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 Ptkt 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 genes 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-Pthl-F/Xpk(QS) or pMTL-Pthl-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-Pthl-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-Pfba-F/Xpk(BD) and pMTL-Ptkt-F/Xpk(BD) plasmids

Our results showed that the strain with Ptkt to drive the expression of F/Xpk(BD) had better growth and product yields than those with Pfba or Pthl 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	Pthl-F/Xpk(BD), expression of F/Xpk from Clostridium acetobutylicum ATCC824 with Pthl promotor	3036
3	BBa_K4886002	Composite	Pthl-F/Xpk(QS), expression of optimized F/Xpk from Bifidobacterium adolescentis ATCC 15703 with Pthl promotor	3123
4	BBa_K4886003	basic	Pfba, derived from Clostridium tyrobutyricum	300
5	BBa_K4886004	composite	Pfba-F/Xpk(BD), expressing F/Xpk from Clostridium acetobutylicum with fba promotor	2739
6	BBa_K4886005	basic	Ptkt, promoter derived from Clostridium tyrobutyricum	300
7	BBa_K4886006	composite	Ptkt-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). 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).

 

 

 

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

 

 

 

 

 

 

 

 

 

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-Pthl-F/Xpk(QS)  plasmid and pMTL-Pthl-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.

 

 

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

 

 

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

Target 3 (Test) Carbon 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.

 

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  

 

Target 4 (Build and test) Construction of NOG pathway in C. tyrobutyricum: F/Xpk(BD); Pfba or Ptkt

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, Pfba and Ptkt, 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-Ptkt-F and P-Ptkt-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-Pfba-F/Xpk(BD) and pMTL-Ptkt-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-Pfba-F/Xpk(BD) and pMTL-Ptkt-F/Xpk(BD).

 

 

 

 

Figure 6 Genetic circuits of pMTL-Pfba-F/Xpk(BD) and pMTL-Ptkt-F/Xpk(BD)

 

 

 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-Pfba-F/Xpk(BD) and pMTL-Ptkt-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 the 5.21 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.

 

 

 

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

 

 

 

 

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-Pthl-F/Xpk(BD), F/Xpk(BD) fragment was replaced with F/Xpk(ASR). Using pMTL-Pthl-F/Xpk(BD) as the template and X-pMTL-F and X-pMTL-R as the primers, X-pMTL-Pthl 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-Pthl 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-Pthl-F/Xpk(ASR).

 

 

 

 

Figure 10 Genetic circuit of pMTL-Pthl-F/Xpk(ASR)

 

Test:  Transfection and function analysis

By using E. coli CA434 as a donor strain, pMTL-Pthl-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 yield increasing 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.

 

 

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

 

 

Table 3 Product yields of C. tyrobutyricum transfected with pMTL-Pthl-F/Xpk(ASR) and that transfected with pMTL-Pthl-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 Ptkt 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 Ptkt 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.