Microbial Fermentation for Astaxanthin Synthesis

Astaxanthin is a high-value carotenoid with strong antioxidant activity, as well as various benefits such as anticancer, anti-inflammatory, and eye protection effects. With the continuous development of synthetic biology technology, microbial fermentation is one of the most effective ways to achieve industrial production of astaxanthin. It also better meets consumers' demand for natural compounds. Currently, microorganisms involved in astaxanthin production include bacteria, fungi, algae, and more. Through genetic engineering, fermentation process regulation, metabolic engineering, methods such as overexpression of astaxanthin synthesis genes, use of high-intensity promoters, and optimization of metabolic pathways can increase astaxanthin production. This enhances the applications of astaxanthin in food, cosmetics, and nutraceuticals and supplements.

What is Astaxanthin?

Astaxanthin is an orange-red keto-carotenoid, insoluble in water and lipid-soluble. It consists of 8 isoprene units, forming a tetraterpenoid with conjugated double bonds, and unsaturated hydroxyl and ketone groups at the ends of the conjugated double bonds. As a result, astaxanthin exists in different configurations, including (3S-3'S), (3R-3'R), and meso (3R-3'S). The (3S-3'S) isomer is the most common configuration in nature and has the highest antioxidant activity, followed by the (3R-3'R) and (3S-3'S) configurations. The long conjugated polyene chain of astaxanthin can quench singlet oxygen, eliminate free radicals, enhance cell activity, and protect lipid bodies in the human body. Therefore, it contributes to improving immunity, anti-aging, and preventing various oxidative stress and inflammation-related conditions, including hypertension, cancer, obesity, cardiovascular diseases, inflammatory diseases, bone disorders, skin diseases, and more. Additionally, astaxanthin serves as a natural coloring agent, exhibiting different configurations in various species, giving the organism unique colors. Currently, methods for astaxanthin production mainly include natural extraction, chemical synthesis, and microbial fermentation. Natural extraction involves extracting astaxanthin from shellfish waste like lobsters and crabs, but it has low yield, a complex process, high costs, and is economically unfeasible due to contamination risks. Chemical synthesis has a long and complex production cycle, resulting in a mixture of astaxanthin configurations and various by-products, with lower absorption and utilization rates in the human body compared to naturally extracted astaxanthin, making it not approved for human use. With the continuous development of synthetic biology technology, microbial fermentation for natural product production has shown great potential. Microbial production of astaxanthin offers advantages such as clear configuration, environmental friendliness, and minimal by-products.

Biosynthesis Pathway of Astaxanthin

First, organisms utilize carbon sources like glucose through the glycolytic pathway (Embden-Meyerhof-Parnas, EMP) to produce pyruvate, acetyl-CoA, and other precursors that flow into the mevalonate pathway (MVA) and the methylerythritol phosphate pathway (MEP). The second MVA pathway provides precursors for terpenoid synthesis. Starting from acetyl-CoA, enzymatic reactions generate isopentenyl pyrophosphate (IPP), which is then isomerized into dimethylallyl pyrophosphate (DMAPP) by isopentenyl diphosphate isomerase (IDI). Finally, using IPP and DMAPP as precursors, organisms synthesize precursors for terpenoid compounds. The MEP pathway is another pathway supplying precursors for the synthesis of natural terpenoids and is widespread in bacteria, fungi, plants, and algae. This pathway starts from pyruvate. Third, under the action of farnesyl diphosphate synthase (ispA), IPP and DMAPP produce geranylgeranyl pyrophosphate (GPP). GPP continues to generate farnesyl pyrophosphate (FPP) under the action of GPP synthase (CrtE). FPP, in turn, produces geranylgeranyl diphosphate (GGPP) under the action of geranylgeranyl diphosphate synthase (CrtE). GGPP, with the assistance of phytoene synthase, lycopene cyclase (CrtYB), and phytoene desaturase (CrtI), produces lycopene. Lycopene, under the action of CrtYB, produces β-carotene. The difference in structure between β-carotene and astaxanthin lies in the hydroxyl and carbonyl groups at the ends of the molecule's ring.

Astaxanthin Synthesis Base Cells

Strategies to enhance microbial astaxanthin synthesis typically involve optimization of the fermentation process and metabolic engineering. This includes optimizing fermentation conditions, increasing the supply of precursors in the MVA and MEP pathways, selecting and expressing key genes from different sources, modular engineering to increase gene copy numbers, and targeting different subcellular compartments.

• Algae

Many algae in nature can produce astaxanthin, such as Haematococcus pluvialis, Chlamydomonas, Acetabularia, and Euglena. H. pluvialis is a freshwater single-cell green alga, and its astaxanthin content can reach 5% of the cell dry weight, making it a major alga for astaxanthin production. The astaxanthin produced by H. pluvialis is predominantly the strongest antioxidant (3S-3'S) configuration. However, H. pluvialis has a long growth cycle, high cultivation requirements, needs light, and astaxanthin is present in thick-walled cysts, resulting in low extraction efficiency. Therefore, new processes need to be developed to achieve commercial applications by reducing production costs and increasing astaxanthin content in H. pluvialis. For example, adding some exogenous substances such as plant hormone analogs to enhance astaxanthin production and using hydrophilic solvents to extract astaxanthin from H. pluvialis.

• Yeast

Yeast that naturally produces astaxanthin in nature includes Phaffia rhodozyma, Rhodotorula rubra, Rhodotorula benthica, and Rhodotrula glutinis. With the development of synthetic biology, engineered yeasts constructed based on genetic engineering can also produce astaxanthin, such as Saccharomyces cerevisiae and Kluyveromyces marxianus. Compared to microalgae, yeast has a wide range of substrate sources, fast growth, short fermentation cycles, and relatively mature genetic modification tools. Therefore, yeast is one of the most promising chassis cells for industrial astaxanthin production. P. rhodozyma is considered the most suitable microorganism, aside from H. pluvialis, for astaxanthin production in nature. It can use various sugars as carbon sources for fermentation to synthesize astaxanthin. Additionally, it can achieve high-density cultivation, and the astaxanthin it produces is predominantly the (3R-3'R) configuration, which is easily absorbed by the human body. Optimizing fermentation conditions is the easiest and most direct way to increase astaxanthin production. pH has an impact on both cell growth and astaxanthin accumulation in P. rhodozyma. Research indicates that the optimal initial pH for cell growth is 6.0, the optimal pH for astaxanthin formation is 4.0, and the optimal pH for astaxanthin accumulation is 5.0.

• Bacteria

Although the astaxanthin content in most bacteria is much lower than some algae and fungi, introducing relevant genes for synthetic astaxanthin production in bacteria can greatly increase astaxanthin production. Moreover, compared to fungi and algae, extracting astaxanthin from bacteria through fermentation is easier, simplifying subsequent extraction processes. Gram-negative bacteria, in particular, have thin and easily breakable cell walls, facilitating the extraction of astaxanthin from inside the cells. Escherichia coli is a Gram-negative, facultative anaerobic bacterium that is easy to culture, simple to operate, cost-effective, and has mature molecular genetic modification tools. It has become one of the best hosts for metabolic engineering and synthetic biology. As a non-carotenoid-producing strain, E. coli can synthesize the terpenoid compound precursors IPP and DMAPP through the MEP pathway. In wild-type E. coli, FPP synthase is present in the body, an enzyme that can condense IPP and DMAPP to generate GPP and FPP. However, it lacks enzymes to convert FPP into the final astaxanthin. Therefore, by introducing an external astaxanthin synthesis module into E. coli, it is relatively easy to achieve astaxanthin synthesis in E. coli, and the synthesized astaxanthin is predominantly the (3S-3'S) configuration. Determining the key genes in the astaxanthin metabolic synthesis pathway provides possibilities for constructing high-yield engineered bacteria for astaxanthin production.

Astaxanthin Extraction Process

Astaxanthin is an intracellular product, so extracting astaxanthin from microorganisms involves two steps: cell disruption and astaxanthin collection. Compared to bacteria, the cell walls of algae and yeast are tough and thick, making them less prone to breakage. Therefore, the focus of astaxanthin extraction lies in the cell wall disruption. Traditional cell wall disruption methods include physical methods, chemical methods, and enzymatic methods. Physical methods include mechanical crushing, ultrasonic crushing, and supercritical fluid extraction. Mechanical crushing is a simple operation that tears the cell wall through mechanical stirring, releasing astaxanthin from inside the cells. However, mechanical crushing may cause localized high temperatures, leading to some damage to astaxanthin. Ultrasonic crushing can effectively break the cell wall in the solute, but with increasing ultrasonic intensity and duration, the production of strong oxidative free radicals increases, reducing the extraction efficiency of astaxanthin. Additionally, it can generate some noise pollution. Supercritical fluid extraction is the most effective extraction method for various algae products in recent years. Compared to traditional liquid solvents, it has some unique physicochemical properties, such as high diffusion rate, high compressibility, low surface tension, low viscosity, and easy penetration of cell walls, improving product extraction efficiency. Chemical methods mainly include organic solvent extraction, acid-base treatment, and dimethyl sulfoxide (DMSO) method. As astaxanthin is a lipophilic natural product, the choice of organic solvent must consider whether astaxanthin is soluble and the solvent's polarity. DMSO, as a polar solvent soluble in both water and organic solvents, has become a commonly used cell wall-breaking solvent in laboratories. It can quickly and efficiently break the cell wall of microorganisms without causing significant damage to astaxanthin. Mixing DMSO with acetone in an appropriate ratio can more completely extract astaxanthin. Due to the strong antioxidant properties of astaxanthin, if exposed to air for a long time, oxygen in the air will undergo oxidation reactions with astaxanthin, causing it to lose its antioxidant capabilities. Therefore, an anaerobic (nitrogen-filled) extraction process is one of the steps that cannot be ignored in current industrial production. Enzymatic extraction of astaxanthin has the advantages of mild conditions, low energy consumption, and short processing time. It can not only rapidly and efficiently break the cell wall, releasing intracellular astaxanthin but also inhibit cell activity, preventing denaturation of intracellular substances. For example, β-glucosidase can hydrolyze β-glucan in the cell wall, avoiding the overflow of astaxanthin from the cells and reducing losses. However, enzymatic methods require a large amount of enzymes, undoubtedly increasing production costs.