iGEM 2023 — Team nanjing-biox

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Notebook

# **10/7-14/7****Construction of recombinant plasmid pMTL-*****Pthl-F/Xpk(BD)***

1. **PCR amplification on the genome of** ***Clostridium acetobutylicum*****to obtain the fragment** ***F/Xpk(BD)*****.**

2. **Linearized vector pMTL-*****Pthl*****was obtained by PCR amplification using plasmid pMTL82151-*****Pthl-adhE2*****as template;**

3. **pMTL-*****Pthl-F/Xpk(BD)*****was obtained by Gibson assembly**

**Experimenter**：Pengfei Bai,Huaize Zhu,Ruige Li,Junjie
Zheng,Youchang Zhang,Yuyao Cao,Yuntian Yang,Shufan Wu

**Recorder：**Huaize Zhu

[PDF1- Construction of recombinant plasmid pMTL-Pthl-F/Xpk(BD)](https://static.igem.wiki/teams/4883/wiki/pdf1-construction-of-recombinant-plasmid-pmtl-pthl-f-xpk-bd.pdf)

# **17/7-20/7****Construction of recombinant plasmid pMTL-*****Pthl-F/Xpk(QS)***

1. **The fragment** ***F/Xpk(QS)*****was obtained by PCR amplification on** ***Bifidobacterium adolescentis*****genome**

2. **Linearized vector pMTL-*****Pthl*****was obtained by PCR amplification using plasmid pMTL82151-*****Pthl-adhE2*****as template;**

3. **pMTL-*****Pthl-F/Xpk(QS)*****was obtained by Gibson assembly**

**Experimenter：**Pengfei Bai,Huaize Zhu,Junjie Zheng,Youchang Zhang,Yuyao Cao

**Recorder**：Huaize Zhu

[PDF2-Construction of recombinant plasmid pMTL-Pthl-F/Xpk(QS)](https://static.igem.wiki/teams/4883/wiki/pdf2-construction-of-recombinant-plasmid-pmtl-pthl-f-xpk-qs.pdf)

# **21/7-1/8****Construction of recombinant plasmids** ：**pMTL-*****Pfba-F/Xpk*****、pMTL-*****Ptkt-F/Xpk***

1. **PCR amplification on the genome of** ***Clostridium tyrobutyricum*****to obtain the fragment** ***Pfba******、Ptkt******)*****.**

2. **Linearized vector pMTL-*****F/Xpk(BD)*****was obtained by PCR amplification using plasmid pMTL-*****Pthl-F/Xpk(BD)*****as template;**

3. **pMTL-*****Pfba-F/Xpk(BD)******、*****pMTL-*****Ptkt-F/Xpk(BD)*****was obtained by Gibson assembly**

**Experimenter：**Leyi Zhang,Yijiu Lu,Xiaoran Zhao,Yixuan Liu,Qingyue Feng,Ruoxi Liu,Jiale
Li

**Recorder：**Leyi Zhang

[PDF3-Construction of recombinant plasmids ：pMTL-Pfba-F/Xpk、pMTL-Ptkt-F/Xpk](https://static.igem.wiki/teams/4883/wiki/pdf3-construction-of-recombinant-plasmids-pmtl-pfba-f-xpk-pmtl-ptkt-f-xpk.pdf)

# **7/8-17/8****Replacement of carbon sources and determination of butyric acid**

1. **Culture of** ***Clostridium tyrobutyricum*****engineered bacteria** ***Ct (Pthl F/Xpk-BD)*** **using TGY medium supplemented with different carbon sources**

2. **Fermentation broths of Ct (Pthl F/Xpk-BD) in media containing different
carbon sources were collected and assayed for product content using
HPLC**

3. **Analysis of HPLC data**

**Experimenter：**Yijiu Lu,Pengfei Bai,Huaize Zhu,Leyi Zhang，Shuyan Zhou,Keyu Chen,Zexi Zheng,Yixuan Liu

**Recorder:**Yijiu Lu

[PDF4-Replacement of carbon sources and determination of butyric
acid](https://static.igem.wiki/teams/4883/wiki/pdf4-replacement-of-carbon-sources-and-determination-of-butyric-acid.pdf)

# **18****/8-****25****/8****Ancestral sequence reconstruction (ASR)****and** **Construction of recombinant plasmids****pMTL-P*****thl-F/Xpk(ASR)***

1. **Using PUC57 vector plasmid as the template, F/Xpk(ASR) fragment (2436
bp) was amplifie;**

**2.Linearized vector pMTL-Pthl****was obtained by PCR amplification using plasmid pMTL-Pthl-F/Xpk(BD)
as template;**

**3.The F/Xpk (ASR) gene fragment and the X-pMTL-Pthl linearized vector
were ligated by Gibson assembly****.**

**Experimenter：**Yijiu Lu,Pengfei Bai,Huaize Zhu

**Recorder:**Pengfei Bai

[PDF5-Construction of recombinant plasmids ：pMTL-Pthl-F\_Xpk-ASR.pdf](https://static.igem.wiki/teams/4886/wiki/pdf5-construction-of-recombinant-plasmids-pmtl-pthl-f-xpk-asr.pdf)

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Engineering success

Engineering success

## **Abstract**

This project is aimed to reduce the carbon emission during the fermentation of
a valuable industrial strain, *Clostridium tyrobutyricum* (*C. tyrobutyricum*). Phosphoketolase（*F/Xpk*） gene sequences well fitted for *C. tyrobutyricum* L319 were obtained by strain screening and sequence
optimization. Plasmids of the gene were constructed and
transferred into *C. tyrobutyricum* L319 to construct a non-oxidative glycolysis (NOG)
pathway. Unlike the native Embden-Meyerhof-Parnas (EMP) pathway
which emits CO2 during glycolysis, this alternative NOG pathway has no
carbon emission while metabolizing glucose. We constructed nine parts, BBa\_K4886000-BBa\_K4886008, to express phosphoketolase using different combinations of *F/Xpk* sequence and promoter and construct the NOG pathway in C.
tyrobutyricum.  The constructed NOG pathway was evaluated
by experiments such as PCR, HPLC, AcP assay and fermentation
experiment and the best combination of gene sequence and
promoter was obtained.

## **1 Project Overview**

Our project aims to reduce carbon emission of *Clostridium tyrobutyricum* (*C. tyrobutyricum*) through the construction of a non-oxidizing glycolysis (NOG) pathway
by expressing phosphoketolase encoded by *F/Xpk* gene in the bacteria. We successfully constructed five *C. tyrobutyricum* strains with the NOG pathway which had reduced carbon
loss, and found that the best carbon source for these strains
was xylose. With PCR, SDS-PAGE, fermentation experiment and HPLC
to analyze DNA, protein, growth and product yields, we found
that ancestral *F/Xpk* gene from *Clostridium acetobutylicum* predicted by ASR  was the fittest gene sequence and
P*tkt* promoter was the best promoter to construct *C. tyrobutyricum* with the best growth, highest butyric acid yield and least carbon loss.

To achieve these, we fulfilled five targets as follows.

**Target 1:** **We selected two promising** ***F/Xpk*****g****enes from two strains**

*F/Xpk(BD)*(BBa\_K4119076) from *Bifidobacterium adolescentis*(*B**.**adolescentis*), and *F/Xpk(QS)* ( BBa\_K4886000)  from*Clostridium acetobutylicum* (*C**.**acetobutylicum*).

**Target 2: We constructed two*****C. tyrobutyricum*****strains with NOG pathway by transfection with
pMTL-P*****thl-F/Xpk(QS)*****or pMTL-P*****thl-F/Xpk(BD)*****plasmids**

Our results showed that both strains have better butyric acid
yield and less carbon loss compared to the native strain.
Between the two strain, the strain with pMTL-P*thl-F/Xpk(BD)* had better growth and product yield. Therefore, *F/Xpk(BD)* gene was the optimal gene for constructing NOG
pathway.

**Target 3: We found that among glucose, fructose and xylose,
the optimal carbon source for the above*****C. tyrobutyricum*****strains with NOG pathway was xylose.**

**Target 4: We constructed two** ***C. tyrobutyricum*****strains transfected with pMTL-P*****fba-F/Xpk(BD)*** **and pMTL-P*****tkt-F/Xpk(BD)*****plasmids**

Our results showed that the strain with P*tkt* to drive the expression of *F/Xpk(BD)* had better growth and product yields than those with
P*fba* or P*thl* promoter.

**Target 5: We further improved** ***F/Xpk(BD)*****gene sequence by ancestral sequence reconstruction
(ASR)**

Our results showed that the*C. tyrobutyricum* strain expressing ancestral *F/Xpk* gene predicted by ASR had better growth and product yields than
that expressing*F/Xpk(BD)*.

Here is the part list we constructed this year.

Table 1 Part list

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| No. | Name | Type | Description | Length |
| 1 | BBa\_K4886000 | Basic | *F/Xpk**(**QS**)**,* optimized*F/Xpk*gene derived from *Bifidobacterium adolescentis* ATCC 15703 | 2478 |
| 2 | BBa\_K4886001 | Composite | P*thl*-*F/Xpk(BD)*, expression of *F/Xpk* from *Clostridium acetobutylicum* ATCC824 with P*thl* promotor | 3036 |
| 3 | BBa\_K4886002 | Composite | P*thl*-*F/Xpk(**QS**)*, expression of optimized *F/Xpk* from *Bifidobacterium adolescentis* ATCC 15703 with P*thl* promotor | 3123 |
| 4 | BBa\_K4886003 | basic | P*fba**,* derived from *Clostridium tyrobutyricum* | 300 |
| 5 | BBa\_K4886004 | composite | P*fba*-*F**/**Xpk(BD)*, expressing *F/Xpk* from *Clostridium acetobutylicum* with *fba* promotor | 2739 |
| 6 | BBa\_K4886005 | basic | P*tkt**,* promoter derived from *Clostridium tyrobutyricum* | 300 |
| 7 | BBa\_K4886006 | composite | P*tkt*-*F**/**Xpk(BD)*  It is a part that is responsible for expressing F/Xpk from *Clostridium tyrobutyricum*. with tkt promotor. | 2739 |
| 8 | BBa\_K4886007 | basic | F/Xpk(ASR) | 2433 |
| 9 | BBa\_K4886008 | Composite | Pthl-F/Xpk (ASR) | 3036 |

## **2 Detailed Targets**

**Target 1*****F/Xpk*****gene screening**

Transition from EMP pathway to NOG pathway in *C. tyrobutyricum* requires importing *F/Xpk* gene from other strains. To obtain a highly fitted *F/Xpk* gene for *C. tyrobutyricum*, we selected two phosphoketolase-derived strains, *B. adolescentis* and *C. acetobutylicum*. *B. adolescentis* is used widely as a common source for phosphoketolase,
therefore its *F/Xpk* gene sequence has a high fitness for a wide range of
strains. *C. acetobutylicum* is closely related to our host strain, *C. tyrobutyricum*.  Considering the kinship, *F/Xpk* gene from *C. acetobutylicum* may have a better fitness in *C. tyrobutyricum*. *F/Xpk* gene derived from *C. acetobutylicum* is notated as *F/Xpk(BD)*. *F/Xpk* sequence derived from *B. adolescentis* was optimized by codon optimization ([https://www.genscript.com.cn](https://www.genscript.com.cn/)). The optimized sequence is notated as *F/Xpk(QS)*.

**Target 2****(Build and test)****Construction of NOG pathway in** ***C. tyrobutyricum*****:** ***F/Xpk(QS)*** ***vs******F/Xpk(BD)*****;**

**Build****: Plasmid Construction**

By using a recombinant plasmid *Pthl-adhE2* as a template, and X-pMTL-F and X-pMTL-R as primers, we
obtained a X-pMTL-*Pthl* vector (5461bp). *F/Xpk(QS)* (2478 bp) and *F/Xpk(BD)* (2391 bp) gene fragments were amplified from the genome of *C. acetobutylicum* and the genome of *B. adolescentis*, respectively, by PCR. DNA electrophoresis confirmed the
lengths of the PCR products. *F/Xpk(QS)* and *F/Xpk(BD)* gene fragments were ligated with the X-pMTL-*Pthl* vector into a pMTL-*Pthl-**F/Xpk(QS)* recombinant plasmid and a pMTL-*Pthl-**F/Xpk(BD)* recombinant plasmid, respectively, by Gibson assembly. Each
plasmid was transformed into *E. coli* JM109. After verification by colony PCR and DNA
electrophoresis (745 bp), positive colonies were transferred and
expanded. Gene sequencing was used to verify that the plasmids
extracted from the colonies were pMTL-*Pthl-**F/Xpk(QS)*and pMTL-*Pthl-**F/Xpk(BD)*.

![](https://static.igem.wiki/teams/4886/wiki/home/enginering1.png)

Figure 1 Genetic circuits of pMTL-*Pthl-F/Xpk(QS)* and pMTL-*Pthl-F/Xpk(BD)*

![](https://static.igem.wiki/teams/4883/wiki/eng6225.png)

Note: a)*F/Xpk(QS)*, b) *F/Xpk(BD)*, c) X-pMTL-*Pthl* vector

Figure 2 Verification of *F/Xpk(QS)* (2478 bp) and *F/Xpk(BD)* (2391 bp) gene fragments and X-*pMTL-Pthl* vector (5461bp) by gel electrophoresis

**Test****:  Transfection and function analysis**

By using *E. coli* CA434 as a donor strain, pMTL-P*thl*-*F/Xpk(QS)*  plasmid and pMTL-P*thl*-*F/Xpk(BD)* plasmid were transferred to *C. tyrobutyricum*, notated as Ct(*Pthl* *F/Xpk-QS*) and Ct(*Pthl* *F/Xpk-BD*), respectively.

*Ct(Pthl F/Xpk-QS)*, *Ct(Pthl F/Xpk-BD)* and native *C. tyrobutyricum* (the control) were fermented using glucose as carbon source.
Fermentation experiment showed that the growth of *Ct(Pthl F/Xpk-BD)* was better than that of *Ct(Pthl F/Xpk-**QS**)*, Figure 3.

Acetyl phosphate (AcP) is the final product of NOG pathway. AcP assay showed that the levels of AcP in *Ct(Pthl F/Xpk-BD)* and *Ct(Pthl F/Xpk-QS)* were both higher than the control, which was in accordance
with the growth of the strains. This indicated that NOG
pathway was open in the engineered strains, Figure 4.

HPLC experiment showed that after fermentation for 26h, the
yields of butyric acid were 3.35 g/L and 3.31 g/L in *Ct(Pthl F/Xpk-BD)* and *Ct(Pthl F/Xpk-QS)*, both higher than the yield in the control. The yields of
acetic acid were 1.36 g/L and 1.28 g/L in *Ct(Pthl F/Xpk-BD)* and *Ct(Pthl F/Xpk-QS)*, both lower than that in the control. Glucose consumption was
much higher in *Ct(Pthl F/Xpk-BD)* and *Ct(Pthl F/Xpk-QS)* compared with the control (Table 1). *Ct(Pthl F/Xpk-BD)* showed higher glucose consumption and butyric acid yield
than *Ct(Pthl F/Xpk-QS)*.

Butyric acid is a 4-carbon molecule, while acetic acid is a
2-carbon molecule. The increase in the butyric acid production
and glucose consumption and decrease in the by-product acetic
acid yield suggested that *Ct(Pthl F/Xpk-BD)* and *Ct(Pthl F/Xpk-QS)* both had higher efficiency of using glucose and less carbon loss
in glycosis compared with the native strain. In addition, *Ct(Pthl F/Xpk-BD)*  was better in reducing carbon loss than *Ct(Pthl F/Xpk-QS)*. Based on these results, we selected *F/Xpk(BD)* gene for our final engineered strain.

![](https://static.igem.wiki/teams/4883/wiki/eng8325.png)

Figure 3 Growth comparison of *Ct(Pthl F/Xpk-BD)* and *Ct(Pthl F/Xpk-QS)*

Figure 4 Levels of acetyl phosphate in*Ct(Pthl F/Xpk-BD)* and *Ct(Pthl F/Xpk-QS)*

 Table 1 Metabolite level in*Ct(Pthl F/Xpk-BD)* and *Ct(Pthl F/Xpk-QS)* after 26h fermentation

|  |  |  |  |
| --- | --- | --- | --- |
| Strains | Glucose（g/L） | Acetic acid（g/L） | Butyric acid（g/L） |
| Control | 0.82±0.82 | 1.43±0.21 | 3.06±0.24 |
| *Ct(Pthl F/Xpk-BD)* | 0 | 1.36±0.26 | 3.35±0.22 |
| *Ct(Pthl F/Xpk-QS)* | 0.09±0.09 | 1.28±0.24 | 3.31±0.24 |

Due to limited experimental conditions, it was not possible to
directly measure specific emissions of CO2 We estimated the value of carbon dioxide being fixed from
the yield of the obtained product butyric acid, based on the
principle of carbon conservation. The experimental results show
that the introduction of the NOG pathway increases the
production of butyric acid by 9.5% compared to the control
group. Therefore, assuming that the global demand for butyric
acid production is 80,000 tons, the formula calculates that the
carbon dioxide emissions can be reduced by approximately 6,941
tons. The detailed calculation is shown in the following PDF
file.

[CO2 emission reduction models.pdf](https://static.igem.wiki/teams/4883/wiki/co2-emission-reduction-models.pdf)

**Target 3****(Test)****C****arbon source****selection****for engineered** ***C. tyrobutyricum***

To find the best carbon source to grow the engineered *C. tyrobutyricum*, we compared the growth of *Ct(Pthl F/Xpk-BD)* and *Ct(Pthl F/Xpk-QS)* on different carbon sources, including glucose, fructose
and xylose. Fermentation experiment found that both strains had
better growth than the native strain (control) on all the carbon
sources, and fructose was the best carbon source for the growth of *Ct(Pthl F/Xpk-BD)* and *Ct(Pthl F/Xpk-QS)*, Figure 5.

HPLC was used to compare the product yields and carbon source
consumption of the strains cultured on different carbon sources
for 45h (Table 2). The yields of acetic acid in *Ct(Pthl F/Xpk-BD)* and *Ct(Pthl F/Xpk-QS)* were both higher than the control when cultured on
fructose, indicating a low flow in NOG pathway. In *Ct(Pthl F/Xpk-BD)* and *Ct(Pthl F/Xpk-QS)* cultured on xylose, the yields of acetic acid were both
lower than the control, and the xylose consumption was higher
than the control. Considering both the product yields of butyric
acid and acetic acid and the consumption of carbon source,
xylose was the best carbon source for NOG pathway in the
engineered strains.

These experimental results of carbon source selection showed
that engineered *C. tyrobutyricum* with NOG pathway had an enhanced ability to utilize
xylose, which is more beneficial to the utilization of cheap
substrates like plant straw in subsequent industrial
applications of the strain.

 ![](https://static.igem.wiki/teams/4883/wiki/eng10936.png)

Note: a) Glucose,b) Fructose, c) Xylose

Figure 5 Growth of *Ct(Pthl F/Xpk-BD)* and *Ct(Pthl F/Xpk-QS)* cultured on glucose, fructose and xylose

Table 2 Metabolite level in *Ct(Pthl F/Xpk-BD)* and *Ct(Pthl F/Xpk-QS)* cultured on different carbon sources for 45h

|  |  |  |  |
| --- | --- | --- | --- |
| Carbon sources and Products | Native strain | *Ct(Pthl F/Xpk-BD)* | *Ct(Pthl F/Xpk-QS)* |
| Glucose (g/L) | 0.41±0.41 | 0 | 0.09±0.09 |
| Acetic acid (g/L) | 1.43±0.21 | 1.36±0.26 | 1.28±0.24 |
| Butyric acid (g/L) | 3.06±0.24 | 3.31±0.22 | 3.35±0.24 |
| Fructose (g/L) | 3.75±0.35 | 2.71±0.32 | 2.53±0. 21 |
| Acetic acid (g/L) | 1.57±0.20 | 1.77±0.22 | 1.87±0.20 |
| Butyric acid (g/L) | 3.18±0.23 | 3.31±0.20 | 3.41±0.25 |
| Xylose (g/L) | 12.69±1.35 | 8.31±0.43 | 7.71±0.58 |
| Acetic acid (g/L) | 0.62±0.01 | 0.63±0.01 | 0.63±0.01 |
| Butyric acid (g/L) | 2.61±0.26 | 2.97±0.22 | 2.91±0.26 |

**Target 4****(Build and test)****Construction of NOG pathway in*****C. tyrobutyricum*****:** ***F/Xpk(BD)*****; P*****fba*****or** **P*****tkt***

**Build****:  Plasmid Construction**

The above experiments showed that *Ct(**Pthl* *F/Xpk-BD)* had better growth, higher butyric acid yield and lower carbon
loss than *Ct(**Pthl**F/Xpk-QS)*. So*F/Xpk(BD)* is a more fitted gene for *C. tyrobutyricum*. Therefore, we selected *F/Xpk(BD)* gene for our final engineered strain*.*

Since promoters can affect the transcription strength of genes,
we tried two other promoters, P*fba* and P*tkt*, to see if they were more suitable for expressing *F/Xpk* in *C. tyrobutyricum*.

By using a recombinant plasmid pMTL-*Pthl-**F/Xpk(BD)* as a template, and X-PN-F and X-PN-R as primers, we obtained a
X-*F/Xpk(BD)* vector (7670 bp). *Pfba* fragment (300bp) was amplified from the genome template
of *C.* *tyrobutyricum* using P-*Pfba*-F and P-*Pfba*-R as primers, by PCR. *Ptkt* fragment (300bp) was amplified from the genome template
of *C.* *tyrobutyricum* using P-*P**tkt*-F and P-*P**tkt*-R as primers, by PCR. DNA electrophoresis confirmed the
lengths of the PCR products. *Pfba*and *Ptkt* fragments were ligated with X-*F/Xpk(BD)* vector into a pMTL-*P**fba**-**F/Xpk(BD)* and pMTL-*P**tkt**-**F/Xpk(BD)* recombinant plasmids, respectively, by Gibson assembly. The
plasmid was transformed into *E. coli* JM109. After verification by colony PCR and DNA
electrophoresis (623 bp, 1396 bp), positive colonies were
transferred and expanded. Gene sequencing was used to verify
that the plasmid extracted from the colonies was pMTL-*P**fba**-**F/Xpk(BD)* and pMTL-*P**tkt**-**F/Xpk(BD)*.

![](https://static.igem.wiki/teams/4886/wiki/home/enginering6.png)

Figure 6 Genetic circuits of pMTL-P*fba*-*F/Xpk(BD)* andpMTL-P*tkt*-*F/Xpk(BD)*

![](https://static.igem.wiki/teams/4883/wiki/eng13300.png)

 Note: a) *Pfba* and *Ptkt* fragments, b) X-*F/Xpk(BD)* vector

Figure 7 Verification of *Pfba* and *Ptkt* fragments (both 300bp) and *X-F/Xpk(BD)* vector (7670 bp) by gel electrophoresis

**Test****:  Transfection and function analysis**

By using *E. coli* CA434 as a donor strain, pMTL-P*fba*-*F/Xpk(BD)* andpMTL-P*tkt*-*F/Xpk(BD)* plasmids were transferred to *C. tyrobutyricum*, notated as *Ct**(Pfba F/Xpk**-**BD)* and *Ct(Ptkt F/Xpk**-**BD)*, respectively.  *Ct**(Pfba F/Xpk-BD)*, *Ct(Ptkt F/Xpk-BD)* and *Ct(Pthl F/Xpk-BD)* were fermented using xylose as carbon source.

Fermentation experiment showed that the growth of *Ct**(Ptkt F/Xpk-BD)* was better than that of *Ct(Pthl F/Xpk-BD)*, and the growth of *Ct(Pfba F/Xpk-BD)* was worse than that of *Ct(Pthl F/Xpk-BD)* (Figure 8).

HPLC experiment showed that after culturing on xylose for
60.5h, the yield of butyric acid was 5.89 g/L in *Ct**(Ptkt F/Xpk-BD)*, higher than the 5.19 g/L yield in *Ct(Pthl F/Xpk-BD)* and the5.21 g/L in *Ct(Pfba F/Xpk-BD)*. *Ct**(Ptkt F/Xpk-BD)* showed higher xylose consumption than *Ct(Pthl F/Xpk-BD)*and*Ct(Pfba F/Xpk-BD)* (Figure 9).

The results implied that among *Pthl*, *Ptkt* and *Pfba*, *Ptkt* was the best promoter for *F/Xpk(BD)* gene to construct NOG pathway in *C. tyrobutyricum*, which had satisfactory growth, butyric acid production and carbon
conservation.

![](https://static.igem.wiki/teams/4883/wiki/eng14582.png)

Figure 8 Growth performance of *Ct**(Pfba F/Xpk-BD)*, *Ct(Ptkt F/Xpk-BD)* and *Ct(Pthl F/Xpk-BD)* on xylose

![](https://static.igem.wiki/teams/4883/wiki/eng14688.png)

Figure 9 Butyric acid yield and xylose consumption of *Ct**(Pfba F/Xpk-BD)*, *Ct(Ptkt F/Xpk-BD)* and *Ct(Pthl F/Xpk-BD)*

**Target 5 (Build and test): Improvement of** ***F/Xpk(BD)*****gene sequence by ancestral sequence reconstruction
(ASR)**

**Build: Ancestral sequence reconstruction (ASR) of*****F/Xpk(BD)*****and plasmid construction**

FireProt-ASR (<https://loschmidt.chemi.muni.cz/fireprotasr/>) was used by Ms. Yang to carry out ASR to further optimize the *F/Xpk(BD)* sequence. The phosphoketolase (*FXpk*) sequence (BBa\_K4119076) derived from *Clostridium acetobutylicum* was used as the input sequence. No essential residues were selected. Percent sequence identity was set to 30%-70%. Clustering identity was set to 0.9. Evolutionary model was set to WAG. RAxML (Randomized Axelerated Maximum Likelihood) was chosen as the phylogenetic tree inference tool.
Bootstraps were set to 500. The ancestral sequence of
phosphoketolase (*FXpk*) predicted by ASR was named *F/Xpk(ASR)*.

On the basis of plasmid pMTL-P*thl-F/Xpk(BD)*, *F/Xpk(BD)* fragment was replaced with*F/Xpk(ASR)*. Using pMTL-P*thl-F/Xpk(BD)* as the template and X-pMTL-F and X-pMTL-R as the primers, X-pMTL-P*thl* linearized vector (5461 bp) was amplified. Using PUC57 vector plasmid as the template and P-*F/Xpk(ASR)*-F and P-*F/Xpk(ASR)*-R as the primers, *F/Xpk(ASR)* fragment (2436 bp) was amplified. The*F/Xpk (ASR)* gene fragment and the X-pMTL-P*thl* linearized vector were ligated by Gibson assembly. Colony PCR was performed on the transformed
colonies using CX-FXpk-F-1 and CX-FXpk(BD)-R JP750 as the primers (957 bp). The positive colonies were transferred and the plasmid was
extracted. After gene sequencing verification, the recombinant plasmid was obtained:
pMTL-P*thl-F/Xpk(ASR)*.

![](https://static.igem.wiki/teams/4886/wiki/home/enginering10.png)

Figure 10 Genetic circuit of
pMTL-P*thl-F/Xpk(ASR)*

**Test****:  Transfection and function analysis**

By using *E. coli* CA434 as a donor strain, pMTL-P*thl-F/Xpk(ASR)*  plasmid was transferred to *C. tyrobutyricum*, notated as *Ct(Pthl F/Xpk-ASR)*.

Our experiment showed that*Ct(Pthl F/Xpk-ASR)* had slightly better growth than*Ct(Pthl F/Xpk-BD)* (Figure 11)*.* After fermentation for 42 hours,
*Ct(Pthl F/Xpk-ASR)* produced more butyrate than
*Ct(Pthl F/Xpk-BD),*with the average yieldincreasing from 3.52 g/L to 4.22 g/L by 20% (Table
3). *Ct(Pthl F/Xpk-ASR)*had reduced acetate synthesis. Our results indicated better product yield and more efficient carbon conservation in
*Ct(Pthl F/Xpk-ASR)*. So F/Xpk(ASR) sequence was more suitable than F/Xpk(BD)
in constructing NOG pathway in *C. tyrobutyricum.*

![](https://static.igem.wiki/teams/4883/wiki/eng17251.png)

Figure 11 Growth comparison of *C. tyrobutyricum* transfected with pMTL-P*thl-F/Xpk(ASR)* and that transfected with
pMTL-P*thl-F/Xpk(BD)*

Table 3 Product yields of *C. tyrobutyricum* transfected with pMTL-P*thl-F/Xpk(ASR)* and that transfected with
pMTL-P*thl-F/Xpk(BD)* after fermentation for
42 hours

|  |  |  |  |
| --- | --- | --- | --- |
| Strain | Glucose（g/L） | Acetate（g/L） | Butyrate（g/L） |
| *Ct(Pthl F/Xpk-BD)* | 0.03±0.03 | 1.52±0.22 | 3.52±0.24 |
| *Ct(Pthl F/Xpk-ASR)* | 0.05±0.02 | 1.35±0.24 | 4.22±0.23 |

## 3 **Conclusion**

Our project aims to reduce carbon emission of *C. tyrobutyricum* through non-oxidizing glycolysis, and also provide ideas
for carbon reduction of other strains. In the experiment, we
selected two sources of phosphoketoolase sequences and three
promoters to construct NOG pathway. We found that the combination of *F/Xpk* gene from*C. acetobutylicum* and P*tkt*promoter constructed the best *C. tyrobutyricum* strain with NOG pathway. We further optimized this F/Xpk
gene by ASR to achieve even better strain. The engineered
strains had very satisfactory growth, yield of butyric acid and
efficiency in using carbon source. We also found that the engineered strain performed best using xylose as a carbon
source, which is more beneficial to the utilization of cheap
substrates like plant straw in subsequent industrial
applications of the strain. So using the optimized ancestral *F/Xpk* gene and P*tkt* promoter, we can obtain a strain with  reduced carbon emission and high
carbon convervation using cheap carbon source, helping to offer
more economical and environmentally friendly industrial
fermentation for butyric acid production and mitigate the
greenhouse effect.

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Experiments

Experiments

* [1 Strains, plasmids and culture conditions](#inpage_dingwei1)
* [2 Construction and Enhancement](#inpage_dingwei2)

1 Strains, plasmids and culture conditions
:   M 109 constructed with recombinant plasmids and Escherichia coli CA434 used for conjugative transformation of
    Clostridium tyrobutyricum . The Clostridium tyrobutyricum used in the experiment is the strain L319 isolated by
    the laboratory itself, and the preservation number is GDMCC No: 62289 .

    The plasmids mainly constructed in this experiment are shown in the table below:

    Table 1 Mainly used plasmids

    |  |  |
    | --- | --- |
    | Plasmid name | main features |
    | pMTL-P thl - FXpk ( QS) | pMTL 82151, thiolase ( thl ) promoter, phosphoketolase ( FXpk ) from Bifidobacterium adolescentis. |
    | pMTL-P thl - FXpk ( BD) | pMTL 82151, thiolase ( thl ) promoter, phosphoketolase ( FXpk ) from Clostridium acetobutylicum. |
    | pMTL-P fba - FXpk ( BD) | pMTL 82151, aldolase ( fba ) promoter, phosphoketolase ( FXpk ) from Clostridium acetobutylicum. |
    | pMTL-Ptkt - FXpk ( BD ) | pMTL 82151, transketolase ( tkt ) promoter, derived from phosphoketolase ( FXpk ) of Clostridium acetobutylicum. |

    The medium used in this experiment to cultivate Escherichia coli is Luria-Bertani (L B ) medium , the
    composition is 1% peptone, 1% sodium chloride, 0.5% yeast powder; Clostridium tyrobutyricum is used to cultivate
    Clostridium Reinforced medium (R CM ), consisting of 1% peptone, 1% beef extract powder, 0.5% sodium chloride,
    0.5% glucose, 0.3% anhydrous sodium acetate, 0.3% yeast powder, 0.1% soluble starch, 0.05% semi Cystine
    hydrochloride, pH 6.5. The fermentation medium of Clostridium tyrobutyricum used is TGY medium, the composition
    is 50% peptone, 33% glucose, 16% yeast powder, 0.13% cysteine hydrochloride, pH 6.0.

    In this experiment, Escherichia coli was cultured in a Erlenmeyer shaker flask at 37°C at 200 rpm, and
    Clostridium tyrobutyricum was cultured statically in an anaerobic flask at 37°C.

2 Construction and Enhancement
:   PCR amplification system used in this experiment is shown in Table 2:

    Table 2 Experimental PCR amplification system

    |  |  |  |
    | --- | --- | --- |
    | Reagent | Usage amount | Final concentration |
    | PrimeSTAR Max Premix( 2 x ) | 2 5 ul | 1x \_ |
    | Primer 1 | 1 0-15 pmol | 0.2-0.3μM \_ |
    | Primer 2 | 1 0-15 pmol | 0.2-0.3μM \_ |
    | template | ＜ 200 ng |  |
    | Sterilized water | fill up to 50 µ l |  |

    PCR amplification program used in this experiment is shown in Table 3:

    table 3 Experimental PCR amplification procedure

    |  |  |  |
    | --- | --- | --- |
    | temperature | time | cycle |
    | 98 ℃ | 3 minutes |  |
    | 98 ℃ | 1 0 sec | 3 0-35 |
    | 55 °C | 5 sec or 15 sec |
    | 72 °C | 5 sec/kb |
    | 72 °C | 5 minutes |  |

    The plasmid extraction used in this experiment was purchased from Novozyme. The specific method is as follows:

    1. Take 1 - 5 ml of overnight culture (12 - 16 h) bacterial solution, add it to a centrifuge tube (prepared by
    yourself), and centrifuge at 10,000 rpm (11,500 × g) for 1 min. Discard the culture medium, upside down on
    absorbent paper to absorb the residual liquid. 2. Add 250 μl Buffer P1 to the centrifuge tube with the bacterial
    pellet (please check whether RNase A has been added to Buffer P1 first), and mix with a pipette or vortex.
    Thorough resuspension of bacteria at this step is critical to yield, and no bacterial clumps should be visible
    after resuspension. If there are bacteria lumps that are not thoroughly mixed, the lysis will be affected,
    resulting in low extraction volume and purity. 3. Add 250 μl Buffer P2 to step 2 , and gently invert up and down
    for 8-10 times to fully lyse the bacteria. Gently invert to mix. Vortexing can cause genomic DNA fragmentation,
    resulting in mixed genomic DNA fragments in the extracted plasmid. At this point the solution becomes viscous
    and clear, indicating that the bacteria have been fully lysed. Do not take more than 5 minutes to avoid damage
    to the plasmid. If the solution does not become clear, the lysis may be incomplete due to too many cells, and
    the amount of cells should be appropriately reduced. 4. Add 350 μl Buffer P3 to step 3 , immediately invert 8 -
    10 times gently to neutralize Buffer P2 completely. At this point a white flocculent precipitate should appear.
    Centrifuge at 12,000 rpm (13,400 × g) for 10 min. After Buffer P3 is added, it should be inverted and mixed
    immediately to prevent local precipitation from affecting the neutralization effect. If there are tiny white
    precipitates in the supernatant, centrifuge again and take the supernatant. 5. Place the spin column in a 2 ml
    collection tube. Carefully transfer the supernatant from step 4 to the adsorption column with a pipette, taking
    care not to absorb the precipitate, and centrifuge at 12,000 rpm (13,400 × g) for 30 - 60 sec. Pour off the
    waste liquid in the collection tube, and put the adsorption column back into the collection tube. 6. Add 500 μl
    Buffer PW1 to the adsorption column. Centrifuge at 12000 rpm (13400 × g) for 30 - 60 sec. Discard the waste
    liquid and put the adsorption column back into the collection tube. If the host bacteria are end A + (TG1, BL21,
    HB101, JM series, ET12567, etc.), these host bacteria contain a large number of nucleases, which are easy to
    degrade plasmid DNA, and this step is recommended. If the host bacteria are endA - host bacteria (DH5α, TOP10,
    etc.), this step can be omitted. 7. Add 600 μl Buffer PW2 (please check whether it has been diluted with
    absolute ethanol) to the adsorption column. Centrifuge at 12 0 00 rpm (13,400 × g) for 30 - 60 sec. Discard the
    waste liquid and put the adsorption column back into the collection tube. 8. Repeat step 7. 9. Return the Spin
    Column to the Collection Tube. Centrifuge at 12,000 rpm (13,400 × g) for 1 min to dry the adsorption column, in
    order to completely remove the residual rinse solution in the adsorption column. Residual ethanol in the rinsing
    solution will affect downstream enzyme reactions, such as enzyme digestion, enzyme chain, PCR, etc. This step
    cannot be omitted. 10. Place the spin column in a new sterile 1.5 ml centrifuge tube. Add 30 - 100 μl Elution
    Buffer to the center of the membrane of the column adsorption column. Let stand at room temperature for 2
    minutes, then centrifuge at 12,000 rpm (13,400 × g) for 1 minute to elute the DNA. The elution volume should not
    be less than 30 μl , less than 30 μl will lead to a decrease in elution efficiency. To obtain the highest yield,
    preheat the Elution Buffer to 55°C to improve the elution efficiency. In addition, the solution obtained by
    centrifugation can be added to the centrifugal adsorption column again, and step 10 is repeated . If the
    subsequent sequencing needs to be eluted with ddH 2 O, make sure that the pH of ddH 2 O is in the range of 7.0 -
    8.5, and the pH will decrease if the pH is lower than 7.0. Elution efficiency. 11. Discard the adsorption
    column, and store the DNA product at -20°C to prevent DNA degradation.

    Competent preparation method used in this experiment: 1. Streak on LB plates with strains stored in ultra-low
    temperature refrigerators, and place them in a 37°C incubator for static culture for 12-16 hours until the
    colonies grow to a diameter of 1-2mm. 2. Pick a colony with normal growth, inoculate it into 10-15mL liquid LB
    medium, and culture it in a shaker at 37°C (≥250rpm) for 12-16h. 3. Take 0.5mL of the above-mentioned activated
    bacterial solution and inoculate it into the prepared 50mL BTMedia , and fully shake the culture on a shaker at
    37°C until the bacterial solution OD600=0.5-0.6. For 50mL culture, please use a conical tube with a capacity
    greater than 250mL bottle, while the shaker speed exceeds 250rpm to ensure adequate ventilation. ¸Pay attention
    to monitoring the OD value of the bacterial solution. The time required to cultivate to OD600 of 0.5~0.6 is
    usually 2~3h. 4. Transfer the bacterial solution to a 50mL polypropylene plastic centrifuge tube and place it on
    ice for 5-10min. ¸ Do not put the bacterial solution in the glass Erlenmeyer flask directly on ice.

    The Escherichia coli transformation method used in this experiment :

    1. Take out the competent E. coli from the -80°C refrigerator , and quickly insert it into the ice box to
    dissolve it. 2. Add the DNA sample and mix gently, and place it on ice for 30 minutes. 3. Heat shock in a water
    bath at 42°C for 45 seconds, then quickly put it back into the ice for about 2 minutes, and be careful not to
    shake it. Add 700 μl of sterile medium without antibiotics and mix well. 4. Shake culture at 37°C for 1 hour
    (160~225rpm), draw an appropriate volume and evenly spread it on the LB agar medium plate containing the
    corresponding antibiotics. Incubate overnight at 37°C until the liquid is absorbed.

    The method for transforming Clostridium tyrobutyricum in this experiment:

    using a recombinant plasmid takes E. coli CA434 as the donor strain and Clostridium tyrobutyricum as the
    recipient strain. The specific conjugation method is: add 30 μl / mL chloramphenicol to the strain E. coli CA434
    carrying the target plasmid Cultivate in LB culture medium until the optical density (OD600 ) is 1.5 -2.0 , take
    3 mL of E. coli CA434 cells, centrifuge at 4000 x g for 2 min, wash with 1 mL of 50 mM phosphate buffer saline
    (PBS, pH 7.0) Wash 2 times. Then , resuspend the collected donor cells with 0.3 mL of recipient cells (OD600
    2.0-3.0). Spread the cell mixture on a dry RCM agar plate and incubate in an anaerobic bag at 37°C for 20 Use
    600 after h Dissolve the colony grown on the plate with mL PBS (7.0), and then re-apply on the RCM plate
    containing 15 μl/ mL thiamphenicol and 250 μg /mL D-cycloserine , culture anaerobically at 37 °C , To remove
    residual Escherichia coli CA434. Culture the plate for 48-96 hours until obvious colony growth is observed.
    Randomly pick 10 single colonies on the plate in RCM medium containing 15 μg / ml thiamphenicol, culture
    anaerobically at 37 ° C for 8-10 h, take about 1 μl for colony PCR identification, and identify After being
    correct, take an appropriate amount of bacterial liquid and expand it in fresh RCM medium containing 15 μg /mL
    thiamphenicol to obtain engineering bacteria .

    The experimental method of nucleic acid electrophoresis in this experiment :

    Weigh 0.8g agarose, dissolve in 100mL 1×TAE solution, heat in microwave until the solution is warm, shake
    evenly, continue heating until the solution boils, repeat 3 times. Add 5 µL of nucleic acid dye to the
    completely melted solution and shake well. Pour it into the well-arranged nucleic acid gel mold (comb and base
    included), and cool it for later use.

    Cell Metabolite Determination :

    The metabolites acetic acid and butyric acid were detected by high-performance liquid chromatography. Take 700
    µL of the bacterial solution in the culture medium and add it to a 2 mL centrifuge tube, centrifuge at 10,000
    rpm for 1 min in a centrifuge, and take it out. Take the supernatant and filter it through a membrane with a
    pore size of 0.22 micron into a chromatographic sample bottle, use high performance liquid chromatography (HPLC)
    to measure the concentration of sugars, butyric acid and acetic acid, and use an Aminex HPX-87H organic acid
    analysis column (100 mm×7.8 mm, BioRad , Marnes -la-Coquette) and maintained at 65°C with a mobile phase of 5 mM
    H 2 SO 4 and a flow rate of 0.6 mL/min to determine the product. Sampling was taken after a certain period of
    time and repeated several times. After obtaining the data, the cell metabolite curve was made with the sampling
    time as the abscissa and the concentration converted from the peak area as the ordinate.

    2.1 Construction of phosphoketolase recombinant expression plasmid derived from Bifidobacterium
    adolescentis

    The primers used in this experiment are listed in Table 4 :

    Table 4 Primers for constructing recombinant plasmids

    |  |  |
    | --- | --- |
    | Primer name | Primer sequence |
    | QS-FX-F | CATAAATATTTAGGAGGAATAGTCATGACGAGTCCTGTTATTGG |
    | QS-FX-R | ggacgcgtgacgtcgactctagagTCACTCGTTATCGCCAGC |
    | X- pMTL -F | GACTATTCTCCTAAATATTTTATGGATCC |
    | X- pMTL -R | ctctagagtcgacgtcacgc |
    | J-QS-FX-F | ATGACGAGTCCTGTTATTGGC |
    | J-QS-FX-R | CGACGAACTCGTACGGCT |

    P thl -adhE2 constructed by B B a\_ K4408008 in the database as a template, and X- pMTL -F and X- pMTL -R as
    primers, the X- pthl vector ( 5461 bp) was amplified . Using the Bifidobacterium adolescentis genome as a
    template and QS-FX-F and QS-FX-R as primers, a phosphoketolase ( Phosphoketolase , FXpk (QS) ) gene fragment (
    2478 bp) was amplified . The FXpk (QS) gene fragment and the X- pthl linearized vector were connected by Gibson
    assembly method . Carry out colony PCR (745 bp) on the transformed colonies , and the primers are J-QS-FX-F and
    J-QS-FX-R . The positive colonies with correct colony PCR were transferred and the plasmid was extracted, and
    the recombinant plasmid was obtained after sequencing verification : pMTL-P thl - FXpk (QS) .

    ![](https://static.igem.wiki/teams/4886/wiki/6382900980485411693090601.jpg "wps6.jpg")

    figure 1 Construction of recombinant plasmid pMTL-P thl - FXpk (QS)

    2.2 Construction of Recombinant Expression Plasmid of Phosphoketolase from Clostridium
    acetobutylicum

    The primers used in this experiment are shown in 5:

    table 5 Primers for constructing recombinant plasmids

    |  |  |
    | --- | --- |
    | Primer name | Primer sequence |
    | X- pMTL -F | GACTATTCTCCTAAATATTTTATGGATCC |
    | X- pMTL -R | ctctagagtcgacgtcacgc |
    | FXpk ( BD )-F | ATAAATATTTAGGAGGAATAGTCATGCAAAGTATAATAGGAAAACAT |
    | FXpk ( BD)-R | cgcgtgacgtcgactctagagTTATACATGCCACTGCCAATTAG |
    | CX-FXpk-BD-F3 | ATGAAGCCTTTCCTTCGTATTG |
    | CX- FXpk -R | gtcacgacgttgtaaaacgac |

    P thl -adhE2 constructed by B B a\_ K4408008 in the database as a template, and X- pMTL -F and X- pMTL -R as
    primers, the X- pthl vector ( 5461 bp) was amplified . Using the Clostridium acetobutylicum genome as a template
    and FXpk (BD)-F and FXpk (BD)-R as primers, the FXpk (BD) gene fragment ( 2391 bp) was amplified . The FXpk (BD)
    gene fragment and the X- pthl linearized vector were connected by Gibson assembly method . Carry out colony PCR
    (1000 bp) , the primers were CX-FXpk-BD- F3 and CX- FXpk -R . The positive colonies with correct colony PCR
    were transferred and the plasmid was extracted, and the recombinant plasmid was obtained after sequencing
    verification : pMTL-P thl - FXpk (BD) .

    ![](https://static.igem.wiki/teams/4886/wiki/6382900981832265948009613.jpg "wps7.jpg")

    figure 2 Construction of recombinant plasmid pMTL-P thl - FXpk (BD)

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Results

Experimental results

Construction of NOG pathway strain in Clostridium tyrobutyricum

![](https://static.igem.wiki/teams/4886/wiki/6382901029327559983241863.jpg "wps1.jpg")

Figure 5 Nucleic acid gel verification of engineering Clostridium Ct ( FXpk ) recombinant plasmids (a. FXpk fragment
derived from Bifidobacterium adolescent b. FXpk fragment derived from Clostridium acetobutylicum c. Linear vector X- pthl )

![](https://static.igem.wiki/teams/4886/wiki/6382901029344750161956250.jpg "wps2.jpg")

Figure 6 Comparison of Growth Performance of Engineering Strains Ct( FXpk -QS ) and Ct( FXpk -BD )

|  |  |  |  |
| --- | --- | --- | --- |
| strain | Glucose content (g/L) | Acetic acid (g/L) | Butyric acid (g/L) |
| control | 0.82±0.82 | 1.43±0.21 | 3.06±0.24 |
| Acetin source engineering bacteria | 0 | 1.36±0.26 | 3.3 5 ±0.22 |
| blue double source engineering bacteria | 0.09±0.09 | 1.28±0.24 | 3.3 1 ±0.24 |

Table 3 Product comparison of engineering strains Ct ( FXpk -QS ) and Ct ( FXpk -BD )

According to the growth curve in Figure 6, it can be seen that the growth of the engineering bacteria derived from Clostridium
acetobutylicum is better than that of the engineered bacteria derived from Bifidobacterium adolescentis. From the product
analysis in Figure 7, it can be found that the butyric acid production of the engineered bacteria derived from acetone butanol
has increased by 9.5% compared with the control. In addition, the production of by-product acetic acid decreased from 1.43 g /L
to 1.36 g/L, it can be concluded that the phosphoketolase derived from Clostridium acetobutylicum is a better strain.

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