Blakeslea trispora is a mould and member of the division Zygomycota. This species has been well studied for its ability to produce carotenoids, particularly, β-carotene and lycopene. β-carotene and lycopene are the precursors of vitamin A and play a significant role in the inhibition of oxidative stress. Blakeslea trispora is named in honor of Albert Francis Blakeslee. While A.F. Blakeslee was studying another fungus called Botrytis rileyi, he collaborated with Roland Thaxter to further study this fungus. In 1914, while Thaxter was doing further research on Botrytis rileyi, Blakeslea trispora was accidentally isolated from a contaminated caterpillar infected with Botrytis rileyi. Blakeslea trispora was first identified from the infected larvae of the caterpillar, which was growing on the cowpea plant. The caterpillar was infected by the fungus Botrytis rileyi; however, Blakeslea trispora was unexpectedly transferred onto the diseased caterpillar which was growing on the faded flower on the cowpea plant and thus, Blakeslea trispora was able to be identified. Blakeslea trispora is commonly seen in soil samples throughout the Southern United States and Southern Asia. B. trispora is a known pathogen for tropical plants. However, this fungus has not been the subject of many studies to determine whether it is a cause or an agent of animal or human diseases. In-vivo pathogenicity test in animal models did not suggest a potential cause for animal and human pathogenicity.
Growth and morphology
Blakeslea trispora is in the genus of Blakeslea, which was first identified by Roland Thaxter in 1914. Blakeslea trispora undergoes both sexual and asexual reproduction. When Blakeslea trispora undergoes asexual reproduction, it produces sporangiospores. When Thaxter first identified B. trispora, he considered B. trispora to be very closely related to the genus of Choanephora because of the relatively identical characteristics between the sporangiospores of the genus Blakeslea and of the genus Choanephora. Their sporangiospores both have a peculiar brown colour with an indistinguishable, longitudinally striated sporangiolum wall. The shape of the large spherical heads of their sporangiola are also similar. However, Choanephora and Blakeslea are considered to be 2 separate genera and can be distinguishable by the spore wall and its separation from the sporangiolum wall. The species of the genus Choanephora have a sporangiolum wall highly attached to the spore wall. At maturity, their sporangiolum wall and spore wall are inseparable. In contrast, Blakeslea species are able to separate the sporangiolum wall from the spore wall at the underlying spore maturity. The colonies of Blakeslea trispora can grow rapidly on the agar medium at 25 °C. The colonies are white at first but they become yellow to pale brown and very dark brown as they mature. The hyphae of the fungus Blakeslea trispora are aseptate and the mycelium becomes very dense and highly branched as the colonies mature.
Sporangiospores of Blakeslea trispora, which are produced in sporangia during asexual reproduction can germinate in the presence of free water. On the other hand, zygospores, which are the results of sexual reproduction of Blakeslea trispora, can exist for a long time without germinating. Their germination depends on the state of cytoplasmic regulatory system. Zygospores can exist for a long time without germinating when they are under unfavorable condition, therefore, B. trispora can possess an adaptive process through which they can transit their fungal cells into a dormancy state to survive. They are able to exist in the dormancy state for a long period of time due to the dormancy state's high lipid contents, composed of triacylglycerols and phosphatidylcholine.
Blakeslea trispora produce zygospores during their sexual reproduction and zygospores can grow below the surface of the agar medium. The zygospores range in size from 40-80μm. They are spherical or slightly flattened in shape. Blakeslea trispora has a heterothallic mating system, having (+) and (-) mating types. Contact and interchange between the opposite mating types is a necessary precursor to induce sexual reproduction and development of zygospores. Extensions called gametangia are formed from each of the opposite mating type's mycelium, which contains haploid nuclei. Together, they form heterokaryotic zygosporangium to further develop zygospores. During its sexual reproduction, carotenoids are produced from both of the mating types. Carotenoids are precursors of many apocarotenoids that contain very important sex-specific precursors, trisporic acid (TSA) for the sexual reproduction of Blakeslea trispora. Carotenes produced from carotenoids are further processed by carotene oxygenase to synthesize TSA. TSA produced from carotene stimulates both sexually complementary cells to make contact with each other. Therefore, TSA acts as an important signalling molecule to initiate and control the sexual reaction between the (+) and (-) mycelia.
At the beginning of the sexual reproduction cycle of Blakeslea trispora, the initial step is the production of carotenes from carotenoids. Carotenes are further processed by carotene oxygenase, which is encoded in the tsp3 gene of the Blakeslea trispora, to produce trisporic acid (TSA). TSA is produced by both of the mating types: (+) and (-) strains, and it is explosively produced especially when the 2 different sex mycelia grow together. As these two different sex types produce TSA, they sense sexually complimentary cells and form gametangia. Eventually, those gametangia merge together to assemble into the zygosporangium. As these two different mating types meet each other, each mating type transfers the sex-specific precursor of the trisporoid TSA, and acts as a signal for the synthesis of the surface protein agglutinin. Agglutinin allows the two TSAs to recognize each other. It then causes rapid contact and efficient interactions between those two different mating types. Additionally, stimulating both of the mating types by TSA promotes synthesis of β-carotene. As β-carotene is produced, it becomes a precursor of trisporoid, which is a pheromone for B. trispora. Production of β-carotene promotes a positive feedback process that further stimulates carotenogenesis and the production of trisporoid which serves as a β-carotene increasing substance. Furthermore, it act as a hormone stimulator of its biosynthesis. Thus, Blakeslea trispora requires certain concentrations of TSA to activate carotenogenesis and produce more carotenoids (about 0.5% of its dry weight) which can be accumulated in the zygospores of B. trispora. Therefore, both TSA and trisporoid acts as sex hormones in Blakeslea trispora, which triggers the sexual reproduction and controls intimate contact between heterothallic strains, further governing the formation of sex structures, zygospores.
Carotenoids are absolutely necessary not only for the production of trisporic acid, but also for the process of zygote formation, as significant factors for the production of sporopollenin, a structural component of the zygospore cell wall. It is consequential to regulate this feedback-type synthesis with carotenoids and further synthesis of TSA. Therefore, the formation of the zygospores can be prevented by the inhibition of carotenogenesis in Blakeslea trispora.
The sexual interaction between (+) and (-) mating type mycelia in Blakeslea trispora forms different shapes of zygospores, which are filled with lipid-carotene. It is typically more characterized that the (-) mating type mycellium is playing the more important role in carotenogenesis; however, further studies are needed to clarify this role.
Relevance to human health
Current research has pointed towards the role of β-carotene and its precursor molecule, lycopene, in human health. Lycopene is the predominant compound found in human plasma and tissues. Lycopene is also found in fungal cells such as Blakeslea trispora. The production of lycopene primarily requires some interaction between the mating strains. Blakeslea trispora needs both mating types to synthesize lycopene on a commercially applicable scale. The (-) strain is twice proportionally as important as the (+) strain in determining the productivity of the synthesis of the lycopene. To produce an optimal amount of lycopene, excess (-) mating type at a 1:2 (+/-) ratio with inoculum ages 36 and 48 hours respectively is favorable. Lycopene and β-carotene are both found to be important carotenes, which play a significant role in anti-oxidative activities.
Blakeslea trispora is known to be the most effective producer of lycopene. Lycopene is processed by lycopene cyclase and leads to the production of β-carotene. Therefore, lycopene can be harvested industrially using lycopene cyclase inhibitor which can be introduced into the fermentation process of Blakeslea trispora. In the zygospores of Blakeslea trispora tend to contain a maximum amount of lycopene. Lycopene is an intermediate in the biosynthesis of all dicyclic carotenoids including β-carotene. Lycopene is one of the most important carotene molecules because it is capable of producing both β-carotene and other carotenoids, well known for their potent anti-oxidant activities. As such, β-carotene and other carotenoids play crucial roles for oxidative stress reduction and cardiovascular protection. Carotenoids have highly efficient antioxidant scavenging activities against ROS (reactive oxygen species), such as singlet-oxygen and free radicals. Therefore, they have the ability to prevent chronic diseases such as cancer, cerebrovascular and cardiovascular diseases and myocardial infarction. Lycopene is considered a very important and relevant source to human health. A case study by Weilian Hu and his colleagues in 2013 showed that the administration of lycopene in adult mice appeared to improve the activity of antioxidant enzyme. They have reported that, the administration of Blakeslea trispora powder, which contains high amounts of lycopene has the potential to protect the liver, brain, kidney and skin against oxidative stress. This is done by reducing the concentration of ROS and by enhancing the activities of the antioxidant enzyme. Furthermore, they are further investigating whether the fungus Blakeslea trispora could be a potent effector of anti-aging because of its ability to efficiently mass produce amounts of lycopene.
β-carotene is also a relevant component for human health and chronic diseases. β-carotene is a molecule which displays a red-orange pigment. Therefore, it is used as a coloring agent for food products. β-carotene is a member of carotenes which are highly unsaturated isoprene derivatives. Because Blakeslea trispora has an effective ability to produce great amount of β-carotene from lycopene, Blakeslea trispora is the main organism used for its production on an industrial scale. β-carotenes are known to be a powerful stimulant of the human immune system and play significant roles in the prevention of degenerative diseases and cancers. All cells are capable of producing and regulating ROS. However, dysregulation of ROS can lead to DNA damaging, inactivation of enzymes and proteins, disruption of membranes. This ultimately causes cell death, becoming very toxic to the individuals. Further investigations of β-carotene usage collected from Blakeslea trispora may lead to great improvements to human health in the treatment and prevention of certain chronic diseases such as cancer.
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