Human Practices

Overview:

In order to cope with the energy problems in today's society, countries are generally promoting energy transition, increasing the development and utilization of renewable energy sources, and promoting energy diversification and technological innovation in the energy field, so as to improve the efficiency of energy utilization and to reduce carbon emissions in order to promote sustainable development and environmental protection.
Butyl butyrate has a broad application prospect in the field of biofuel, based on the research of 2022iGEM team, we have done some new work on the development of butyl butyrate.
In order to solve the problem of the yield ratio of butyric acid and butanol, the two precursor products of butyl butyrate, we consulted Prof. Zhijun Zhang from East China University of Science and Technology (ECUST) in the field of bioengineering and Prof. Jiachang Lian, an expert in metabolic engineering from Zhejiang University, and got suggestions for modification in both metabolic engineering and promoter engineering.
In terms of Butyl butyrate as biofuel, after talking with Prof. Lian and reading the policies related to energy structure transformation, we also have a clearer idea of the appropriate direction of use and the direction we need to work on in the future.

1 Energy Crisis and Low Carbon Demand

Energy markets began to tighten in 2021 due to a number of factors, including a soaring economic rebound after the epidemic. Natural gas prices, electricity prices in some markets are at record highs, and oil prices have reached their highest levels since 2008. The current energy crisis is different from the oil crisis of the 1970s in that it involves all types of fossil fuels. Increasing the supply of clean energy and technology has a protective effect on consumers and reduces upward pressure on fuel prices to some extent.
According to the IEA, Biofuel demand in 2022 reached a record high of 4.3 EJ (170 000 million litres). Then, if net zero emissions are to be achieved by 2050, biofuel production would need to grow by an average of about 11% per year to reach more than 10 EJ by 2030. This is not a small increase, and not a small challenge for countries.

2 Current Status of Biofuel Utilization

According to the IEA, biofuels account for more than 3.5% of global transportation energy demand in 2022, with a 7% increase in global transportation biofuel capacity, the largest annual increase in more than a decade. Biofuel use has grown at a rate of nearly 5% per year over the past six years.
At the same time, investment in biofuels has also increased dramatically in 2022, and new capacity has reached its highest point in a decade at around 260 kg/d [2]. Several countries, including the United States, have also announced support for biofuel development [5]. For example, the U.S. Department of Energy (DOE) has announced that it will provide up to $118 million for 17 biofuel research projects at universities and companies working on advanced biofuels, with the aim of increasing local biofuel production and enhancing the carbon reduction role of biofuels in transportation and manufacturing [6].
In order to achieve the "dual-carbon goal" [4], the Chinese government issued the "Peak Carbon Action Plan by 2030", which mentions the need to optimize the energy structure, increase the proportion of non-fossil energy sources in the supply of energy structure, and strictly control the growth of coal consumption. Moreover, the document also mentions the need to strongly promote the substitution of traditional fuels by advanced bio-liquid fuels and sustainable aviation fuels.
It can be seen that while the demand for traditional energy sources such as coal and oil will gradually decrease, the biofuel market will also usher in the wind of demand.

In order to understand the prevalence of the energy shortage problem, the impact of the use of traditional fuels, and the views on biofuels, we also created a questionnaire, and analyzed the results of 1,208 responses to find that a relatively large number of people have a certain understanding of the problem of energy shortages, and agree that public awareness and action can alleviate the current situation of energy shortages. Moreover, most people are aware of new energy sources such as biofuels, and the number of people who advocate the development and use of new energy sources and support the development of biofuels is quite large.

Questionnaire

We have learned through communication with the 2022iGEM team (Worldshaper-NJBIOX) that Butyl butyrate can be used as an excellent fuel additive and even a valuable alternative fuel source, especially in the aviation industry [1]. However, the current conventional synthesis method for industrial Butyl butyrate is the esterification catalyst method using concentrated sulfuric acid as a catalyst, which inevitably adds burden to the environment and even to the economy. Therefore, the use of Clostridium tyrobutyricum as a chassis cell, which can produce Butyl butyrate from waste materials such as cellulose, shrimp and crab shells [3], can effectively solve these problems.
In the original synthesis pathway, there is no butanol synthesis pathway, so the Worldshaper-NJBIOX team gave Clostridium tyrobutyricum the ability to produce butanol by introducing an enzyme, adhE2, which means that the new pathway produces both butyrate and butanol from the same substrate.Worldshaper-NJBIOX found that the transfer of adhE2 to construct the butanol pathway resulted in a severe imbalance in the ratio of butanol to butyrate production, leading to a decrease in productivity, which they did not address at the time. This aroused our interest.

3 Control of the Yield Ratio of Butyl Butyrate Precursor Products Butyrate and Butanol

3.1 Metabolic Engineering Modification

We consulted Prof. Zhang on this issue.
The simultaneous production of butyrate and butanol will lead to the problem of decreased productivity, which can be tried to solve from two aspects. One is to try to adjust the production ratio of Butyrate and butanol to 1:1 for the same total amount, then theoretically the production of butyl butyrate will be maximized at this time. The second is to increase the metabolic flow so that the total amount of butyrate and butanol produced becomes larger, then the yield of butyl butyrate will also increase.
Regarding the method to reduce the by-product acetate, Prof. Zhang suggested that in the inhibition of the competitive pathway, the metabolic inhibition of the by-product acetate can be achieved by strengthening CoA transferase (cat1) in Clostridium tyrobutyricum, which indirectly increases the metabolic flow of butyrate and butanol.
Prof. Zhang said that we can also consider the conversion of acetate to acetyl CoA from the existing route. however, this route needs to consume ATP, and we need to consider whether there is enough ATP in the cell to maintain the cell's life activities, and if there is not enough, we need to look for a more efficient enzyme, or modify the enzyme to improve the efficiency.

Through the study of butyrate metabolic pathway in Clostridium tyrobutyricum, we found that there are a lot of genes related to butyrate production, including Dac, thl, hbd, bcd, crt. When faced with the choice of which pathway and gene to select, we sought help from Prof. Lian, an expert in metabolic engineering. Prof. Lian suggested that the acetylation of butyl butyrate can be regulated by strengthening the expression of deacetylase (Dac) to achieve the metabolic synthesis of butyrate and butanol, the precursors of butyl butyrate synthesis; and that the synthesis efficiency of the precursors can be enhanced by strengthening the expression of key genes in the synthesis pathway of butyrate and butanol, such as crt and bcd.

Inspired by Prof. Lian, we discussed with instructor and finally chose three methods to solve the problem of butyrate butanol yield ratio.
According to the experimental results, in the enhancement of the competitive pathway, the butyrate yield was increased with no change in butanol production by strengthening deacetylase (Dac); the synthesis efficiency of both precursors was simultaneously increased by strengthening the expression of key genes (bcd,crt). In the competitive pathway of inhibition, CoA transferase was enhanced in Clostridium tyrobutyricum to achieve metabolic inhibition of the by-product acetate.

3.2 Promoter Engineering

Since we conducted experiments in the same lab as Nanjing-BioX 2023iGEM and were inspired by their promoter engineering, we also tried to optimize the promoter for adhE2 expression. Here we chose the promoter Ptkt, which is also derived from the transketolase gene of Clostridium tyrobutyricum itself, for transcriptional expression control of adhE2 based on its weaker promoter properties than the thl promoter. Thus, it is expected that the resulting engineered strain will not have too large a gap in the production of butyrate and butanol, converging as much as possible to the optimally catalyzed 1:1 ratio of butyrate to butanol.

4 Future application for Butyl Butyrate

Butyl butyrate is commonly used as a flavor, solvent or in organic synthesis. However, in view of its special combustion properties, it can also be considered to play a role in the field of energy and fuel.
We found that biofuels are generally used in transportation, power generation, heat production, etc. In order to further clarify the direction of the fuel market in which butyl butyrate can be employed, we interviewed Prof. Lian Jiachang of Zhejiang University.
The professor pointed out that butyl butyrate has better engine suitability than other biofuels such as ethanol, so it is one of the directions worth considering for the development of aviation fuel or military fuel.
In general, the combustion of organic esters usually produces fewer pollutants, especially the greenhouse gas CO2, in the heat method and power generation, which means that butyl butyrate has a more excellent advantage in environmental protection, especially low-carbon emissions, and helps to combat climate change. In addition, Clostridium tyrobutyricum, as a chassis organism, can utilize waste such as shrimp and crab shells, straw, etc., which also helps to reduce dependence on fossil fuels and has the advantage of being a renewable energy source.
In vehicles and ships, butyl butyrate is mainly seen as an alternative to biofuels. In addition to its environmental and renewable characteristics, butyl butyrate can be mixed with petroleum fuels, and the ratio can be adjusted to suit different types of engines and vehicles without the need for large-scale equipment modification or replacement.
In aviation, according to the International Air Transport Association (IATA), it is predicted that 65% of carbon reduction will be achieved through the use of sustainable aviation fuels (SAF) by 2050 [7]. The potential of butyl butyrate as an aviation fuel is being researched and developed as a potential aviation fuel or aviation fuel additive with higher energy density and more environmentally friendly properties that can help reduce carbon emissions and dependence on fossil fuels in the aviation industry.

Proposed implementation direction

When we noticed that Clostridium tyrobutyricum has the characteristic of consuming shrimp and crab shell powder, we also found in our communication with the Nanjing-BioX team that Clostridium tyrobutyricum has a good ability to consume cheap carbon sources such as xylose after being modified by NOG pathway. This inspired us to link energy production with kitchen waste disposal. At present, the treatment of kitchen waste is time-consuming and laborious, and it also consumes a lot of energy. We have communicated with the sustainable development experts on this issue.

Mr. Yang introduced us the goal of sustainable development. At present, the disposal of kitchen waste is mostly carried out by landfill and incineration, which still consumes a lot of energy and resources.
Mr. Yang shared with us a drying device for kitchen waste. It is smaller and more efficient, but still requires the consumption of electrical energy. Through crushing and tumble drying, the moisture content of kitchen waste is quickly reduced, enabling its resourceful, minimized, and harmless treatment. But it still needs to consume energy.
This has given us great inspiration, and we expect that the Clostridium tyrobutyricum we have constructed can achieve the repeated use of biological energy supply, kitchen waste crushing and drying, and substrate supply for cultivation. That is, it can reduce energy consumption, instantly solve the waste generated by restaurants, and achieve the reuse of kitchen waste. The following is a product video we referenced. Of course, due to time constraints, our journey was forced to stop. In the future, we still hope to continue this.

With the development of renewable energy technologies and the advancement of energy transition, butyl butyrate has a better prospect as a renewable biofuel. However, there are still challenges to realize its wide application, such as production cost, marketing, supply chain construction and compliance certification. Through technological innovation and policy support, the prospects for the application of butyl butyrate as a fuel are expected to be sustained and promoted.

Reference

[1]Xin F, et al. Bioprocessing butanol into more valuable butyl butyrate. Trends Biotechnol. 2019, 37, 923–926.
[2]https://www.iea.org/energy-system/low-emission-fuels/biofuels
[3]https://2022.igem.wiki/worldshaper-njbiox/engineering
[4]https://www.gov.cn/zhengce/2021-10/24/content_5644613.htm
[5]LiMin Li, Biofuel market sees windfall in demand[N]. China Energy News，2023-2-13（12）.http://paper.people.com.cn/zgnyb/html/2023-02/13/content_25966076.htm
[6]U.S. Department of Energy Awards $118 Million to Accelerate Domestic Biofuel Production. https://www.energy.gov/articles/us-department-energy-awards-118-million-accelerate-domestic-biofuel-production
[7]International Air Transport Association,Net-zero carbon emissions by 2050.https://www.iata.org/contentassets/dcd25da635cd4c3697b5d0d8ae32e159/2021-10-04-03-cn.pdf
[8] https://new.qq.com/rain/a/20220310V02F2900