Purdue University team successfully analyzes the synthetic pathway of benzaldehyde in plants

  As early as the mid-19th century, people recognized the important role of aromatic compound monomers in aroma contribution, and began to use chemical methods to synthesize them to replace some rare or expensive natural fragrances, but it was not until the 20th century that synthetic fragrances were realized. mass production. Benzaldehyde is one of the earliest synthetic important aromatic substances.
  Benzaldehyde is the simplest structure in nature, and it is also the most commonly used aromatic aldehyde in industry. It is often described as having a special almond or cherry aroma. It exists widely in the flowers, fruits and leaves of many families and genera, and has the functions of attracting insect pollinators and inhibiting the growth of fungi.
  Although widely used as a commercial food flavoring and industrial solvent, benzaldehyde’s main use is in the synthesis of a variety of other compounds ranging from pharmaceuticals to plastic additives, and an important intermediate in the production of perfumes, fragrances, and in the synthesis of certain aniline dyes .
  Generally speaking, benzaldehyde is mainly obtained through chemical synthesis and natural extraction, which in turn includes plant and animal sources. The current main preparation route is the liquid chlorination or oxidation of toluene. Preparation methods that have been eliminated include incomplete oxidation of benzyl alcohol, alkaline hydrolysis of benzoyl chloride and addition of benzene to carbon monoxide. Chemical synthesis has many disadvantages such as unfriendly environment and complex reaction conditions.
  ”Benzaldehyde can also be produced by insects, non-insect arthropods and microorganisms. Compared with animal sources, the advantages of plant production of benzaldehyde are that the potential yield is huge, the production process is relatively convenient, the production conditions are not high, and it is easy to achieve green production. , and plant extract products directly used in the food industry have high safety and added value, and consumers are very easy to accept.” Huang Xingqi said.

  For many years, researchers only know that benzaldehyde in plants is synthesized from phenylalanine, but the gene for the direct synthesis of benzaldehyde has not been cloned. Many research groups have carried out correlation analysis from the transcriptome of multiple tissues and multiple time points, trying to screen some candidate genes and verify their functions, but they have not been successful.
  In February 2022, a study published in the journal “Nature Communications” successfully analyzed the biosynthetic pathway of benzaldehyde in plants.
  The first author of the article, Dr. Huang Xingqi, was admitted to Nanjing University in 2007. After graduating from an undergraduate degree, he continued his postgraduate study in Professor Lu Shan’s laboratory at the School of Biological Sciences of NTU, and received his Ph.D. in 2018. He then went to Purdue University for postdoctoral research. Under the leadership of Prof. Natalia Dudareva, Department of Biochemistry, College of Agriculture, he studied the secondary metabolism and regulation of plant volatiles.

Huang Xingqi
01Analysis of the synthetic pathway of benzaldehyde in plants

  To attract pollinators, petunias synthesize and release large amounts of volatile organic compounds containing benzene rings at night, of which benzaldehyde is the second highest. At present, it is generally believed that benzaldehyde can be synthesized through the β-oxidation or non-β-oxidation pathway of cinnamic acid. Huang Xingqi and his collaborators first used isotope labeling to prove that benzaldehyde in petunia is synthesized through the β-oxidation pathway of cinnamic acid. .
  ”Theoretically, the easiest way to synthesize benzaldehyde through the β-oxidation mechanism is to directly reduce benzoyl-CoA to benzaldehyde, so we tried to detect the activity of benzaldehyde synthase with crude petunia petal protein extract. Luckily Yes, the first coenzyme we tested, NADPH, was the reducing agent used by benzaldehyde synthase, so it was determined that the two substrates of petunia benzaldehyde synthase were benzoyl-CoA and reduced coenzyme II.” Huang Xingqi said.
  Although correlation analysis has helped identify a large number of secondary metabolic genes over the past five to ten years, many metabolic steps remain unresolved. Huang Xingqi and the others decided to use the substrates and reaction conditions discovered by the team for enzyme purification.

Partial purification of native PhBS from petunia

  Huang Xingqi pointed out that the enzyme purification was the most difficult step in this study. This enzyme showed extremely high in vitro enzymatic activity in previous experiments, so although benzaldehyde is the second most abundant compound in petunia fragrance, unlike other floral fragrance synthases, the enzyme directly responsible for the synthesis of benzaldehyde is in total The proportion of the protein was very low, so several previous purifications did not identify the correct gene. “We were able to see the corresponding bands on the protein gel by increasing the amount of starting plant material and optimizing the purification method.
  ” The components of enzymatic activity were identified by protein profiling, and two candidate proteins with NADPH-binding domains were finally locked. Since the sum of the molecular weights of these two proteins is just close to the apparent molecular weight of petunia petal benzaldehyde synthase ( When we got this result, we highly suspected that these two proteins are the enzymes we are looking for, and they may function by forming heterodimers,” said Huang Xingqi.
  Subsequent in vitro and in vivo experiments further verified their conjecture, that these two proteins have no benzaldehyde synthase activity when they exist alone, and only have enzymatic activity when they form heterodimers, and this heterodimerase responsible for the major flux of benzaldehyde synthesis in petunia.
  In addition, the team also proved through in vitro enzyme activities that the homologous proteins of these two subunits in other species, including Arabidopsis thaliana, tomato and amygdala, also form active benzaldehyde synthase by forming heterodimers. Moreover, most of the α and β subunits between species can also form functional heterodimers, indicating that the heterodimerase may be a relatively conserved way of synthesizing benzaldehyde in the evolutionary process.
  The vast majority of biochemical reactions in plant cells are catalyzed by enzymes encoded by a single gene, and there are only a handful of cases where heterodimeric enzymes are involved in metabolism. “The heterodimeric benzaldehyde synthase identified in this work is probably the first identified enzyme involved in the secondary metabolism of plant volatiles, which suggests that the secondary metabolism of our plants is likely to be stronger than we previously thought. It needs to be more complicated.”
  Huang Xingqi learned from it that, in the future, if he encounters any metabolic steps that are difficult to identify through correlation analysis, the idea cannot be limited to the level of single genes, and it is very likely that heterodimers or even multiple enzymes will appear again. complex situation.
  In view of this research, limited by the length and focus of the article, there are still many questions, such as whether the two subunits have catalytic activity? Or is only one of the subunits catalytically active? Or do the two subunits together form the active center of the enzyme? What is the structural basis for the dimerization of two subunits? Which key amino acids are involved in the interaction between proteins?
  In this regard, Huang Xingqi said, “These questions can only be revealed by the data of structural biology. Currently, the protein crystal structure of petunia benzaldehyde synthase is being analyzed together with the collaborating laboratory.”
02It is of great significance in actual production

  At present, the benzaldehyde used in the food flavor industry is mainly produced by semi-chemical synthesis methods, and the proportion of benzaldehyde from natural sources in practical applications is very low. The traditional natural source of benzaldehyde is mainly produced by the hydrolysis of amygdalin, but the benzaldehyde obtained by this method is usually polluted with a small amount of toxic compound hydrocyanic acid, which is difficult to remove.
  There are similar difficulties or disadvantages in plant synthesis of benzaldehyde. Higher concentrations of benzaldehyde have obvious toxicity to cells, and it is difficult to accumulate to a high concentration in cells. Plant cells will oxidize benzaldehyde to benzoic acid or reduce it to benzyl alcohol to reduce cytotoxicity, while benzoic acid and benzyl alcohol can be easily glycosylated for storage and further reduce toxicity. In addition, due to the high volatility of benzaldehyde itself, it may also be lost through volatilization when the anabolism is very strong.

Biosynthetic pathway of benzaldehyde in plants

  According to Huang Xingqi, “benzaldehyde is synthesized by heterodimeric benzaldehyde synthase without any toxic and harmful by-products, and can be safely used as a flavor additive in the food industry. This biosynthetic pathway may also be transferred to In yeast or other microorganisms, it has become a fermentation process widely used in food and beverage production.”
  At the same time, the research has an important potential application direction. Considering that the reduction reaction of benzaldehyde easily occurs in plant cells to generate benzyl alcohol, and benzyl alcohol can be further stored in cells after further glycosylation derivatization, this method can also be used as a production method of natural source benzyl alcohol.
  ”In fact, in this study, it was easy to obtain about 140 μg/g fresh weight by simply transiently transforming the petunia benzaldehyde synthesis pathway gene in tobacco leaves, and feeding it with phenylalanine substrate. It is entirely possible to obtain higher yields in tobacco leaves if deep synthetic biology optimization is carried out with yield as the primary goal.”