Overview

Through searching and consulting, we have learned about our country's emphasis and determination to reduce carbon emissions and develop green and low-carbon industries. Therefore, constructing the non-oxidative glycolysis (NOG) pathway in microbial cell factories to improve efficiency and reduce CO ₂  production is a favorable way to promote the low-carbon development of biomanufacturing industry.

At the suggestion of Prof. Yongkun Lv, we chose C. acetobutylicum to obtain the key enzyme F/Xpk , which proved to be more homologous to the host strain (C. tyrobutyricum) and provided a more efficient enzyme than that provided by the common source bacterium B. adolescentisis.

Prof. Wenming Zhang and Prof. Lidan Ye helped us in optimizing the pathway by providing advice on trying to change the promoter, and the results showed that the use of the fba promoter promoted the growth of the engineered bacteria and the production of butyric acid more than the thl promoter. When we encountered difficulties in verifying the NOG pathway by direct measurement of CO ₂ , Prof. Zhang and Prof. Lv gave us guidance, and we successfully conducted the experiment by measuring butyric acid and obtained the theoretical amount of "carbon conservation" by calculation.

Finally, we also collected public opinions, and most of the public are in favor of using new technologies to promote low carbon emissions, and are willing to contribute to the reduction of carbon emissions. However, the number of people who can really practice is not as optimistic as they said, which is  the starting point of our efforts to promote . W e hope to awaken the awareness of the people around us through their own power, so that we can really become a "low-carbon warrior".

1 Why We Care about Carbon Reduction?

It is well known that the emission of greenhouse gases above reasonable levels on a global scale could lead to a cascade of disastrous and permanent consequences.

According to the United Nations report on Africa's vulnerabilities and improvements, sea levels will rise 15-95 centimeters by 2085, leading to the inundation of 30% of coastal buildings, the extinction of one-third of the continent's living species, and the degradation of about 80% of 5,000 plant species due to warming [2].

As the most populous developing country, China's carbon emissions cannot be ignored. China's carbon emissions are visualized on Carbon Monitor's global high spatial resolution , near real-time carbon maps, as  well as from the World Bank's data that China is one of the highest carbon emitters. [3][4].

 

Figure1 Global high-resolution near-real-time carbon map

（ Carbon Monitor ,https://www.carbonmonitor.org.cn/ ）

 

In short, reducing carbon emissions is urgent and significant.

 

2 Project Feasibility Survey

We reviewed the information and found that the Chinese government and enterprises have been trying to control carbon emissions, and the development of low-carbon industries is a very promising measure. According to the OECD's forecast, by 2030, about 35% of chemicals and other industrial products will come from biomanufacturing [10]. The Chinese government believes that the biomanufacturing industry is expected to change the world's industrial manufacturing landscape in the fields of energy, chemicals, and pharmaceuticals, and transform the manufacturing of industrial products to a green, low-carbon, and sustainable development model [6][7][8][9].

In 2020, the Chinese government proposed the goals of "carbon peak" in 2030 and "carbon neutral" in 2060 [13][14], and included them in the national development plan. The State encourages green and low-carbon development in the industrial field, as well as the development of synthetic biology-related bio-economy[11], industry and low-carbon or zero-carbon technological innovation.  [12]. Therefore, the low-carbon innovation of the bio-manufacturing industry is also an important aspect of the realization of the "dual-carbon" plan.

In the study of related policies, we learned about the concept of carbon economy, and China's Ministry of Ecology and Environment also released the national carbon emissions trading quota implementation plan [16].  We were inspired by companies that specialize in providing solutions for carbon emission reduction optimization projects  [15],  i.e.,  the current bio-manufacturing industry is facing the need for transformation.So if we start from the improvement of the technology itself to achieve a more low-carbon way, it is also a feasible solution to help enterprises to achieve low-carbon transformation.

In order to get a more comprehensive and objective point of view, we interviewed Professor Ye Lidan of Zhejiang University and Professor Zhang Wenming of Nanjing Tech University.

Prof. Ye said that biomanufacturing is a solution to reduce carbon emissions in  itself compared to chemical manufacturing. The source of carbon emissions from the biomanufacturing industry generally refers to its upstream and downstream links, such as the the carbon emissions from energy fermentation process using coal to heat.  Instead, we have chosen to address the problem of carbon emissions from a technological route, which is a relatively novel means.

Prof. Zhang said that there are more people studying the NOG pathway, for example, as early as 2013, Bogorad et al. constructed the first in vivo and in vitro non-oxidative fermentation pathway and analyzed it with acetic acid as the target product, and the results showed that the yield rate of acetic acid  exceeded the theoretical rate of pyruvate dehydrogenase. In terms of technical theory, it is feasible to realize the low-carbon transition of industry by constructing the NOG pathway.

3 How Should We Choose the Key Enzymes?

Our instructor told us that the non-oxidative glycolysis (NOG) pathway is a carbon-chain-extended synthetic pathway that utilizes a phosphoketolase ( FXpk )-dependent sugar-phosphate cleavage and carbon rearrangement cycle to generate fully carbon-conservative acetyl-coenzyme A. Therefore, to construct the NOG pathway, the key is to find a suitable source of phosphoketolase.

Generally, phosphoketolase can be obtained from Bifidobacterium adolescentis .  B ut the   instructor suggests that we can find at least one more source of the enzyme. So, how do we find the right source of enzyme ?

In the selection of enzymes, two main issues need to be considered, the activity of the enzyme and its suitability for the chassis. From the point of view of suitability, it is possible to choose to use enzymes that are homogenous to the chassis, Clostridium tyrobutyrium.

 

We contacted Professor Lv from Zhengzhou University and he  gave us several suggestions。

Prof. Lv said that there are two main issues to consider when choosing an enzyme, the activity of the enzyme and its suitability for the chassis. We can choose to use the endogenous one from the chassis ( Clostridium tyrobutyrium ), or use the promoter, terminator and RBS that have been validated in the chassis.

With the suggestion of Prof.Lv and the  help of our instructor,in the end we chose the F/Xpk  gene  from  Clostridium acetobutylicum ,  which has high homology with the host strain (C. tyrobutyricum)：

 

4 How to increase flow in the NOG pathway?

We envisioned whether the NOG pathway could be enhanced in some way to achieve increased efficiency and effectiveness.

The general optimization approach can be achieved by directed evolution of enzymes or promoter engineering, therefor e we plan to achieve by directed evolution of enzymes , and our instructor advised us to select the Ancestral sequence reconstruction to optimize the enzyme which is relatively simple . To verify this idea, we consulted Prof. Lidan Ye of Zhejiang University.

 

"The NOG can be made the mainstream pathway by replacing the inducible promoter and adding inhibitors to down-regulate the EMP when the cells are growing relatively well."

However, Prof. Ye did not recommend us to do directed evolution of the enzyme because there may be no suitable high-throughput screening method for this enzyme (lack of obvious traits such as growth or color change of the bacteria), but we can consider introducing the key enzyme in the case of  down-regulating the natural pathway or inhibition of the growth of the bacterial strain to make the bacterium mainly rely on NOG for survival, and thus screen for the mutants corresponding to the best-growing bacterial strains.

For promoter engineering, Prof. Ye is more recognized.  She believes that inhibitors can be added when cell growth is better, thus utilizing the inducible promoter to make NOG the mainstream pathway. It is also possible to try to knock out part of the gene of EMP and observe the butyric acid yield and growth rate, and then make a choice.

 

 

Different promoters with different strengths were used to weaken the EMP pathway on the one hand and strengthen the NOG pathway on the other hand.

Instead of knocking out the EMP pathway, Prof. Zhang suggested us to adopt promoters with different strengths and weaknesses respectively to weaken the EMP pathway on one hand and strengthen the NOG pathway on the other.

Inspired by the two professors, we decided to adopt the promoter optimization scheme. The aldolase promoter (fba) and the transketolase promoter (tkt)   were used   respectively to replace the thiolase (thl) promoter to try to see if the NOG pathway could be enhanced .

5 How to Verify the Carbon Reduction Effect of the NOG Pathway?

Once the NOG pathway has been successfully constructed in the cell, we need to consider how to verify the carbon reduction effect of the pathway. The most direct way is to measure the amount of CO₂ produced by the engineered bacteria during the growth process, but during the actual measurement, we could not collect CO₂ completely, which would lead to errors in the measurement, and our lab did not have a more accurate measuring instrument.So we envisioned if we could directly measure butyric acid, the product of the strain. To verify this conjecture, we consulted with several professors.

 

Prof. Zhang from Nanjing Tech University said that it is feasible to measure the product butyric acid, in addition to detecting the product precursor - acetyl coenzyme A with a kit.

Prof. Lv from Zhengzhou University also said that CO ₂  emissions can be calculated by stoichiometry, which will simplify the experiment.

With a positive answer, we finally decided to measure the yield of butyric acid by HPLC to verify the specific effect of the NOG pathway.

6 Future application pr ospects

Currently, carbon reduction is in full swing and the manufacture of industrial products is facing the dilemma of transformation As a low-carbon industry, the biomanufacturing industry has been widely recognized and promoted in recent years. Clostridium typhimurium has a strong capacity to produce butyric acid and is also a production strain of fine chemicals such as butanol, butyl butyrate, ethyl acetate, isopropanol and hydrogen [10]. Compared to the traditional production of fine chemicals which is more dependent on petroleum industry products, microbial fermentation method can utilize some cheap substrates or wastes to further control the cost and pollution.The production of carbon dioxide from pyruvate decarboxylation in the glycolysis pathway is  the major sources of carbon loss in biorefining and microbial carbon metabolism as well as one of the major causes of carbon emissions [17] .  The construction of the NOG pathway in Clostridium typhimurium or similarly engineered strains not only achieves complete carbon conservation, but also significantly reduces microbial carbon dioxide emissions during fermentation and production, making it an effective way to achieve carbon reduction goals in various microbial manufacturing industries. In addition, the introduction of the NOG pathway have potential to give more acetyl-CoA which will further enhanced the fatty acid synthesis resulted into  high production of  many compounds or drug precursors with high value,such as lipstatin.

 

Carbon markets around the world have attempted to reduce greenhouse gas emissions through market-based measures in recent years. Currently, companies usually operate by investing in research and development of more environmentally friendly technologies [19]. The introduction of the NOG pathway can help biomanufacturing companies to achieve low-cost carbon emissions reductions due to its simplicity and economy of approach. At the same time, the remaining carbon allowances of enterprises can be sold in the carbon trading market, attracting more green investments and accomplishing the dual goals of economic development and emission reduction and energy saving [20]

 

 

8 Public Attitudes

We conducted a public survey in July on people's awareness of carbon emissions. We collected 1,507 valid responses from people of all ages, educational backgrounds and genders.

It is clear that the issue of carbon emissions has been recognized by the majority of people and that there is a strong willingness to contribute to carbon reduction. But it will take a lot more effort to get more people to actually take action. Because of this, we have incorporated the promotion of carbon reduction strategies and policy popularization in almost every event at Education.

Questionnaire results

Reference

[1] AR6 Synthesis Report: Climate Change 2023

[2] United Nations report on Africa's weaknesses and improvements

[3]https://carbonmonitor.org.cn/

[4] https://data.worldbank.org.cn/indicator/EN.ATM.CO2E.KT?end=2020&most_recent_value_desc=true&start=2020&type=shaded&view=map&year=2020

[5] https://news.cnstock.com/news,qy-202211-4986289.htm?continueFlag=901573bcdd0049eb6413d02cdd15cab7

[6] https://www.ndrc.gov.cn/xxgk/jd/jd/202205/t20220510_1324454.html

[7] http://www.csteelnews.com/special/1311/2022061002/202206/t20220615_63975.html

[8] https://www.gov.cn/zhengce/2021-10/27/content_5646697.htm

[9] https://link.springer.com/article/10.1007/s43979-022-00010-y

[10] JiayuLIU,ZhihanYANG,LeiYANG,LiyingZHU,ZhengmingZHU,LingJIANG. AdvancesinthedevelopmentofClostridiumtyrobutyricumcellfactoriesdrivenbysyntheticbiotechnology[J]. SyntheticBiologyJournal,2022,3(6):1174-1200,doi:10.12211/2096-8280.2022-022.

[11] 中国政府网，《“十四五”生物经济发展规划》,https://www.gov.cn/xinwen/2022-05/10/content_5689561.htm

[12] Zhihan YANG,et al,Production of Novel Single Cell Protein Feed Using Clostridium tyrobutyricum Fed on Discarded Shrimp Shells[J].Journal of Food Science and Biotechnology,2023(2):25-30

[13] 中国政府网，国务院关于印发2030年前碳达峰行动方案的通知，https://www.gov.cn/zhengce/zhengceku/2021-10/26/content_5644984.htm?eqid=f712a9940002632100000005646397d0

[14] 中国政府网，力争2030年前实现碳达峰，2060年前实现碳中和——打赢低碳转型硬仗，https://www.gov.cn/xinwen/2021-04/02/content_5597403.htm

[15] https://www.vitolenergy.com.cn/?bd_vid=12573275744524451454

[16] https://www.mee.gov.cn/ywdt/zbft/202303/t20230316_1019719.shtml

[17]  Bogorad I W, Lin T S, Liao J C. Synthetic non-oxidative glycolysis enables complete carbon conservation[J]. Nature, 2013, 502(7473): 693-697.

[18] Jatain I, Yadav K, Nitharwal R G, et al. A system biology approach for engineering non-oxidative glycolysis pathway in Streptomyces toxytricini for high lipstatin biosynthesis[J]. Bioresource Technology Reports, 2022, 19: 101188

[19] Wara M. Is the global carbon market working?[J]. Nature, 2007, 445(7128): 595-596.

[20] Bai C, Liu H, Zhang R, et al. Blessing or curse? Market-driven environmental regulation and enterprises' total factor productivity: Evidence from China's carbon market pilots[J]. Energy Economics, 2023, 117: 106432.