Recently, Professor Tan Zaigao from Shanghai Jiao Tong University and his collaborators successfully bypassed the inherent drawbacks of the natural pathway, achieving efficient synthesis of malonyl-CoA and its derivatives.

"Up to now, many domestic and international peers are very interested in the new pathway we have constructed, and they all hope to apply it to their own research," said Tan Zaigao.

In the study, the new pathway for malonyl-CoA synthesis constructed by Tan Zaigao and others effectively overcame the traditional problems existing in the natural pathway.

This not only increased the yield of malonyl-CoA in various chassis cells but also provided great possibilities for the development of malonyl-CoA and its related products.

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Since malonyl-CoA is a precursor for the synthesis of all fatty acid-like, polyketide compounds, and flavonoid compounds, this new pathway can be used for the efficient synthesis of these compounds.

At present, more than 10,000 different polyketide compounds have been discovered, and the new products derived from them are countless. According to statistics, more than 20 types of polyketide compounds (such as erythromycin, tetracycline, lovastatin, avermectin, etc.) have become commercialized drugs.

Among them, erythromycin is the most commonly used macrolide antibiotic in the pharmaceutical and veterinary industries. Statin drugs, including lovastatin, account for more than 80% of the total sales of lipid-lowering drug markets. Currently, the global annual sales of the aforementioned more than 20 drugs have exceeded 20 billion US dollars.

Flavonoid compounds are also widely present secondary metabolites, and more than 8,000 types of flavonoids have been discovered, most of which have good physiological activities such as antiviral, antibacterial, anti-inflammatory, anticancer, and anti-obesity.It is projected that by 2024, the flavonoid market will reach $1.2 billion. Therefore, the research group believes that the newly proposed biosynthetic pathway has a broad application prospect.

Natural pathways are not perfect

As mentioned earlier, malonyl-CoA is a common precursor for the synthesis of over 30,000 high-value compounds such as fatty acids, polyketides, and flavonoids.

However, many studies have shown that the intracellular malonyl-CoA is in a state of insufficient supply, which limits the efficient biosynthesis of high-value derivatives.

In almost all organisms, the synthesis of malonyl-CoA is very conservative. That is, under the catalysis of pyruvate dehydrogenase (PDH), pyruvate can form acetyl-CoA.

And under the catalysis of acetyl-CoA carboxylase (ACC), acetyl-CoA can form malonyl-CoA.

However, from the perspective of efficient biosynthesis, although the PDH-ACC pathway is a natural pathway, it is not perfect.

The catalytic rate of this pathway is often low, with activity only at the level of nmol/min/mg. Decarboxylation reactions produce the greenhouse gas carbon dioxide, leading to carbon source waste. And carboxylation requires the consumption of adenosine triphosphate, thus causing energy waste.In addition, the decarboxylation reaction is subject to strict regulation by a variety of intracellular metabolites. The existence of these drawbacks is the main factor leading to the insufficient supply of malonyl-CoA.

So, how can the supply of malonyl-CoA be increased? Previously, the academic community focused on improving the expression of the natural pathway.

There are mainly two methods adopted: first, overexpression of acetyl-CoA carboxylase; second, mutation of acetyl-CoA carboxylase to enhance its activity.

However, this cannot solve the inherent defects of low carbon efficiency and low energy efficiency in the natural pathway. Moreover, many studies have reported that overexpression of acetyl-CoA carboxylase can inhibit cell growth.

Avoiding the drawbacks of the natural pathway

Therefore, Tan Zai Gao's team did not continue to focus on optimizing the natural synthetic pathway.

Instead, they took a different approach and tried to construct a new synthetic pathway for malonyl-CoA, hoping to increase the synthesis rate of malonyl-CoA while avoiding the drawbacks of the natural pathway.

In the research, the first thing in front of them was to select the substrate for malonyl-CoA synthesis. Whether the intracellular metabolic flux is large enough is the main selection principle.In cells, pyruvate is not only a very common central metabolite but also has a very large synthetic flux. Therefore, the team chose it as the synthetic substrate.

However, the generation of malonyl-CoA from pyruvate cannot be achieved in one step, and it needs to go through several steps in between.

To avoid the waste of carbon sources caused by the release of carbon dioxide, they no longer consider using decarboxylation reactions, so they chose a C3 type on the intermediate.

Among many C3 compounds, they found that 3-oxopropionic acid is very likely to be synthesized into malonyl-CoA in one step, and pyruvate can also be obtained by a one-step transamination reaction to 3-oxopropionic acid.

That is to say, under ideal conditions, for pyruvate, it only needs to go through two steps to synthesize malonyl-CoA.

After determining the intermediate product, the research group searched the database for possible reactions involved. The results showed that for the first step of the above reaction, it can be completed by a transamination reaction; for the second step of the above reaction, there has been no relevant report before.

However, some studies have shown that under the action of MCR-C (the C-terminal fragment of malonyl-CoA reductase), 3-oxopropionic acid can be synthesized into malonyl-CoA.

The research group speculated: catalysis for the related reverse reaction may also be a feasible way. After some calculations, they found that the reaction from 3-oxopropionic acid to malonyl-CoA can find some basis in thermodynamics.

Therefore, they selected this type of enzyme as a candidate enzyme for the second step of the reaction. For the natural pathway, it adopts a "C3 (pyruvate) - C2 (acetyl-CoA) - C3 (malonyl-CoA)" pathway that first decarboxylates and then carboxylates.

The team designed a new pathway of "C3 (pyruvate) - C3 (3-oxopropionic acid) - C3 (malonyl-CoA)", which gets rid of the drawbacks of carbon source waste and energy consumption.Later, they named this pathway the NCM pathway (non-carboxylative malonyl-CoA formation pathway).

Next, they began to explore enzymes, using candidate enzymes for in vitro reactions. And by the increase in the absorption value at 340nM of the reduced coenzyme II synthesized by the reductase-catalyzed reduction reaction, they judged the strength of the candidate enzymes.

And by using liquid chromatography-mass spectrometry technology to detect the products of in vitro reactions. Under this dual verification, the feasibility of the NCM pathway in vitro was proven, and the activity was very high, reaching the level of micromol/min/mg, which is 1000 times that of the natural synthetic pathway (nmol/min/mg level).

Received strong support from two external teams

In fact, in the initial detection of liquid chromatography-mass spectrometry technology, they always could not detect malonyl-CoA. The first author of this paper, Li Jian, who is also the first doctoral student recruited by Tan Zai Gao at Shanghai Jiao Tong University, thought there might be the following two reasons:

One is that the detection signal of malonyl-CoA is not strong, and the malonyl-CoA synthesized by in vitro reaction is too little, so it cannot be detected;

The second is that malonyl-CoA is too unstable and has decomposed a lot during the processing process.To address these issues, they decided to conduct most of the experiments under conditions at 4°C and to enrich the in vitro reaction products. After these optimizations, they finally successfully detected the target products.

The in vitro experimental results demonstrated that the NCM pathway has a certain degree of feasibility. But can it also be applied within cells?

To accurately measure the synthesis flux of malonyl-CoA within cells, C13 metabolic flux analysis is required.

Considering that Dr. Yang Chen from the Excellence Innovation Center for Molecular Plant Sciences, Chinese Academy of Sciences, is an extremely authoritative expert in C13 metabolic flux analysis.

Therefore, the Tan Zaigao team began to seek cooperation with Dr. Yang Chen's team. Under the guidance of Dr. Yang Chen, the research by Ph.D. student Dong Wen Yue showed that after introducing the NCM pathway into E. coli, 57% of the malonyl-CoA inside the cells was produced through the NCM pathway, which directly proves that the NCM pathway is feasible within cells.

In addition, overexpression of the natural pathway enzyme acetyl-CoA carboxylase will inhibit cell growth. Will overexpression of the NCM pathway have a similar situation?

After a series of studies, they found that overexpression of the NCM pathway did not significantly inhibit cell growth.

So, will the introduction of the NCM pathway affect the robustness of the cells? Experiments have shown that after introducing the NCM pathway into E. coli, even in a growth-inhibiting environment caused by unfavorable growth conditions such as organic acids, toxic polyketide compounds, and high osmotic pressure, the cells' tolerance to this environment can be improved.

Under these stress conditions, the strains overexpressing acetyl-CoA carboxylase grow very slowly.

Subsequently, the Tan Zaigao team also tested the application of the NCM pathway in the synthesis of malonyl-CoA derivatives.In the model strain Escherichia coli, they utilized the NCM pathway for the synthesis of malonyl-CoA derivatives such as caprylic acid, resorcinol, flavomycin, and pentadecenyl, and found that the yields of these products were significantly improved.

So, can the application of the NCM pathway be more extensive? Can it play a role in different chassis cells?

Later, they planned to test the effect of the NCM pathway in Streptomyces because Streptomyces is a natural polyketide synthesis chassis, and many commercial drugs are polyketide compounds.

To this end, Tan Zaigao's team began to seek cooperation with Professor Liu Tiange's team from Shanghai Jiao Tong University. Under the guidance of Professor Liu Tiange, Ph.D. candidate Mu Xin from Wuhan University introduced the NCM pathway into Streptomyces fulvissimus to produce natamycin, and introduced the NCM pathway into Saccharopolyspora erythraea to produce spinosad.

The results showed that the yields of both natamycin and spinosad were significantly improved. Among them, the yield of spinosad in the shake flask reached 4.6g/L, which is the highest level reported so far, and also means that they have achieved industrial mass production.

Finally, the related paper was published in Nature Catalysis with the title "A non-carboxylative route for the efficient synthesis of central metabolite malonyl-CoA and its derived products" [1].

Shanghai Jiao Tong University Ph.D. student Li Jian, Wuhan University Ph.D. student Mu Xin, and Ph.D. student Dong Wen from the Chinese Academy of Sciences' Center for Excellence in Molecular Plant Sciences are the co-first authors.

Tan Zaigao, a researcher at Shanghai Jiao Tong University, Professor Liu Tiange, and Researcher Yang Chen from the Chinese Academy of Sciences' Center for Excellence in Molecular Plant Sciences are the co-corresponding authors.

At present, Researcher Tan Zaigao has received support from the National Natural Science Foundation of China in 2024. Therefore, in addition to the NCM pathway, they also plan to use other types of substrates to achieve efficient synthesis of malonyl-CoA.At present, they have successfully created artificial synthetic pathways from various substrates to malonyl-CoA.

Compared to the natural pathways, these pathways exhibit significant catalytic advantages. Therefore, the next step will be to carry out the efficient synthesis of malonyl-CoA derivatives.