,
resulting in the successful expression of the desired genes. Ultimately, the engineered strain po1g-3
displayed a
remarkable erythritol yield, reaching 5.9 g/L.

### Reference

1. Islam, Z.U., et al., Microbial conversion of pyrolytic products to biofuels: a novel and sustainable
approach
toward second-generation biofuels. J Ind Microbiol Biotechnol, 2015. 42(12): p. 1557-79.

2. Xu, Y., et al., Sustainable bioproduction of natural sugar substitutes: Strategies and challenges.
Trends in Food
Science & Technology, 2022. 129: p. 512-527.

3. Biewenga, L., B. Rosier, and M. Merkx, Engineering with NanoLuc: a playground for the development of
bioluminescent protein switches and sensors. Biochem Soc Trans, 2020. 48(6): p. 2643-2655.

4. Wang, X., et al., Reversible thermal regulation for bifunctional dynamic control of gene expression in
Escherichia
coli. Nat Commun, 2021. 12(1): p. 1411.

5. Qiu, C., H. Zhai, and J. Hou, Biosensors design in yeast and applications in metabolic engineering. FEMS
Yeast
Res, 2019. 19(8).

6. Trassaert, M., et al., New inducible promoter for gene expression and synthetic biology in Yarrowia
lipolytica.
Microb Cell Fact, 2017. 16(1): p. 141.

7. Carly, F., et al., Enhancing erythritol productivity in Yarrowia lipolytica using metabolic engineering.
Metab
Eng, 2017. 42: p. 19-24.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiment{% endblock %}
{% block page\_content %}

# Experiments

##### Overview

In this project, we constructed xylose reductase-expressing strains with different enzyme sources to
catalyze xylose for
xylitol production in Escherichia coli. The strategy of promoter engineering was utilized to improve the
efficiency of
conversion. We overexpressed the erythritol synthesis pathway enzyme in Yarrowia lipolytica. Based on a
high-throughput
screening strategy, we constructed fluorescent reporter system for characterizing erythritol production and
improving
erythritol production in Y.lipolytica.

##### Shake flask culture of E. coli

All engineering strains were optimized and screened before shake flask culture. For shake flask culture, 500
uL of seed
solution was transferred to 30 mL LB+kana+Cm medium, incubated at 37℃ and 220 rpm for 3 h, added 0.1 mmol/L
inducer
IPTG, incubated at 28℃ and 220 rpm under shaking bed for 20 h. 1.5/3 mL of 100 g/L xylose precursor was
added to 30 mL
of fermentation broth, transformed at 30℃ and 220 rpm for 36 h. Samples were taken every 12 h, OD600 was
measured, and
samples were retained for HPLC to determine the xylitol yield.
The seed medium used in this study was LB+kana medium: tryptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L,
and kana 50
mg/L. The fermentation medium was TB+kana+Cm medium: tryptone 12 g/L, yeast extract 24 g/L, glycerol 4 ml/L,
KH2PO4 2.31
g/L and K2HPO4 12.54 g/L.

##### Shake flask culture of yeast

The engineered strain was selected from the plate po1g-pylxp- ylER-EcYida, and the control strain po1g was
selected from
the po1g plate, both of which were transferred to CSM-URA seed solution, and the seed solution was obtained
by shaking
bed culture at 30℃ for 48 h. Transfer 500 uL seed solution in 30 mL YNB-URA fermentation medium, 30℃ and 220
rpm shaker
culture 120 h, during the period of every 24 h sampling, and detection of OD600.
Preparation of YNB-Leu fermentation broth: glucose 40 g/L, YNB (no ammonium sulfate and no amino acid yeast
nitrogen
source) 1.7 g/L, CSM-Leu (leu-free) 0.74 g/L, (NH4)2SO4 1.1 g/L.
Preparation of YNB-URA fermentation broth: glucose 40 g/L, YNB (no ammonium sulfate and no amino acid yeast
nitrogen
source) 1.7 g/L, CSM-URA (URA -free) 0.74 g/L, (NH4)2SO4 1.1 g/L.

##### E.coli transformation

The pYLXP' recombinant plasmid was transformed into E. coli DH5α, incubated on ice for 30 min and then
heat-excited at
42°C for 1 min. The bacterial solution was spread on LB+amp plates and incubated at 37°C in an incubator for
14-16h. The
pET-28a recombinant plasmid transformation procedure is the same, only the antibiotic (chloramphenicol)
needs to be
changed.

##### Yeast transformation

The standard protocol for transformation of defatted yeast by lithium acetate method was as previously
reported (Islam,
Zhisheng et al. 2015, Xu, Wu et al. 2022). Briefly, during the exponential growth period (16-24 h), 1 mL of
the
bacterium was extracted from 2 mL of YPD medium (yeast extract 10 g/L, peptone 20 g/L, and dextrose 20 g/L)
in a 14 mL
shaker tube and washed twice with 100 mM phosphate buffer (pH 7.0). Then, the cell were resuspended in 105
µL
transformation solution, containing 90 uL of 50% PEG 4000 solution, 5 μL lithium acetate (2 mol/L), 5 uL
boiled single
strand DNA (salmon spermatozoa, denatured), and 5 μL DNA products (including 200-500 ng of plasmid, liner
plasmid, or
DNA fragment), which were mixed well and then heated and incubated for 1 h at 37°C in a water bath, and
finally coated
on selected solid plates. It should be noted that the transformation mixtures needed to be oscillated every
15 min for
15s during the process at 37°C incubation. The selected markers chosen in this study were leucine or uracil.

##### Expression vector construction and plasmid assembly

The YaliBrick plasmid pYLXP' and plasmid pET-28a were used as the expression vectors in this study (Wong,
Engel et al.
2017). Plasmid constructions were performed by using preciously described methods (Lv, Edwards et al. 2019).
In brief,
utilized linearized pYLXP' and the corresponding PCR-amplified DNA fragments, and the recombinant plasmid of
pYLXP ' -XX
(single-gene expression) was obtained by golden gate assembly. The recombinant plasmid of pYLXP ' -XX-XX was
obtained by
restriction endonuclease and T4 ligase for multi-gene assembly (Wong, Engel et al. 2017). The modified DNA
fragments and
plasmids were sequenced by Sangon Biotech (Shanghai, China). The endonucleases used in this study were
purchased from
Thermo Fisher Scientific or Takara.

The relevant plasmid construction steps for pET-28a were as follows: linearized pET-28a and the
corresponding
PCR-amplified DNA fragments were obtained by PCR amplification, and the recombinant plasmid of pET-28a -XX
(single gene
expression) was obtained by Gibson assembly method. Sequencing was performed by Sangon Biotech (Shanghai,
China).

##### Fluorescence intensity detection

The reaction mixture for luciferase whole-cell assay was prepared according to the operating procedure of
Nano-Glo
Luciferase Assay System Kit, including 100 μL of luciferase buffer, 2 μL of substrate, 10 μL of cell
suspension and 88
μL of sterile water. The program of multifunctional microtiter plate detector was set up for 60 cycles of
90s each, in
which the plate was vibrated for 10s, the detection was performed for 80s, and the total time of each
detection was
90min. The total time of each assay was 90min. The data of each assay were integrated to obtain the
luciferase activity
data, and then divided by OD600 to obtain the unit fluorescence intensity (RUL/OD600).

##### GFP fluorescence intensity measurement

The samples of 200 μL were transferred into the 96-well transparent microplate for GFP fluorescence
intensity. The
fluorescence intensity was measured at 485 nm excitation wavelength and 520 nm emission wavelength. Measured
the
fluorescence intensity to screening high-yield strains.

##### HPLC detection of xylitol and erythritol content

1ml of the sample was centrifuged at 12000 rpm for 15 min, and the liquid was passed through an water-based
filter
membrane and HPLC was performed to detect the content of xylitol/erythritol in the sample. Gradient
concentrations (1g,
2g, 3g, 4g, 5g) of xylitol and erythritol were prepared and the standard curve was plotted. The
concentrations of
xylitol and erythritol were measured by Agilent HPLC 1220 equipped with a SupelcogelTM Carbohydrate column
(Sigma, USA)
and a refractive index detector. H2SO4 (5 mM) was used as the mobile phase at a flow rate of 0.6 mL/min at
35°C.

##### Reference

Islam, Z. U., Y. Zhisheng, B. Hassan el, C. Dongdong and Z. Hongxun (2015). "Microbial
conversion of pyrolytic products
to biofuels: a novel and sustainable approach toward second-generation biofuels." J Ind Microbiol Biotechnol
42(12):
1557-1579.

Lv, Y., H. Edwards, J. Zhou and P. Xu (2019). "Combining 26s rDNA and the Cre-loxP System for Iterative Gene
Integration
and Efficient Marker Curation in Yarrowia lipolytica." ACS Synth Biol 8(3): 568-576.

Wong, L., J. Engel, E. Jin, B. Holdridge and P. Xu (2017). "YaliBricks, a versatile genetic toolkit for
streamlined and
rapid pathway engineering in Yarrowia lipolytica." Metab Eng Commun 5: 68-77.

Xu, Y., Y. Wu, Y. Liu, J. Li, G. Du, J. Chen, X. Lv and L. Liu (2022). "Sustainable bioproduction of natural
sugar
substitutes: Strategies and challenges." Trends in Food Science & Technology 129: 512-527.

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# Results

### Result 1. Screening of xylose reductases

Xylose reductase (XR) is responsible for encoding xylose reductase and can increase the expression of
xylose
reductase. It can realize the conversion from xylose to xylitol. Therefore, we chose three different sources
of XR to
construct pET28a-PsXR ([BBa\_K4941100](http://parts.igem.org/Part%3ABBa_K4941100)), pET28a-DnXR ([BBa\_K4941099](http://parts.igem.org/Part%3ABBa_K4941099)) and pET28a-KsXR ([BBa\_K4941098](http://parts.igem.org/Part%3ABBa_K4941098)). They were
transformed
into *E. coli* BL21. And xylose reductase was expressed to catalyze the reaction of xylose to xylitol
in TB
medium supplemented with 5 g/L xylose precursor, using glycerol as the carbon source. The xylitol yield in
the
fermentation broth was detected by HPLC.

The correctly sequenced xylose reductase expression plasmids pET28a-KsXR-kana ([BBa\_K4941034](http://parts.igem.org/Part%3ABBa_K4941034)),
pET28a-PsXR-kana
([BBa\_K4941033](http://parts.igem.org/Part%3ABBa_K4941033)), pET28a-DnXR-kana ([BBa\_K4941035](http://parts.igem.org/Part%3ABBa_K4941035))), and T7 RNAP expression plasmids ([BBa\_K4941063](http://parts.igem.org/Part%3ABBa_K4941063)) were
transferred into
*E. coli* BL21. The transformants were selected and transferred into the liquid medium and cultured
in a shaker
at 37 ℃ and 220 rpm for 12 hours. The seed solution was then transferred into 30 ml TB medium and cultured
at 37 ℃ and
220 rpm for 3 hours. After that, the expression of xylose reductase was induced by adding 30 ul of IPTG
inducer and
the culture was continued at 28 ℃ and 160 rpm for 16 hours. Subsequently, xylose was added to a
concentration of 5
g/L, and the reaction was carried out at 30 ℃ and 220 rpm for 36 hours. Samples were taken every 12 hours
for HPLC
detection.

The results showed that BL21-pET28a-PsXR catalyzed the production of 4.6 g/L xylitol from the substrate 5
g/L xylose
after 36 hours. It was demonstrated that the xylose reductase derived from Pichia stipitis has the ability
to catalyze
xylose to produce xylitol in the *E. coli* BL21 protein expression system, and xylitol production
through
fermentation in *E. coli* is achieved.

![](https://static.igem.wiki/teams/4941/wiki/pages/results/image1.png)

Figure 1. a. Xylitol yields of xylitol-expressing strains expressing three xylose reductase enzymes; b.
Standard curve
of xylitol in HPLC assay.

### Result 2. promoter engineering to enhance xylose reductase expression

By replacing promoters of T7RNAP with different strengths, the regulation of protein expression levels can
be realized.
Based on promoter engineering, the optimal promoter is selected to further enhance the expression of xylose
reductase,
resulting in more efficient catalytic efficiency.

![](https://static.igem.wiki/teams/4941/wiki/pages/results/image2.png)

Figure 2. promoter engineering to enhance xylose reductase expression

Three transformants containing different lacUV5 promoters were selected and transfected into 96-well
culture plates
containing LB liquid medium. The plates were then cultured in a shaker at 37 °C and 220 rpm for 12 hours.
The seed
solution was further transferred into 48-well culture plates containing 2 ml of fermentation medium and
cultured at 37
°C and 220 rpm for 3 hours. Afterwards, 30 μl of IPTG was added as an inducer, and the culture was continued
at 28 °C
and 160 rpm for 16 hours to allow for the expression of xylose reductase. For xylose reductase expression,
the plates
were incubated at 16°C and 160 rpm for 16 hours. Subsequently, xylose was added to a final concentration of
8 g/L, and
the reaction was carried out at 30°C and 220 rpm for 36 hours. HPLC detection was performed to analyze the
reaction.

The results showed that MB7-PsXR successfully catalyzed the production of 6.8 g/L of xylitol from the
substrate of 8
g/L xylose after 36 hours. This outcome demonstrates the feasibility of the strategy employed, which
involved
optimizing the pET expression system through the construction of a promoter library for T7RNAP expression.

![](https://static.igem.wiki/teams/4941/wiki/pages/results/image3.png)

Figure 3. Xylitol production under different promoter variants.

### Result 3. De novo synthesis of erythritol in Y. lipolytica

A natural erythritol synthesis pathway exists in *Y. lipolytica*. E4PP/Yida is the gene encoding
4-phosphate
erythritol phosphatase, which catalyzes the conversion of erythrose 4-phosphate to erythrose, and ER is the
gene
encoding erythritol reductase, which catalyzes the conversion of erythrose to erythritol. However, the yield
of
erythritol was too low to be detected under HPLC conditions. It was speculated that the expression levels of
erythritol 4-phosphate phosphatase (Yida) and erythritol reductase (ER) might be too weak, resulting in
undetectable
erythritol production. *LhYida* ([BBa\_K4941041](http://parts.igem.org/Part%3ABBa_K4941041)) is an erythritol 4-phosphate phosphatase derived from
*Lactobacillus helveticus*. Based on this, we attempted to overexpress *LhYida* ([BBa\_K4941041](http://parts.igem.org/Part%3ABBa_K4941041))
and
*ylER* ([BBa\_K4941037](http://parts.igem.org/Part%3ABBa_K4941037)) in *Y. lipolytica* to enhance the expression of erythrose 4-phosphate
(E4P) to
erythritol, thus realizing the de novo synthesis of erythritol yield.

We proceeded to integrate the LhYida-ylER expression fragment ([BBa\_K4941106](http://parts.igem.org/Part%3ABBa_K4941106)) into the genome of *Y.
lipolytica* po1g in order to achieve de novo synthesis of erythritol. For successful integration, we
employed
the lithium acetate transformation method. Subsequently, we selected positive transformants for fermentation
experiments. The strain was transferred to YNB-URA medium and cultured for 48 hours to obtain the seed
liquid. Samples
were taken after transferring 500 µL of seed liquid to 30 mL of fermentation medium, which was then cultured
at 30°C
and 220 rpm on a shaker for 120 hours. The content of erythritol was detected using HPLC. The results
revealed that
the engineered strain po1g-1 overexpressing *LhYida* and *ylER* achieved a yield of 2.1 g/L of
erythritol, thus successfully realizing de novo synthesis of erythritol in *Y. lipolytica*.

![](https://static.igem.wiki/teams/4941/wiki/pages/results/image4.png)

Figure 4. a. Integration of LhYida-ylER into the genome for erythritol production; b. Erythritol
production of
po1g-LhYida-ylER (po1g-1).

### Result 4. Construction of the fluorescent reporter system for characterizing erythritol

The fluorescent reporter system has been widely used to characterize the intensity of protein expression
[1] [2].
Inducible promoters produce different intensities depending on the concentration of the inducer, thus
altering the
level of protein expression [3]. Jean Marc Nicaud [4]combines an erythritol-inducible promoter with a
luciferase
reporter system that couples fluorescence intensity to erythritol concentration. Thus, the erythritol
concentration
was obtained by a more intuitive and simple method. Based on this, we used an erythritol-inducible promoter
to express
the luciferase-encoding gene. We attempted to build an erythritol-luciferase reporter system in *Y.
lipolytica*, and characterized the erythritol yield using fluorescence intensity.

We proceeded to transform the pylxp-pEYK1-Nluc ([BBa\_K4941036](http://parts.igem.org/Part%3ABBa_K4941036)) plasmid into the *Y. lipolytica* po1g
strain.
The transformation was successful, resulting in the creation of the engineered strain po1g pylxp-pEYK1-Nluc
(po1g-2).
Subsequently, we proceeded to select positive transformants for fermentation experiments. The strain was
transferred
to YNB-leu medium and cultured for 48 hours to obtain the seed liquid. Then, 500 μl of the seed liquid was
transferred
to 30 ml of fermentation medium with varying concentrations of erythritol (0 g/L, 1 g/L, 2 g/L, 3 g/L, 4
g/L, 5 g/L, 6
g/L). The samples were taken after 48 hours of culturing in a shaking bed at 30°C and 220 rpm. The
luciferase
substrate was added to initiate the reaction, and a kinetic assay was performed to compare the fluorescence
intensity
of the fermentation broths with different erythritol concentrations.

The results demonstrated a notable variation in the fluorescence intensity of the fermentation broth upon
the
addition of different concentrations of erythritol, as depicted in Figure 5. The fluorescence intensity
provided a
visual representation of the varying erythritol concentrations within the fermentation broth.

![](https://static.igem.wiki/teams/4941/wiki/pages/results/image5.png)

Figure 5. Construction of the fluorescent reporter system for characterizing erythritol production

### Result 5. Screening of Erythrose-4P phosphatase and construction of the optimal strain

The successful construction of the fluorescent reporter system for characterizing erythritol production
helped us to
better screen erythritol-producing strains. To further enhance the expression of E4P to erythritol, we
selected
Erythrose-4P phosphatase (Yida) from different sources. We selected *EcYida* ([BBa\_K4297032](http://parts.igem.org/Part%3ABBa_K4297032)) from
*E. coli*[1] and *StYida* from *Streptococcus thermophilus* ([BBa\_K4297032](http://parts.igem.org/Part%3ABBa_K4297032)). The expression of
pylxp-pEYK1-Nluc ([BBa\_K4941036](http://parts.igem.org/Part%3ABBa_K4941036)) was used to assess the erythritol production of each engineered strain by
fluorescence
intensity. we proceeded to transform the expression fragments EcYida-ylER ([BBa\_K4941103](http://parts.igem.org/Part%3ABBa_K4941103)) and StYida-ylER
([BBa\_K4941108](http://parts.igem.org/Part%3ABBa_K4941108)) into the genome of *Y. lipolytica* po1g, enabling integration of their expression [1,
2]. In our
project, the selection marker used was Uracil. Consequently, we successfully integrated the EcYida-ylER and
StYida-ylER expression fragments into the genome of *Y. lipolytica* po1g, resulting in the engineered
strains
po1g-EcYida-ylER plate (po1g-3) and po1g-StYida-ylER plate (po1g-4).

Subsequently, we utilized the lithium acetate transformation method to introduce pylxp-pEYK1-Nluc into the
mixed
bacteria of po1g-1, po1g-3, and po1g-4 separately. The strains were then transferred to YNB-URA medium and
cultured
for 48 hours to obtain the fermentation broth. The fermentation broth was cultivated at 30°C and 220rpm. In
order to
initiate the reaction, the luciferase substrate was added, and a kinetic assay was performed to compare the
fluorescence intensity of the fermentation broths of the three engineered strains. The strain with the
highest yield
was selected for further analysis. The chosen engineered strains were subsequently transferred into YNB-leu
fermentation broth and allowed to ferment for 120 hours. The fermentation broth was then collected for HPLC
detetion.
According to the results, strain po1g-3, expressing EcYida-ylER, exhibited the highest erythritol yield, as
depicted
in Figure 6. The HPLC assay confirmed that the erythritol yield reached 5.9 g/L.

![](https://static.igem.wiki/teams/4941/wiki/pages/results/image6.png)

Figure 6. a. Screening of Erythrose-4P phosphatase, b. Fluorescence results of high throughput screening,
c. Erythritol
yields of control po1g-3 after 120h.

### Conclusion

Finally, we produced xylitol by fermentation in E. coli. The optimal promoter mutant MB7 was utilized to
initiate the
expression of T7RNAP, which directed xylose reductase transcription. the MB7-PsXR strain was fermented for
36h in TB
medium with 8g/L xylose as substrate to obtain 6.8g/L xylitol. We also successfully constructed the
erythritol de novo
synthesis pathway in *Y. lipolytica* po1g. A fluorescent reporter system for characterizing
erythritol was
constructed to screen the source of Erythrose-4P phosphatase. The engineered strain po1g-3 (po1g-
EcYida-ylER) was
fermented in YNB-Leu medium for 120 h. The yield of erythritol in the fermentation broth was 5.9 g/L by
HPLC.

### Reference

1. Biewenga, L., B. Rosier, and M. Merkx, *Engineering with NanoLuc: a playground for the development of
bioluminescent protein switches and sensors.* Biochem Soc Trans, 2020. **48**(6): p.
2643-2655.

2. Wang, X., et al., *Reversible thermal regulation for bifunctional dynamic control of gene expression
in
Escherichia coli.* Nat Commun, 2021. **12**(1): p. 1411.

3. Qiu, C., H. Zhai, and J. Hou, *Biosensors design in yeast and applications in metabolic
engineering.* FEMS
Yeast Res, 2019. **19**(8).

4. Trassaert, M., et al., *New inducible promoter for gene expression and synthetic biology in Yarrowia
lipolytica.* Microb Cell Fact, 2017. **16**(1): p. 141.

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