This project aims to improve the use of longan(Dimocarpus longan Lour.), by genetically modifying longan to produce astaxanthin, and astaxanthin development of a skin care products.

1.1 Introduction to Astaxantn

Astaxanthin is a ketone secondary carotenoid, which is a fat-soluble pigment and is divided into two forms: free state and esterified state. Astaxanthin has a unique molecular structure, the molecule contains thirteen carbon-carbon conjugated double bonds, and there is an oxygen-containing six-membered ring at each end, each six-membered ring is connected with a hydroxyl group and a ketone group, and the hydroxyl group is located at the A position of the carbonyl group, forming a hydroxyketone conjugated structure. Astaxanthin has a very long conjugated double bond and -hydroxy ketone structure, which has a more active electronic effect, which can provide electrons to free radicals and react with free radicals, thereby scavenging free radicals, terminating the chain reaction of free radicals, and playing an antioxidant role. Unlike other carotenoids, astaxanthin has a "polar-non-polar monopolar" linear molecular structure, and the phospholipid bilayer is also a "polar-non-polar monopolar" molecular structure. These unique chemical structures explain some of its properties[1].

1.2 Nature of Astaxanthin

Antioxidant active

Natural astaxanthin has an active structure of electron-effector molecules that can bring electrons to free radicals or attract unpaired electrons to free radicals, this in turn stabilizes the free radicals and returns them to normal atoms without further damaging the body’s cells. Therefore, astaxanthin molecules are both lipophilic and hydrophilic. Astaxanthin molecules can penetrate the blood-brain barrier and enter the cell membrane or between the hydrophilic and hydrophobic layers of the mitochondrial membrane. The transmembrane is embedded in the membrane’s Lipid bilayer. This unique structure allows it to bind to the cell membrane from the outside in. In the cell membrane, the long polyene chain of Astaxanthin can interact with the radical to convert free radicals into more stable products. Outside the cell membrane, the terminal rings also stop the free radical chain reaction. Therefore, astaxanthin can effectively eliminate the free radicals inside and outside the cell membrane, protecting the cell membrane from oxidative damage[16]. This is why astaxanthin has strong antioxidant activity compared with other Carotenoid.

It is worth mentioning that under certain conditions, the common antioxidants at low doses of the condition of antioxidant, once reached a certain dose, antioxidant has a pro-oxidation, resulting in adverse effects on the body caused by the oxidation reaction. But astaxanthin only has antioxidant, will not become an oxidant, no side effects, obviously superior to other antioxidants. In astaxanthin molecule, there are mainly conjugated double bond, hydroxyl group, and unsaturated brewing group which connect the end of dry conjugated double bond chain. The hydroxyl group and ketone group constitute α-hydroxyketone. These structures have active electronic effect, so they can more effectively quench the singlet reactive oxygen species with high oxidation ability and other free radicals in the environment. Astaxanthin is 800 times more effective at scavenging free radicals than coenzyme Q10,6,000 times more effective than vitamin C and 500 times more effective than vitamin E, known as “super vitamin E”[18][19].

Enhance immune function

Researchers at the Kanazawa University Health Care Research Center gave mice a Steatosis high-cholesterol diet for three months, they were compared with those fed astaxanthin mixtures in high-cholesterol diets[2]. The results showed that compared with the non-mixed Astaxanthin Group, the mixed astaxanthin group reduced liver fat deposition by about 50% and was not easy to get fatty liver. In addition, the researchers believe that the Lipid peroxidation formed by the accumulation of lipids is inhibited by more than 80% , through the transformation of inflammatory macrophages into anti-inflammatory macrophages by inflammatory macrophages, it suppresses fatty liver to Steatosis[2].

Heart health

High blood Low-density lipoprotein levels are associated with an increased risk of arteriosclerosis. The Low-density lipoprotein in the plasma is not fully oxidized, but the oxidation of the Low-density lipoprotein is thought to contribute to the formation of arteriosclerosis, so astaxanthin supplementation leads to higher levels of high-density lipoprotein in the plasma, which increases the levels of Low-density lipoprotein and high-density lipoprotein cholesterol, which also contributes to heart health[2].

Protection of eyesight

Age-related macular degeneration and senile cataract are two common causes of visual impairment and blindness. These two kinds of diseases, and by the human eye light-induced oxidation. An animal experiment showed that astaxanthin was able to accumulate in the mammalian retina through the blood-brain barrier, providing effective protection against UV damage to the eye and oxidation in retinal cells [2] .

The anti-inflammatory effect

The anti-inflammatory effect of astaxanthin is closely related to its antioxidant activity. As mentioned earlier, when skin is exposed to ultraviolet light for a long time, it will become dry and rough, and wrinkles will worsen. This is because of the excessive accumulation of reactive oxygen species in the epidermis and dermis, iL-1, IL-6, IL-8 and inflammatory cytokines such as Tumor necrosis factors (TNF) are released, leading to Keratinocyte, the gene expression and enzyme activity of the matrix metalloproteinase family, including MMP1, MMP3 and MMP9, were increased. Through the further accumulation of MMPs, the activity of elastase increased[9] , collagen and elastic fiber degradation, wrinkles gradually formed, loss of skin elasticity. Elevated levels of L-1a in the skin induce other pro-inflammatory cytokines, which can lead to dry skin. Astaxanthin can effectively inhibit the expression of inflammatory factors such as Interleukin-8 and Tumor necrosis factors (TNF) through a variety of mechanisms, thus enhancing the body’s ability to prevent and resist inflammation. Astaxanthin, either directly or indirectly, inhibits matrix metalloproteinase expression and protects dermal extracellular matrix (i.e. collagen, elastic fibers and glycosaminoglycans) from degradation, thus delaying aging[3].

Anti-photoaging effect

Anti-photoaging effect astaxanthin in addition to anti-oxidation, anti-inflammation, there is a function of anti-photoaging. The ultraviolet rays that cause skin photoaging are UVB and UVB. UVB wavelength shorter, acting on the surface of the skin, easy to make the skin sunburn and blackening. The wavelength of UVB is long, 380 ~ 420nm, it can penetrate the dermis layer of skin and cause certain damage to collagen and elastin. Astaxanthin has a unique molecular structure, its absorption peak is about 470 nm. The wavelength of astaxanthin molecule is close to that of UVB, which can block UVB radiation, effectively absorb UVB, prevent skin photooxidation damage and improve skin photoaging [3] .

1.3 Application of astaxanthin

Astaxanthin has strong antioxidant activity, strong ability to scavenge free radicals and quench reactive oxygen species. Its antioxidant activity is 10 times that of Beta-carotene and 500 times that of Ve, hailed as a“Super antioxidant”, which makes it in food, medicine, cosmetics and other industries have a wide range of applications [4] . In the cosmetic industry, Astaxanthin is a powerful scavenger of free radicals, reactive oxygen species and reactive nitrogen, which can significantly reduce the damage of ROS and NMP to collagen and elastin in dermis. At the same time, the skin’s own repair mechanism can rebuild damaged collagen, ensure the normal metabolism of the skin. Therefore, Astaxanthin plays an anti-oxidation, anti-wrinkle effect. At the same time, Astaxanthin has its unique molecular structure, and the absorption peak of Astaxanthin is about 470 nm, which is similar to the wavelength of UVA (380 ~ 420 nm) . Therefore, a small amount of astaxanthin can absorb a large amount of UVA, used as a sunscreen. However, astaxanthin antioxidant, anti-wrinkle effect is more prominent, so the cosmetics on the market mainly to astaxanthin antioxidant, anti-wrinkle effect as claimed. Astaxanthin powerful antioxidant, many global cosmetic brands add natural astaxanthin as its super antioxidant ingredients [5] .

Superoxide dismutase (SOD) is the first enzyme to eliminate free radicals in the organism. Superoxide anion free radicals are one of the common free radicals in the body. The Thioredoxin reductase keeps the endogenous substrate thioredoxin in a reduced state, participating in redox-based signaling pathways that protect Low-density lipoprotein from oxidation. Astaxanthin, for example, inhibits the production of reactive oxygen species by increasing the expression of Superoxide dismutase 2(SOD2) during ultraviolet radiation. The results showed that Astaxanthin was added into the feed of rabbits and the antioxidant enzyme activity in the serum of rabbits was increased, oxidation induces an increase in Superoxide dismutase and Thioredoxin reductase activity in rabbits[16] . This laid a foundation for the antioxidant and anti-aging properties of astaxanthin, which made astaxanthin can be widely used in cosmetics and adapt to organisms.

1.4 Source

Astaxanthin is widely found in animals (e.g. , Aquatic animal, birds) , bacteria, fungi, algae, and plants. It is common in the feathers of marine animals such as shrimp, salmon, crabs, lobsters and crayfish, as well as certain birds such as flamingos and quails. At present, Astaxanthin is divided into natural astaxanthin and synthetic astaxanthin according to their sources. Synthetic Astaxanthin is the racemic mixture of three isomers in a ratio of about 1:2:1. Natural astaxanthin mainly comes from algae and crustaceans, and is extracted in many different ways. At the same time, the chemical composition of natural astaxanthin depends on the natural source and even on the extraction method used.

Synthetic method

In the late 1980s, Swiss Roche company successfully synthesized astaxanthin, the astaxanthin content is 5% ~ 10% . There are many problems in artificial chemical synthesis, such as high cost, complex process, functional effect and natural astaxanthin have a certain gap. In addition, astaxanthin is particularly susceptible to contamination by other substances when it is synthesized, which can lead to a range of problems and limitations in its application. Nowadays people pay more and more attention to food safety, and natural products make customers prefer them.

Chemical extraction method

Using polymer to extract astaxanthin ester and astaxanthin from the waste of aquatic products, such as shrimp, crab, fish, etc. . The yield was as high as 153 μg/g. The yield of astaxanthin is affected by many materials in the waste, so the extraction technology is strict, the cost is high and the yield is low, which is not suitable for large-scale production of Astaxanthin.

Microbial culture

Fermentative culture was carried out by using fusiform yeast, flavobacterium, yeast and other fungi. The yeast could be cultured under aerobic and anoxic conditions, and the astaxanthin content could reach 0.5% under the condition of no light. The astaxanthin content in the mycelium is low, so it needs more carbon and nitrogen sources to increase the astaxanthin production and increase the cost. In addition to fungi, astaxanthin can be produced by two kinds of bacteria, mycobacterium lactis and bacillus brevis, which are difficult to be applied in biotechnology because of their harsh culture conditions and low availability.In microalgae culture, many algae can produce astaxanthin, such as Chlamydomonas polar, Chlorella sp., Haematococcus pluvialis, etc. Studies have learned that Haematococcus pluvialis accumulates astaxanthin at the fastest rate and the highest content, accounting for about 3% of the dry weight of cells, and the astaxanthin extracted from Haematococcus pluvialis has high biological activity. It is currently the most widely used algae for the production of natural astaxanthin[6].

1.5 Nature of Astaxanthin

Astaxanthin although has good antioxidant, anti-aging, but Astaxanthin production is more difficult, raw materials can not meet the market demand, in addition, the price of Astaxanthin is also more expensive, so the application of Astaxanthin in the cosmetic industry has become a pain point.

2.1 Synthesis of astaxanthin

Astaxanthin precursor synthesis

Astaxanthin biosynthesis begins with isoprenes within plants, including methollyl alcohol (GGPP) synthesized by vinyl acrylic acid or non-methacrylic acid pathways [7].

Carotenoid synthesis

In plants, carotenoid synthesis occurs mainly in chloroplasts, through a series of enzymatic reactions that convert simple carbon sources (such as glucose) into complex carotenoid molecules. These reactions include the synthesis of IPP (isoprene pyrophosphate), the generation of GGPP (Yak Pyrophosphate), and the construction of a carotenoid backbone. GGPP is converted into precursors to carotenoids, such as early carotene. This process involves a variety of enzymes, such as carotene synthetase (PSY) and carotene synthesis isomerase (PDS) [8].Astaxanthin synthesis is mainly carried out by key enzymes such as β-carotene ketonase (BKT) and β-carotene hydroxylase (CrtR-B)[9][10]. These enzymes are expressed in plant cells and catalyze the conversion of β-carotene to astaxanthin. In Chlamydomonas reinhardtiformas, astaxanthin is produced by transforming chloroplasts, which involves β-lycopene cyclase, β-carotene carbonylase, and β-carotene hydroxylase [11].

Astaxanthin synthesis

On the basis of carotenoid synthesis, carotenoids are converted into astaxanthin through specific enzymatic reactions, oxidation (ketylation) and hydroxylation. These reactions require the catalysis of specific enzymes, carotene hydroxylase, carotene ketase, and are carried out in chloroplasts, mitochondria. On the basis of carotene synthesis, it is further converted into astaxanthin. This process involves the conversion of lycopene (lycopene) to β-carotene (β-carotene), which is then further produced by enzymes on top of β-carotene catalyzed. Key enzymes include β-carotene-15,15'-dioxygenase (β-carotene-15,15'-dioxygenase)

2.2 Influencing factors of astaxanthin synthesis

Astaxanthin synthesis is influenced by other metabolic pathways, such as the MEP pathway and the MVA pathway, which are important pathways for terpenoid synthesis. Among these pathways, prenylpyrophosphate isomerase (IDI) is an important regulator that regulates the isomerization of IPP and DMAPP, thereby influencing the entire terpenoid metabolic pathway [12].

Regulation of astaxanthin synthesis involves transcription factors, signaling pathways and environmental factors (such as light, temperature, etc.) . These factors can affect the expression level of related enzymes, thus regulating the amount of astaxanthin synthesis. Astaxanthin, an important Carotenoid in plant leaves, may play a role in pigment deposition and photosynthetic efficiency in leaves, in addition to its involvement in light protection and antioxidant defense [13] . Astaxanthin accumulation is closely related to plant response to environmental stress. Water-soluble chlorophyll proteins (WSCPs) play a role in plant resistance to herbivores, which may be related to astaxanthin biosynthesis. Furthermore, Astaxanthin synthesis may also be associated with photosynthesis in plants, and these processes provide the necessary precursors and energy for Astaxanthin synthesis [14][15] . In photoprotection, for example, astaxanthin absorbs and dissipates excess light energy, thereby protecting the photosynthetic system from photoinhibition and photooxidation. The content of astaxanthin often increases under strong light to enhance the light protection ability of plants.

2.3 Solution

Gene screening

The study found that, plants have been found to accumulate Carotenoid on thylakoid membranes and present in high concentrations in lipid droplets within plastids. Beta-carotene is the precursor of Astaxanthin and is widely found in vascular plant. Astaxanthin is produced by β-carotene the β-ionone ring 4, 4’and 3,3’ positions to add ketones and hydroxyl groups to the synthesis. This process requires the catalysis of -carotene ketoenzyme (4,,4’-oxygenase CrtW, BKT or CrtO) and β-carotene hydroxylase (3,3’-hydroxylase, CrtZ or BHY) . Plants generally have the activity of β-carotene hydroxylase, but do not have the activity of β-carotene ketoenzyme(In addition to Adonis annua contains β-carotene ketoenzyme activity). Therefore, iWe think that the production of astaxanthin can be achieved by heterologous expression of β-carotene ketoenzyme gene in plants. Previous studies have shown that the enzyme BKT from microalgae Chlamydomonas reinhardtii is highly active in the production of astaxanthin from zeaxanthin and accounts for up to 85% of astaxanthin production in carotenoid. Co-expression of β--carotene ketoenzyme(BKT) gene from Chlamydomonas reinhardtii and β-carotene hydroxylase(BHY) gene from Haematococcus pluvialis in tomato via chloroplast genetic engineering resulted in the accumulation of free astaxanthin in tomato leaves (3.12 mg /g ) , esterified astaxanthin (16.1 mg /g ) was accumulated in tomato fruit[17].

Based on the above information, in communication with the teacher, we put forward two solutions. we recommend a more in-depth analysis and comparison of the two programmes to ensure their superiority in terms of science, feasibility and expected results. This will help us to make more informed decisions, which will facilitate the smooth progress of research.

Scheme 1

After consulting a large number of related literatures, we preliminarily determined that CrBKT gene and HpBHY gene had significant effects on astaxanthin synthesis. Based on previous studies, corresponding gene sequences were found (GeBank No.AY860820 and GeBank No.BD250390) and analyzed using the online analysis tool ExPASy Web (ARTIMO et al. , 2012)(https://Web.ExPASy.org/translate/)[21] . Translate it into amino acid sequences. A preliminary BLAST alignment of translated amino acid sequences with the third-generation longan genome was performed using TBtools analysis tool, and it was found that the suspected 9 gene members may have homology with them. The phylogenetic tree was constructed by combining the amino acid sequences of 9 suspected homologous genes with those of BKT and BHY genes in MEGA11.0 software. The maximum likelihood of Bootstrap was set at 1000 and the specific parameters were set to WAG + G, the phylogenetic Tree was constructed and beautify with the online landscaping tool iTOL ( Interactive Tree Of Life). Then, according to the selected genes, TBtools software was used to draw the heat map of their expression in each tissue of longan. But through the DNAMAN software analysis, according to the analysis results, exogenous genes and longan endogenous genes, the consistency is only 35.85% . Therefore, no homologous genes were found in longan.So we came up with Scheme two.

Scheme 2

We used pCAMBIA1301+CaMV35S+NOS vector modified by pCAMBIA1301 vector to facil itate the regulation of initiation and termination of transcription. Linearize the vector by designing a reverse primer.By using seamless cloning technology, the CrBKT gene extracted from Chlamydomonas reinhardtii and HpBHY gene from Haematococcus pluvialis were linked to pCAMBIA1301 vector by seamless cloning technique. The recombinant genes pCAMBIA1301-CrBKT-HpBHY were then transformed into dh5α-competent E.coli, resuscitated and plated on LB solid medium containing kanamycins for screening to obtain recombinant genes. Finally, we confirmed whether the recombinant vector was constructed correctly by PCR and sequencing. After confirming that the recombinant gene was correct, we transformed the recombinant gene into the callus of longan to explore the effect of exogenous genes on the synthesis of astaxanthin in longan.

Construction of HpBHY and CrBKT target genes

HpBHY and CrBKT were used as target genes to construct the pCAMBIA1301-CrBKT-HpBHY expression vector. We designed CrBKT-HpBHY seamless cloning primers according to the published sequences through SnapGene software, amplified the target gene by PCR, designed pCAMBIA1301+CaMV35S+NOS reverse primers according to the published vector sequences, and the pCAMBIA1301-CrBKT-HpBHY was obtained after connection, transformation, and screening.