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Blakeslea trispora genes for carotene biosynthesis.

ABSTRACT: We cloned the carB and carRA genes involved in beta-carotene biosynthesis from overproducing and wild-type strains of Blakeslea trispora. The carB gene has a length of 1,955 bp, including two introns of 141 and 68 bp, and encodes a protein of 66.4 kDa with phytoene dehydrogenase activity. The carRA gene contains 1,894 bp, with a single intron of 70 bp, and encodes a protein of 69.6 kDa with separate domains for lycopene cyclase and phytoene synthase. The estimated transcript sizes for carB and carRA were 1.8 and 1.9 kb, respectively. CarB from the beta-carotene-overproducing strain B. trispora F-744 had an S528R mutation and a TAG instead of a TAA stop codon. The overproducing strain also had a P143S mutation in CarRA. Both B. trispora genes could complement mutations in orthologous genes in Mucor circinelloides and could be used to construct transformed strains of M. circinelloides that produced higher levels of beta-carotene than did the nontransformed parent. The results show that these genes are conserved across the zygomycetes and that the B. trispora carB and carRA genes are functional and potentially useable to increase carotenoid production.

SUBMITTER: Rodriguez-Saiz M

PROVIDER: S-EPMC520866 | biostudies-literature | 2004 Sep

REPOSITORIES: biostudies-literature

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Blakeslea trispora genes for carotene biosynthesis.

Rodríguez-Sáiz M M Paz B B De La Fuente J L JL López-Nieto M J MJ Cabri W W Barredo J L JL

Applied and environmental microbiology 20040901 9

We cloned the carB and carRA genes involved in beta-carotene biosynthesis from overproducing and wild-type strains of Blakeslea trispora. The carB gene has a length of 1,955 bp, including two introns of 141 and 68 bp, and encodes a protein of 66.4 kDa with phytoene dehydrogenase activity. The carRA gene contains 1,894 bp, with a single intron of 70 bp, and encodes a protein of 69.6 kDa with separate domains for lycopene cyclase and phytoene synthase. The estimated transcript sizes for carB and c ...[more]

PMID: 15345447

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Project description:Blakeslea trispora is an industrial fungal species used for large-scale production of carotenoids. However, B. trispora light-regulated physiological processes, such as carotenoid biosynthesis and phototropism, are not fully understood. In this study, we isolated and characterized three photoreceptor genes, btwc-1a, btwc-1b, and btwc-1c, in B. trispora Bioinformatics analyses of these genes and their protein sequences revealed that the functional domains (PAS/LOV [Per-ARNT-Sim/light-oxygen-voltage] domain and zinc finger structure) of the proteins have significant homology to those of other fungal blue-light regulator proteins expressed by Mucor circinelloides and Neurospora crassa The photoreceptor proteins were synthesized by heterologous expression in Escherichia coli The chromogenic groups consisting of flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) were detected to accompany BTWC-1 proteins by using high-performance liquid chromatography (HPLC) and fluorescence spectrometry, demonstrating that the proteins may be photosensitive. The absorbance changes of the purified BTWC-1 proteins seen under dark and light conditions indicated that they were light responsive and underwent a characteristic photocycle by light induction. Site-directed mutagenesis of the cysteine residual (Cys) in BTWC-1 did not affect the normal expression of the protein in E. coli but did lead to the loss of photocycle response, indicating that Cys represents a flavin-binding domain for photon detection. We then analyzed the functions of BTWC-1 proteins by complementing btwc-1a, btwc-1b, and btwc-1c into the counterpart knockout strains of M. circinelloides for each mcwc-1 gene. Transformation of the btwc-1a complement into mcwc-1a knockout strains restored the positive phototropism, while the addition of btwc-1c complement remedied the deficiency of carotene biosynthesis in the mcwc-1c knockout strains under conditions of illumination. These results indicate that btwc-1a and btwc-1c are involved in phototropism and light-inducible carotenogenesis. Thus, btwc-1 genes share a conserved flavin-binding domain and act as photoreceptors for control of different light transduction pathways in B. trisporaIMPORTANCE Studies have confirmed that light-regulated carotenogenesis is prevalent in filamentous fungi, especially in mucorales. However, few investigations have been done to understand photoinduced synthesis of carotenoids and related mechanisms in B. trispora, a well-known industrial microbial strains. In the present study, three photoreceptor genes in B. trispora were cloned, expressed, and characterized by bioinformatics and photoreception analyses, and then in vivo functional analyses of these genes were constructed in M. circinelloides The results of this study will lead to a better understanding of photoreception and light-regulated carotenoid synthesis and other physiological responses in B. trispora.

Project description:As an ideal carotenoid producer, Blakeslea trispora has gained much attention due to its large biomass and high production of β-carotene and lycopene. However, carotenogenesis regulation in B. trispora still needs to be clarified, as few investigations have been conducted at the molecular level in B. trispora In this study, a gene homologous to carotenogenesis regulatory gene (crgA) was cloned from the mating type (-) of B. trispora, and the deduced CrgA protein was analyzed for its primary structure and domains. To clarify the crgA-mediated regulation in B. trispora, we used the strategies of gene knockout and complementation to investigate the effect of crgA expression on the phenotype of B. trispora In contrast to the wild-type strain, the crgA null mutant (ΔcrgA) was defective in sporulation but accumulated much more β-carotene (31.2% improvement at the end) accompanied by enhanced transcription of three structural genes (hmgR, carB, and carRA) for carotenoids throughout the culture time. When the wild-type copy of crgA was complemented into the crgA null mutant, sporulation, transcription of structural genes, and carotenoid production were restored to those of the wild-type strain. A gas chromatography-mass spectrometry (GC-MS)-based metabolomic approach and multivariate statistical analyses were performed to investigate the intracellular metabolite profiles. The reduced levels of tricarboxylic acid (TCA) cycle components and some amino acids and enhanced levels of glycolysis intermediates and fatty acids indicate that more metabolic flux was driven into the mevalonate (MVA) pathway; thus, the increase of precursors and fat content contributes to the accumulation of carotenoids.IMPORTANCE The zygomycete Blakeslea trispora is an important strain for the production of carotenoids on a large scale. However, the regulation mechanism of carotenoid biosynthesis is still not well understood in this filamentous fungus. In the present study, we sought to investigate how crgA influences the expression of structural genes for carotenoids, carotenoid biosynthesis, and other anabolic phenotypes. This will lead to a better understanding of the global regulation mechanism of carotenoid biosynthesis and facilitate engineering this strain in the future for enhanced production of carotenoids.

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Blakeslea trispora genes for carotene biosynthesis.

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Blakeslea trispora genes for carotene biosynthesis.

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