PNAS: Endangered "anti-cancer strange wood" is expected to be "reborn" in fermenters

Hainan rough.

Photo courtesy of interviewee

Harringtin alkaloids have broad-spectrum anticancer effects and are mainly used clinically for the treatment
of acute leukemia.
However, plants that naturally produce these alkaloids are almost always endangered
.
Scientists are trying to find a way
to synthesize harringtin alkaloids.

On December 29, 2022, the Proceedings of the National Academy of Sciences of the United States of America (PNAS) published the latest results
of Qiao Fei's team, a researcher at the Institute of Tropical Crop Variety Resources of the Chinese Academy of Tropical Agricultural Sciences (hereinafter referred to as the Chinese Academy of Tropical Agricultural Sciences).
The team first analyzed the biosynthetic pathway of carbon skeleton molecules of Hainan rough harringt alkaloids, and clarified the biosynthetic pathway
of dopamine and 4-hydroxyphenylpropionaldehyde (4-HDCA).
The results of this study will lay the foundation for the
biosynthesis of this broad-spectrum anti-cancer drug.

Endangered "cancer-fighting strange wood"

Qiao Fei, co-first author of the paper, told China Science News that as anti-cancer drugs, homoharringtonine and harringtonine in harringtonic alkaloids have been included in China's pharmacopoeia, and the US FDA has also approved homoharringtine for clinical use
.

However, at present, harringtone alkaloids are mainly extracted from plants of the family Trischund family
.
The plants of this family mainly include tricuspid cedar and coarse beech, both of which grow extremely slowly, have narrow habitats, and are mostly on the verge
of endangerment.

Moreover, the content of harringstone alkaloids in such plants is very low, and it is very difficult
to extract large quantities from plants.
Chemical synthesis has also failed to solve the problem of its commercial production, resulting in the high price of the drug and becoming a major obstacle to its widespread clinical use
.

In order to solve the problem of synthetic harringtine, it is necessary to start from the molecular mechanism of its biosynthesis
.
Hainan Rough entered the sight of Qiao Fei's team in this way
.

Hainan rough beech is mainly distributed in Hainan Island and is an endangered plant
unique to China.
Because Hainan crude beech is the tree species containing the largest variety and highest content of tumor suppressing alkaloids in China, its refined drugs have special effects on various tumors, leukemia and acute lymphoma, and are recognized as the most potential natural anti-cancer drug source and the last line of defense for advanced cancer, so it is known as "anti-cancer strange wood"
.

Li Yide, a researcher at the Institute of Tropical Forestry of the Chinese Academy of Forestry, once wrote that in the 70s of the 20th century, China carried out the extraction and screening of wild plant anticancer drugs, and the Institute of Tropical Forestry of the Chinese Academy of Forestry and the 187 Hospital of the Chinese People's Liberation Army of the People's Republic of China jointly found that the two components of harringtonine and homoharringtine in Hainan rough quince have a good effect of inhibiting cancer cells, and the medicinal effect of Hainan rough bilgee produced in Hainan is much
better than that produced in Yunnan.
This research achievement won the first prize of the National Science and Technology Progress Award in 1985
.

It has been proved that the naturally synthesized ester alkaloids in Hainan rough beech tree contain the largest variety, a total of 11 kinds, especially the 4 ester bases reported at home and abroad that have significant tumor inhibitory effects on animals and clinics: harringtonine, homoharringtonine, deoxyharringrine and isoharringtone, the highest content in Hainan rough beech, animal and clinical trial effects are also more significant than other tree species, and have special curative effects
on various leukemias and acute lymphoma.

However, Hainan rough beech grows slowly, is scarce and scattered in the
tropical rainforest of Hainan Island.
Qiao Fei told China Science News that in the 70s of last century, two Hainan rough trees were planted in the botanical garden of the Academy of Thermal Sciences, but the diameter of the trunk is only 20 centimeters
.

"There are about thousands of plants distributed throughout Hainan, and they have been listed as national second-class protected plants
.
Moreover, it takes more than 200 years of growth to
bloom and mature in Hainan.
"Qiao Fei, who has been working in Hainan for 12 years, has the impression that he has only seen two adult Hainan rough plants
.
Hainan rough dioecious trees, those scattered female trees, often due to the lack of male trees around and unable to fertilize fruit.

How to obtain such anti-cancer drugs in large quantities without destroying wild plant resources? This became a difficult problem
that Qiao Fei's team wanted to solve.

Analysis of the carbon backbone synthesis pathway

According to the data, harringstone alkaloids belong to phenethyl isoquinoline alkaloids (PIAs), PIAs are an important class of natural products, there are three main types, including colchicine, harringtonine and high spinosterone base
.
These compounds have the same carbon skeleton, but the biosynthetic pathway of the carbon backbone has not been described in detail, and the research is still blank
.

Sun Huapeng, co-corresponding author of the paper and assistant researcher of the Institute of Tropical Crop Variety Resources of the Academy of Thermal Sciences, introduced that in order to solve the problem of artificial synthesis of harringtin alkaloids, since 2011, they have selected Hainan crude beech with the highest alkaloid content in tricariaceae as the research object, hoping to analyze its synthesis pathway and find related key genes
.

However, as a gymnosperm, Hainan crude beech genome is large, estimated to be about 20G in size, and no genome map has been obtained for reference
.
The analysis of the biosynthetic pathway of harringtone bioester base is still blank in the world, and there is no reference data
.
These bring great difficulties
to the analysis of synthetic pathways and the search for key genes.

"The biosynthesis of harringtone bioester bases is known to be divided into three main pathways
: upstream, middle and downstream.
" Qiao Fei introduced that the first step is to synthesize the carbon skeleton of the upstream pathway, which is basically the same in PIAs; In the second step, the midstream pathway delambs the carbon skeleton, and "twists" the carbon skeleton into different structures to form a parent nucleus; In the third step, the downstream pathway adds a "small tail" composed of a small molecule to the mother nucleus, so that the structure acquires activity and thus becomes a different type of harringstone bioester base
.

"We started with upstream pathways to search, function annotate, and evolutionary analysis of key amino acids, and speculated that they may belong to a family
of disease-related proteins.
" Qiao Fei explained that the disease-related protein turned out to be a class of proteins with unclear functions, which are often produced in large quantities under the condition that plants suffer from diseases, hence the name
.

With the full-length transcriptome, they validated family members of disease-course-associated proteins one by one
.
After long-term repeated experimental studies, they discovered that PIAs form a key enzyme PSS
in the carbon backbone.
In addition, they verified all the functions of 30 candidate genes in 7 steps in the upstream biosynthesis of PIAs parent nucleus, and opened up the entire synthesis pathway
.

It was found that the carbon backbone of the harringtin alkaloids was condensed by the key nodal compound 4-hydroxyphenylpropionaldehyde (4-HDCA) and dopamine
.

Since its discovery more than half a century ago, it has become clear that dopamine is derived from tyrosine in most plants and animals
.
But in plants that can synthesize alkaloids, no homology gene
has been found to synthesize dopamine.
This makes the biosynthesis of dopamine in alkaloid plants a mystery
to be solved.

Sun Huapeng introduced that after several rounds of screening and verification experiments, they clarified that the polyphenol oxidase PPO family undertakes the role
of catalyzing the synthesis of dopamine in alkaloid plants.
4-Hydroxyphenylpropionaldehyde is formed
by hydroxylation of cinnamic acid.
Catalyzed by the disease-related protein (PR-10), the two condensation formed the carbon backbone of PIAs, completing the upstream pathway
.

From triangular bottles to industrial fermenters

Hainan rough tissue culture seedlings
.
Photo courtesy of interviewee

"The analysis of the upstream synthesis pathway will greatly help the use of bioengineering technology to produce harringtin alkaloid drugs, which can not only reduce the price of drugs, but also reduce the collection of plants from the wild, thereby protecting wild plants
.
" Qiao Fei said
.

However, the midstream and downstream pathways have not yet been resolved, and Qiao Fei believes that this is still far
from the artificial biosynthesis of harringtin alkaloids.
Therefore, they plan to follow the example of the United States and South Korea using yew cell reactors to produce paclitaxel, and develop the use of Hainan crude beech cell engineering technology to create cell lines suitable for liquid suspension culture to induce the production of harringtin alkaloids
.
It is reported that this work has been relatively successful in
the laboratory stage.

"The next step is further research and development on how to increase production
.
In addition, from laboratory studies of triangular bottles to industrial fermenters, there are still many processes that need to be developed
.
Qiao Fei told China Science News that at present, similar fermentation processes are only mastered by a few countries, and there is no information to refer to as a technical secret, and they can only overcome difficulties step by step on their own
.

This work provides new ideas and methods for obtaining such alkaloids, and lays a solid foundation
for the protection and development of Hainan rough bilds.

"China's tropical regions are rich in plant resources and contain rich genetic resources
.
Especially in medicinal plants, many rare genetic resources still need to be tapped
.
After the gene resources and metabolic pathways are clear, modern biotechnology can be combined with modern biotechnology to realize modern drug production
through genetic engineering, cell engineering and other technologies.
Qiao Fei said
.

Related paper information: https://doi.
org/10.
1073/pnas.
2209339120