Global warming, rising sea levels and continued environmental degradation are all linked to carbon emissions. In China's total carbon emissions, microbial carbon emissions account for 7%, and most of the carbon emissions generated by microorganisms come from sugar degradation. Carbon sequestration is conducive to reducing carbon emissions, but it also reduces the efficiency of sugar degradation. This project attempts to introduce a highly active phosphoketolase (F/Xpk) gene into the heterotrophic microbial strain Clostridium butyricum L319, to construct an artificial NOG pathway with high metabolic flux. Compared with the native EMP pathway, this alternative pathway metabolizes glucose into acetyl CoA without carbon loss and carbon emissions. The approach used in this project can be further adopted to a variety of strains with similar metabolic carbon emission issues, thereby contributing to the reduction of global carbon emissions.

Nowadays overfull carbon emission has become a very serious problem because of its harmful impacts on the global environment. Carbon emission is one of the major causes for global temperature rise together with other greenhouse gas emission. Stemming from the immense burning of fossil fuels and tremendous industrial processes such as cement and steel production, carbon emission has increased rapidly over the past years (Figure 1). It potentially leads to melting glaciers and permafrost layer, sea level rise, saline water intrusion, increasing droughts, floods and ecosystem mutation.

Many countries and regions are taking the moves to control carbon emissions. China launches a policy aiming to reach over 1200 GW installed wind and solar power to partially substitute the usage of fossil fuels. US and EU also set up the carbon tariff proposals and legislations, aiming to build new “green trade barriers”. The main measures to reduce emissions include the development of renewable energy, energy conservation and energy recycling. In this project, we focuses on the experimental promotion of microbial carbon sequestration to reduce carbon emission.

 

 

Figure 1 Global annual CO2 emissions (https://ourworldindata.org/co2-emissions)

2.1 Microbial carbon sequestration

Microorganisms play a significant role in reducing carbon emissions by sequestering carbon through carbon fixation. There are three main types of carbon fixation: autotrophic, heterotrophic, and mixotrophic fixation (Figure 2). Autotrophic microorganisms convert CO2 into biomass by deriving energy from light or inorganic electron donors such as H2, H2S, S, NH4+, NO2-, FE2+[1]. Heterotrophic microorganisms are not themselves able of carbon fixation but are able to grow by using the carbon fixed by other organisms. Heterotrophs utilize organic carbon sources. For example, methylotrophs use reduced one-carbon compounds like methanol or methane for their growth. Mixotrophic microorganisms combines autotrophic and heterotrophic fixation, using organic carbon as a supplementary carbon and energy source while utilizing light energy to absorb and convert CO2.

 

 

 

Figure 2 Carbon and energy sources of microorganisms

 

2.2 Microbial carbon emission

While some microorganisms can reduce inorganic carbon from our environment by fixation, microorganisms also emit CO2 during respiration in aerobic and/or anaerobic states to gain energy. In China's total carbon emissions, microbial carbon emissions account for 7%, and most of the carbon emissions generated by microorganisms come from sugar degradation. A study has shown that due to climate change, bacteria can adapt to higher temperatures through enhancing the respiration rate and emitting more CO2 [2]. This could potentially escalate the carbon emission and climate change problems, resulting in a vicious cycle.

In this project, we aim to tackle the issue of microbial carbon emission. We focus on a heterotrophic and anaerobic strain, Clostridium tyrobutyricum. Clostridium tyrobutyricum extract energy from glucose by glycolysis, and release CO2 in the process of glucose metabolism.

3.1 Importance of limiting CO2 emission

Clostridium tyrobutyricum is a mesophilic, Gram-positive and heterotrophic bacterium growing under anaerobic condition. It belongs to the Clostridium genus. It mainly uses glucose and xylose as its carbon and energy source and produces butyric acid as a primary fermentation product and acetate, hydrogen, and carbon dioxide as the main by-products [3]. It is an important industrial microorganism and a novel probiotic. Because of its high yield of butyric acid, its fermentation is widely applied in the fields of fine chemical production and human health [4]. Due to its industrial traits, the control of CO2 emission during the large-scale industrial fermentation of Clostridium tyrobutyricum is quite important.

 

 3.2 CO2 emission during the EMP pathway

One important CO2 emission source of Clostridium tyrobutyricum is the Embden-Meyerhof-Parnas (EMP) pathway. EMP is a pathway of glycolysis. Glycolysis is an ancient metabolic pathway and an anaerobic energy source in practically all living organisms. In the EMP pathway, glucose breaks down into two three-carbon pyruvates, investing two ATPs and harvesting four ATPs and two NADHs. Each pyruvate is converted to a two-carbon acetyl-coenzyme A (acetyl CoA, AcCoA) through oxidative decarboxylation emitting one CO2 molecule (Figure 3). Therefore, for each glucose molecule, two CO2 molecules are released [3]. The enzymatic reactions in this pathway happen in milliseconds. With the large scale of fermentation, the net amount of carbon loss from Clostridium tyrobutyricum is huge. Thus, there is a great significance to reduce carbon emission in the EMP pathway.

 

 

 

Figure 3 Conversion of pyruvate to acetyl CoA through decarboxylation

Non-oxidative glycolysis (NOG) pathway is an artificial pathway proposed by Bogorad et al. to address the carbon loss problem in the EMP pathway. NOG pathway breaks down sugars or sugar phosphates into the maximum amount of C2 metabolites without carbon loss [5].

 

4.1 Structure of NOG pathway

NOG pathway is a cyclic pathway starts with fructose 6-phosphate (F6P) as the input molecule. With two more invested F6P molecules, three F6P molecules split into three acetyl phosphate (AcP) and three erythorse 4-phosphate (E4P) molecules by phosphoketolase. The three E4P molecules form two F6P molecules by carbon rearrangement. These two F6P molecules are the invested F6P in the starting step of NOG pathway thus forming the cyclic pathway. As a result, for each NOG cycle, one F6P molecule is converted into three AcP molecules without carbon loss (Figure 4). The three AcP molecules can then be converted to three AcCoA molecules without carbon loss.

 

4.2 Integration of NOG and EMP pathways

NOG enables complete carbon conservation in sugar catabolism to acetyl-CoA, and can be integrated with EMP pathway.

 

Bogorad first experimented a hybrid of NOG and EMP pathways in E. coli [5]. In the glycolysis of E. coli, glucose is phosphorylated to glucose-6-phosphate which is then rearranged to F6P. Each F6P molecule is converted to two glyceraldehyde 3-phosphate (G3P) molecules. In the EMP pathway, the two G3P molecules eventually convert to two AcCoA molecules and release two CO2 molecules. By adding an alternative NOG pathway, one F6P molecule is converted to three AcP molecules instead of two G3P molecules in the EMP pathway, resulting in producing one more AcCoA molecule without any carbon emission (Figure 4).

 

Integration of NOG pathway has been reported to bring increased carbon fixation in organic products in many microorganisms, such as free fatty acid production in Saccharomyces cerevisiae, P3HB yield in E.coli and acetate and lactam synthesis in E. coli [6-8].

 

 

 

Figure 4 The integration of EMP and NOG pathways [5] 

5.1 Project goal

The purpose of our project is to add an alternative NOG pathway in the heterotrophic microbial strain, Clostridium tyrobutyricum L319, by genetic engineering, so that the carbon emission issue in the EMP pathway can be solved. This project is important for the reduction of global carbon emission, since C. tyrobutyricum is a valuable industrial strain and similar engineering design might be expanded to other microorganisms.

 

5.2 Project feasibility

In the literature survey, we found that the NOG pathway has been preliminary realized in the auto-trophic carbon-fixing syngas-fermenting Clostridium which proves that the NOG pathway is feasible in Clostridium engineering [9].

By comparison, we found that most of the key enzymes in the NOG pathway, as proposed by Bogorad in engineering E. Coli [5], natively exist in Clostridium tyrobutyricum. This means Clostridium tyrobutyricum have the fundamental conditions for the construction of the NOG pathway. The functions of the absent key enzymes in C. tyrobutyricum can be carried out by phosphoketolases, which are known to have either F6P activity (termed Fpk) or xylulose 5-phosphate (X5P) activity (termed Xpk) (Figure 5).

 

 

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

Figure 5 NOG pathway and related enzyme genes (red arrows indicate lack of key enzymes in Clostridium tyrobutyricum)

 

5.3 Project overview

To integrate the NOG pathway into the Clostridium tyrobutyricum, we plan to engineer a highly active F/Xpk gene into Clostridium tyrobutyricum L319 to express phosphoketolases. This will be carried out by screening and optimizing F/Xpk gene sequences first and then constructing and transferring corresponding plasmids into the strain. We will evaluate the constructed NOG pathway in the bacteria through a variety of experiments and strengthen the pathway through providing alternative reducing power.

 

 

 

Figure 6 Scheme of our project design

 

5.4 Prospect

Our approach to reduce carbon emission in microorganisms can be applied to other strains with EMP pathway, or coupled to related pathways such as rGS，MCG. The beneficial effect of the NOG pathway is related to carbon conservation and productivity. NOG can be used in conjunction with CO2 fixation and other one-carbon assimilation pathways to achieve a 100% carbon yield to fuels and industrial chemicals. By using the NOG pathway, it is possible to create carbon conservative and productive systems, which can help reduce the carbon footprint of industrial processes and improve the efficiency of bioproduction.

Overall, the NOG pathway has the potential to provide a more sustainable and environmentally friendly approach to engineering metabolic processes.

1. Claassens N, Sousa D, dos Santos V, et al. Harnessing the power of microbial autotrophy. Nat Rev Microbiol. 2016, 692–706.

2. Smith TP, Thomas TJH, García-Carreras B, et al. Community-level respiration of prokaryotic microbes may rise with global warming. Nat Commun. 2019 Nov 12;10(1):5124.

3. Linger Jeffrey G, Ford Leah R, Kavita R, et al. Development of Clostridium tyrobutyricum as a microbial cell factory for the production of fuel and chemical intermediates from lignocellulosic feedstocks. Front Energy Res. 2020, 8.

4. Liu J, Yang Z, Yang L, et al. Advances in the development of Clostridium tyrobutyricum cell factories driven by synthetic biotechnology. Synthetic Biol J. 2022, 3(6): 1174-1200.

5. Bogorad I, Lin TS, Liao J. Synthetic non-oxidative glycolysis enables complete carbon conservation. Nature. 2013, 502: 693–697.

6. Zheng Y, Yuan Q, Luo H, et al. Engineering NOG-pathway in Escherichia coli for poly-(3-hydroxybutyrate) production from low cost carbon sources. Bioengineered. 2018 Jan 1;9(1):209-213.

7. Yu T, Liu Q, Wang X, et al. Metabolic reconfiguration enables synthetic reductive metabolism in yeast. Nat Metab. 2022, 4:1551–1559.

8. Miyoshi K, Kawai R, Niide T, et al. Functional evaluation of non-oxidative glycolysis in Escherichia coli in the stationary phase under microaerobic conditions. J Biosci Bioeng. 2023 Apr;135(4):291-297.

9. Wu C, Lo J, Urban C, et al. A synthetic acetyl-coa bi-cycle synergizes the Wood-Ljungdahl pathway for efficient carbon conversion in syngas fermentation. 2020. https://assets.researchsquare.com/files/rs-85001/v1_covered.pdf?c=1631843435