CONTROL AND OPERATIONS MANAGEMENT OF EXTRACTION PROCESS IN INDUSTRIAL BIOTECHNOLOGY OF BETA-CAROTINE FROM BLAKESLEA TRISPORA

The technological parameters, namely temperature and duration of β-carotene extraction process from the biomass of filamentous fungus Blakeslea trispora with vegetable oils of various fatty acid compositions and with various contents of natural antioxidants (refined deodorized sunflower, viso-oleic sunflower, corn and sesame) have been investigated. Statistical models of dependences of β-carotene, as well as analytical numbers, characterizing the content of free fatty acids (acid number) and primary products of lipid oxidation (peroxide number) content, in oil extracts of the specified refined deodorized oils, from temperature and extraction duration have been built. Rational parameters of β-carotene extraction from Blakeslea trispora biomass with selected extractants (refined deodorized sunflower, high oleic sunflower, corn and sesame oils) have been determined for the extracts technological properties control. It has been proven that the use of these refined deodorized oils as extractants practically does not affect the content of the target product in oil extracts of biomass, but it does affect the analytical numbers of extracts characterizing the content of free fatty acids, peroxides and hydroperoxides. The highest content of free fatty acids in β-carotene containing biomass extraction with sunflower oil has been observed. The minimum content of free fatty acids in extracts with corn and sesame oils using has been be achieved. The highest content of primary products of lipid oxidation (peroxides and hydroperoxides) during β-carotene containing biomass extraction by sunflower oil has been observed. The minimum content of free fatty acids in extracts with sesame oil using has been achieved. It is possible to predict β-carotene content, as well as acid and peroxide numbers of oil extracts from biomass in specified refined deodorized oils, depending on temperature and extraction process duration using the obtained approximation dependences.

Transcriptome sequencing and global analysis of blue light-responsive genes provide clues for high carotenoid yields in Blakeslea trispora

Blakeslea trispora has great potential uses in industrial production because of the excellent capability of producing a large quantity of carotenoids. However, the mechanism of light induced carotenoid biosynthesis even the structural and regulatory genes in pathways remain unclear. In this paper, we reported the first transcriptome study in B. trispora in which we have carried out global survey of expression changes of genes participated in blue light response. We verified that the yield of β-carotene reaching to 3-fold when transferred from darkness to blue light for 24 h and the enhancement of transcription levels of carRA and carB presented a positive correlation with the increase in carotenoid production. RNA-seq analysis revealed that 1124 genes were upregulated and 740 genes were downregulated respectively after blue light exposure. Annotation through GO, KEGG, Swissprot and COG databases showed 11119 unigenes compared well with known gene sequences, 5514 unigenes were classified into Gene Ontology, and 4675 unigenes were involved in distinct pathways. Among the blue light responsive genes, 4 genes (carG1, carG3, carRA and carB) identified to function in carotenoid metabolic pathways were dominantly upregulated. We also discovered that 142 TF genes belonging to 45 different superfamilies showed significant differential expression (p≤ 0.05), 62 of which were obviously repressed by blue light. The detailed profile of transcription data will not only allow us to conduct further functional genomics study in B. trispora, but also enhance our understanding of potential metabolic pathway and regulatory network involved in light regulated carotenoid synthesis.

Medium optimization and downstream process design for the augmented yield of β-Carotene using fungi Blakeslea trispora

Purpose β-Carotene is the most appropriate and significant precursor of vitamin A. Synthetic carotene supplements have been known to pose a threat to human health, making natural sources such as the indefensible choice for the production and extraction of carotene. Design/methodology/approach This study considers Blakeslea trispora, a filamentous fungus, as a source of production of carotenoids by fermentation and wet and dry mycelium were used to analyse and obtain better extraction results. Findings In this study, natural oils such as soy oil and cottonseed oil were incorporated into fermentation media to increase the production of carotene. For the optimization process, Plackett–Burman and one-factor-at-a-time (OVAT) models were identified as being of great value. Originality/value OVAT was carried out for corn starch because it plays a major role in the production of carotene and the corn starch at 30 g/L concentration has shown the maximum activity of 3.48 mg/gm. After optimizing process variables, submerged fermentation was eventually carried out under highly controlled media conditions. The resulting product was quantified using UV spectroscopy and extraction of carotene has been observed in the presence of various solvents. Among a range of solvents used, the methylene Di chloride produced-carotene at 86% recovery at a significantly lower temperature of 35°C.

Untargeted Global Metabolomic Analysis Reveals the Mechanism of Tripropylamine-Enhanced Lycopene Accumulation in Blakeslea trispora

We previously determined that the cyclase inhibitor tripropylamine (TPA) significantly enhances lycopene accumulation in Blakeslea trispora. To elucidate the mechanism of TPA-enhanced lycopene accumulation, the untargeted metabolome of B. trispora treated with TPA was analyzed by UHPLC-Q-TOF/MS. Forty-two differential metabolites were identified, of which 15 significantly differential metabolites meeting the following parameters were screened: variable importance for the projection > 1, P < 0.05, and fold change > 1.5. The down-regulated metabolites were mainly cyclic dipeptides, bacteriostatic compounds, and lipids, while the up-regulated metabolites were mainly unsaturated fatty acid. Furthermore, the bacteriostatic ability was poor, the extracellular and intracellular pH levels were high, and hyphae with vesicles were swollen locally in B. trispora after treatment with TPA. Our data suggest that the TPA enhances lycopene accumulation not only by inhibiting the cyclization of β-carotene but also by down-regulating cyclic dipeptides for quorum sensing; up-regulating unsaturated fatty acids, 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine, and 4-hydroxybenzoate and down-regulating choline, resulting in locally swelling mycelium with vacuoles; and down-regulating bacteriostatic metabolites for metabolic flux redistribution.

Elevated β-Carotene Production Using Codon-Adapted CarRA&B and Metabolic Balance in Engineered Yarrowia lipolytica

β-carotene is a precursor of vitamin A and has multiple physiological functions. Producing β-carotene by microbial fermentation has attracted much attention to consumers’ preference for natural products. This study focused on improving β-carotene production by constructing codon-adapted genes and minimizing intermediate accumulation. The codon-adapted CarRA and CarB genes from the industrial strain of Blakeslea trispora were integrated into the genome of the Yarrowia lipolytica to construct YL-C0, the baseline strain for producing β-carotene. Thereafter, the β-carotene biosynthetic pathway’s metabolic balance was accurately regulated to reduce the intermediates’ accumulation. Notably, the β-carotene content increased by 21 times to reach 12.5 dry cell weight (DCW) mg/g when minimizing HMG-CoA and FPP accumulation. Further, we improved the expression levels of the CarRA and CarB genes to minimize the accumulation of phytoene and lycopene. Total production of β-carotene of 1.7 g/L and 21.6 mg/g DCW was achieved. These results reveal that the rate-limiting enzymes CarRA and CarB of B. trispora exhibited higher catalytic activity than the same enzymes from other microorganisms. Promoting metabolic balance by minimizing the accumulation of intermediates is a very effective strategy for increasing β-carotene. The β-carotene-producing strain constructed in this study has established the foundation for its potential use in industrial production. These successful engineering strategies also provide a foundation for large-scale production of other terpenoids.