Abstract

This project aims to reduce 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 expressing F/Xpk were constructed and transferred into C. tyrobutyricum L319 to construct 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. The constructed NOG pathway was evaluated by experiments such as HPLC, SDS-PAGE, AcP assay and fermentation experiments.

 

1 Project aim

The purpose of this project is to reduce carbon emission during the fermentation of a valuable industrial strain, Clostridium tyrobutyricum (C. tyrobutyricum). We introduced a screened and optimized phosphoketolase (F/Xpk) gene well fitted for C. tyrobutyricum L319 into this strain by genetic engineering to construct a non-oxidative glycolysis (NOG) pathway. Unlike the native Embden-Meyerhof-Parnas (EMP) pathway which emits CO₂ during the glycolysis of the strain, the alternative NOG pathway enables complete carbon conservation while metabolizing glucose.

 

2 C. Tyrobutyricum and EMP pathway

C. tyrobutyricum is an anaerobic and heterotrophic bacterium. It is widely used in the food, health and chemical industries due to its ability to produce butyric acid through glycolysis. Its glycolysis is done via EMP pathway which is a key source of carbon emission during its fermentation. In EMP pathway, glucose breaks down into two pyruvates. Each pyruvate is converted to an acetyl-coenzyme A (acetyl CoA, AcCoA) through oxidative decarboxylation emitting one CO₂ molecule. For each glucose molecule, two CO₂ molecules are released [1].

 

3 NOG Pathway and phosphoketolase (F/Xpk)

NOG pathway is an artificial pathway proposed by Bogorad et al. to address the carbon loss problem in EMP pathway [2]. NOG pathway enables complete carbon conservation in sugar catabolism to acetyl-CoA without carbon emission, and can be integrated with EMP pathway. NOG pathway functions as a fructose-6-phosphate (F6P) shunt (Figure 1). Through carbon rearrangement, this pathway can convert 1 mol of F6P to 3 mol of AcP without any loss of carbon.

 

By comparison, we found that most of the key enzymes in NOG pathway proposed by Bogorad et al. [2] natively exist in C. tyrobutyricum. The functions of the absent key enzymes in C. tyrobutyricum can be carried out by exogenously introduced phosphoketolase (F/Xpk), which is originated from Bifidobacterium adolescentis and has both the Fpk and Xpk activity [3]. Phosphoketolase catalyzes the conversion of F6P to erythrose-4-phosphate (E4P) and acetyl-phosphate (AcP), as well as the conversion of xylose-5-phosphate (X5P) to glyceraldehyde-3-phosphate (G3P) and AcP.

 

 

 

 Note: 1: Phosphoketolase; 1a: fpk; 1b: xpk; 2: tal; 3: tkt; 4: rpi; 5: rpe; 6: tpi; 7: fba; 8: fbp.

 

Figure 1 NOG pathway and related enzyme genes (red arrows indicate lack of key enzymes in Clostridium tyrobutyricum)

  

4 Integration of NOG and EMP pathways

Here, we propose to construct NOG pathway in C. tyrobutyricum to integrate with the native EMP pathway (Figure 2). In EMP pathway, each F6P molecule is converted to two G3P molecules. The two G3P molecules are eventually converted to two AcCoA molecules releasing two CO₂ molecules.In the alternative NOG pathway we constructed, each F6P molecule is converted to three AcP molecules instead, resulting in producing three AcCoA molecule without any carbon emission.

 

 

 

Figure 2 The integration of EMP and NOG pathways [2]

 

4 Project design overview

Our project is carried out in two stages as shown in Figure 3.

 

Stage 1: Optimization of F/Xpk gene sequence

F/Xpk gene sequences with high fitness for C. tyrobutyricum L319 were obtained through strain screening and sequence optimization using codon optimization.

 

Stage 2: Construction and verification of NOG pathway

Plasmids with F/Xpk gene were constructed using a strong promoter Pthl and transferred into C. tyrobutyricum L319 to construct NOG pathway. Experiments such as SDS-PAGE, HPLC and fermentation experiments were used to evaluate the function of NOG pathway in the engineered strain to find the best gene sequence. Different promoters were tried to express this gene in C. tyrobutyricum L319 to find the best promoter. Ancestral sequence reconstruction (ASR) was used to further optimize this gene sequence.

 

 

 

 

 Figure 3 Overview of the project design  

 

5 Detailed design

Stage 1: Screening and optimization of F/Xpk gene

After the transformation of C. tyrobutyricum, the enzyme encoded by the well-adapted F/Xpk gene should not affect the growth of the strain itself while exerting its own enzyme function to construct NOG pathway. Moreover, due to the fact that one of the characteristics of C. tyrobutyricum is that they have a large industrial use in synthesizing butyric acid, after the well-adapted F/Xpk is transferred into the strain, the strain still needs to  maintain a high level of butyric acid production.

 

Seletion of  F/Xpk gene sequence was achieved via the following two steps:

Step 1: Strain screening

To obtain a highly fitted F/Xpk gene for C. tyrobutyricum, we have a choice of two phosphoketolase-derived strains, Bifidobacterium adolescentis (B. adolescentis) and Clostridium acetobutylicum (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.

 

Step 2: Sequence Optimization

The F/Xpk gene sequence obtained from step 1 was further optimized by codon optimization to improve the activity and fitness of F/Xpk.

  

Stage 2: Construction of NOG pathway

Step 1: Find the best F/Xpk gene

The selected F/Xpk gene sequences from the previous stage were used to construct different pMTL-Pthl-F/Xpk plasmids as shown in Figure 4. Pthl was used as the promoter, and pMTL82151 as the backbone. The plasmids were transferred into C. tyrobutyricum L319. Fermentation experiments, AcP assay and HPLC were used to compare the growth and metabolite levels of the strains. The F/Xpk gene that resulted in the best performance considering growth and carbon conservation was chosen for the final engineered strain.

 

 

 Figure 4  The pMTL-Pthl-F/Xpk plasmid

 

Step 2: Find the best promoter

The best F/Xpk gene sequence selected in step 1 was used to construct plasmids with different promoters, including Pthl, Pfba and Ptkt. These promoters were all native in C. tyrobutyricum L319. The plasmids were transferred into C. tyrobutyricum L319. Fermentation experiments and HPLC were used to compare the growth and metabolite levels of the strains. The promoter that resulted in the best performance considering growth and carbon conservation was chosen for the final engineered strain.

 

Step 3: Further sequence optimization: ancestral sequence reconstruction (ASR)

Ancestral sequence reconstruction (ASR) was employed to reconstruct the ancestral phosphoketolase (F/Xpk), using FireProt-ASR with the best F/Xpk gene sequence as the input sequence. Recombinant plasmid containing the ancestral gene was constructed and transferred into Clostridium tyrobutyricum (C. tyrobutyricum). Fermentation experiments and HPLC were used to compare the growth and metabolite levels of the strains expressing the best gene and the ancestral gene. The gene sequence that resulted in the best performance considering growth and carbon conservation was chosen for the final engineered strain.

 

 

By these steps, we constructed a C. tyrobutyricum strain with NOG pathway using the best combination of F/Xpk gene sequence and promoter. The strain would have excellent growth and carbon conservation. The carbon emission in the strain would be greatly reduced compared with the native strain.

 

6 Conclusion

This project attempts to reduce carbon emission during the fermentation of C. tyrobutyricum by constructing NOG pathway via the introduction of F/Xpk by genetic engineering. This artificial NOG pathway may effectively improve the efficiency of glycolysis of similar strains and reduce the production of carbon dioxide, making it a promising solution to reduce carbon emission in various industries, including biofuels and chemical production.

 

Reference