Transcriptome analysis of β-carotene accumulation by light exposure in Mucor circinelloides

Abstruct: β-carotene is one of the most diverse and effective antioxidants in natural fat-soluble pigments in the food, pharmaceutical, and cosmetic industries. Much attention has been attracted in recent years, due to its potential antibacterial properties and anticancer agents. Among them, microbial synthesis can be expected to serve as the ever-increasingly important source of β-carotene, due to the lower cost, higher safety, and easy large-scale production, compared with animal and plant extraction and industrial synthesis. Light stimulation of carotenoid synthesis is also the earliest and most studied regulatory mechanism in fungi. The genome of Mucor circinelloides can be better used in the β-carotene synthesis mechanism, due to the efficient genetic transformation, gene replacement, and silencing, as well as the ability to express foreign genes. This study aims to clarify the molecular mechanism in the specific metabolic process of β-carotene synthesis, where the microorganisms were stimulated by light. A systematic transcriptomic analysis was then made using high-performance liquid chromatography and Illumina Hiseq 2000. The gene changes and functional annotation was determined in β-carotene accumulation and metabolic pathway of Mucor circinelloides WJ11 after light stimulation, respectively. The fermentation results showed that the cell dry weight was slightly higher under light exposure than under dark conditions, the maximum of which was up to 14.8 g/L under light exposure for 1 440 min. There was a high rate of glucose consumption. The concentration of residual glucose was about 7.0 g/L in the medium at the late fermentation stage. In addition, the lipid content significantly increased, reaching 35.3% of the dry weight of cells. β-carotene was at the lower level under the two cultures before light. There was no difference in production. Mucor circinelloides WJ11 accumulated the maximum pigment amount under 1 440 min of continuous light. The pigment amounts under light and dark conditions were 458.3 and 218.7 μg/g, respectively, and then the β-carotene production slowly decreased. The samples were collected at different times under light and dark conditions for the latter transcriptomic analysis. The annotation of the Kyoto encyclopedia in the genes and genomes (KEGG) database showed that a maximum of 5 598 differential genes were annotated to obtain 119 metabolic pathways. The significantly enriched metabolic pathway "biosynthesis of secondary metabolites" was closely related to β-carotene accumulation in Mucor circinelloides. In addition, accumulated pyruvate by the glycolytic pathway after 120 min of light exposure was converted into acetaldehyde in the cytoplasm, and then activated the pyruvate-acetaldehyde-acetic acid cycle, thus supplying more precursor acetyl-CoA for pigment synthesis. Furthermore, the expression of mevalonate pathway genes was enhanced significantly, leading to a sharp increase in β-carotene synthesis. The lipid accumulation and β-carotene synthesis were stabilized after 1 440 min of light exposure. There was an accumulation of acetyl-CoA to affect the higher activity of acetyl-CoA acetyltransferase (ACAT). However, the activity of the mevalonate pathway decreased instead, and β-carotene accumulation decreased as well. Meantime, the decreased expression of acyl-CoA synthetase (ACS) and ATP: Citrate lyase (ACL) allowed for more carbon sources to flux into the TCA cycle. The transcriptional analysis of differential genes showed that light stimulation increased the utilization rate of glucose, in order to rapidly metabolize into the energy for growth, such as fermentation. The transcription levels of three White collar-1 (WC-1) were significantly down-regulated after 120 min of light exposure, indicating the key regulatory proteins for β-carotene synthesis. The findings can provide theoretical support for the molecular mechanism and genetic modification of β-carotene synthesis.