The Study Of Regulative Mechnism Of Carotenoid Producion By Blakeslea Trispora

Blakeslea trispora was found an excellent strain to produce β-carotene in the level of industrialization. The way of carotenoids production by the microbial fermentation has the advantages of lower cost, high purity, without seasonal restriction, toxic or side effect. Previous researches indicated that the exogenous lipids could enhance the lycopene formation, especially the oils with high content of linoleic and linolenic acid. Compared with the sunflower oil and soybean oil, our results showed that linseed oil had a better effect on promoting the carotenoids.production. However, the mechanism how ALA promoted the carotenoid production was unknown. Linseed oil is rich in a-linolenic acid (ALA). Thus, we chose ALA to study it how to promote carotenoid production at metabolism and transcription level by B. trisporaWe studied the changes of intracellular metabolites induced by ALA addition by using gas chromatography-mass spectrometry (GC-MS) and multivariate analysis method in B. trispora. Based on the PLS-DA (partial least squares-discriminant analysis) method, we found that this method could separate the test group from the control. The results of cluster analysis supported the reliability of PLS-DA. We found that the content of linolenic acid and linoleic acid were much higher than the control with ALA treated, while there were no difference in the content of their precursors between the two groups. Fatty acid and carotenoid have the same precursor, i.e. acetyl-CoA, and this enabled flux from acetyl-CoA to β-carotene biosynthesis increased. ALA made the spore form small pellets and increased the the dissolved oxygen. High dissolved oxygen was beneficial to the growth and nutrient absorption and utilization, and this also induced the increase of oxidative stress. The above conclusions were also supported by measuring the catalase and superoxide dismutase enzyme activities. Thus, oxidative stress was the external factor to promote the carotenoid biosynthesis.As the identified intracellular metabolites were limited, it was difficult to infer the change of metabolic flow by the metabolite instantaneous content. Therefore, we used the RNA-seq technology to global investigate the genes expression at transcriptional level. ALA treatment inhibited the fatty acid and ergosterol biosynthesis and promoted the fatty acid metabolism. This enabled more acetyl-CoA flux to β-carotene biosynthesis. Moreover, ALA significantly promoted the transcription of carRA, carB and carG to produce β-carotene. Besides, we found that the transcription factor Mot3 related to the carotenoid biosynthesis was obvious up-regulated. Mot3 was a multifunctional transcription factor, which played an important part in inhibiting ergosterol biosynthesis and pheromone-induced genes expression. In addition, the foundings related to some transcription factors also suggested that ALA also influences the formation of spores, morphogenesis and the cell wall, and regulated cell division and sexual development (e.g. SteA). Further study is needed to confirm whether the SteA is involved in regulating the β-carotene biosynthesis. Besides, we also found the expression of transcription factor Papl and Prrl were up-regulated, which were sensitive to hydrogen peroxide. This indicated that the oxidative stress was one of the external factors to promote carotenoids biosynthesis.