Molecular analysis and evolution of gibberellin biosynthetic gene clusters in fungi

BETTINA TUDZYNSKI
Institut für Botanik
Westfälische Wilhelms Universität Münster
Schlossgarten 3
48149 Münster
tudzynsb@uni-muenster.de
http://www.uni-muenster.de/Biologie.Botanik/agtudzynski/Tudzynsk.htm

References
Malonek, S., Rojas, M.C., Hedden, P., Gaskin, P., and Tudzynski, B. (2004). The NADPH: cytochrome P450 reductase gene from Gibberella fujikuroi is essential for gibberellin biosynthesis. J. Biol. Chem. 279: 25075-25084.
The fungus Gibberella fujikuroi is used for the commercial production of gibberellins (GAs), which it produces in very large quantities. Four of the seven GA-biosynthetic genes in this species encode cytochrome P450 monooxygenases, which function in association with NADPH: cytochrome P450 reductases (CPRs) that mediate the transfer of electrons from NADPH to the P450 monooxygenases. Only one cpr gene (cpr-Gf) was found in G. fujikuroi and cloned by a PCR approach. The encoded protein contains the conserved CPR functional domains, including the FAD-, FMN-, and NADPH-binding motifs. cpr-Gf disruption mutants were viable but showed a reduced growth rate. Furthermore, disruption resulted in total loss of GA3, GA4 and GA7 production, but low levels of non-hydroxylated C20-GAs (GA15 and GA24) were still detected. In addition, the knock-out mutants were much more sensitive to benzoate than the wild-type due to loss of activity of another P450 monooxygenase, the detoxifying enzyme, benzoate p-hydroxylase. The UV-induced mutant of G. fujikuroi, SG138, which was shown to be blocked at most of the GA-biosynthetic steps catalyzed by P450 monoxygenases, displayed the same phenotype. Sequence analysis of the mutant cpr allele in SG138 revealed a nonsense mutation at amino acid position 627. The mutant was complemented with the cpr-Gf and the Aspergillus niger cprA genes, both genes fully restoring the ability to produce GAs.Northern blot analysis revealed co-regulated expression of the cpr-Gf gene and the GA-biosynthetic genes P450-1, P450-2, P450-4 under GA production conditions (nitrogen starvation). In addition, expression of cpr-Gf is induced by benzoate. These results indicate that CPR-Gf is the main, but not the only electron donor for several P450 monooxygenases from primary and secondary metabolism.

Rojas, M.C., Urrutia, O., Cruz, C., Gaskin, P., Tudzynski, B., Hedden, P. (2004) Kaurenolides and fujenoic acids are side products of the gibberellin P450-1 monooxygenase in Gibberella fujikuroi. Phytochemistry 65 (7): 821-830.
The steps involved in kaurenolide and fujenoic acids biosynthesis, from ent-kauradienoic acid and ent-6alpha,7alpha-dihydroxykaurenoic acid, respectively, are demonstrated in the gibberellin (GA)-deficient Gibberella fujikuroi mutant SG139, which lacks the entire GA-biosynthesis gene cluster, complemented with the P450-1 gene of GA biosynthesis (SG139-P450-1). ent-[2H]Kauradienoic acid was efficiently converted into 7beta-hydroxy[2H]kaurenolide and 7beta,18-dihydroxy[2H]kaurenolide by the cultures while 7beta-hydroxy[2H]kaurenolide was transformed into 7beta,18-dihydroxy[2H]kaurenolide. The limiting step was found to be hydroxylation at C-18. In addition, SG139-P450-1 transformed ent-6alpha,7alpha-dihydroxy [¹⁴C₄]kaurenoic acid into [¹⁴C₄]fujenoic acid and [¹⁴C₄]fujenoic triacid. Fujenal was also converted into the same products but was demonstrated not to be an intermediate in this sequence. All the above reactions were absent in the mutant SG139 and were suppressed in the wild-type strain ACC917 by disruption of the P450-1 gene. Kaurenolide and fujenoic acids synthesis were associated with the microsomal fraction and showed an absolute requirement for NADPH or NADH, all properties of cytochrome P450 monooxygenases. Only 7beta-hydroxy[¹⁴C₄]kaurenolide synthesis and not further 18-hydroxylation was detected in the microsomal fraction. The substrates for the P450-1 monooxygenase, ent-kaurenoic acid and [2H]GA12, efficiently inhibited kaurenolide synthesis with I50 values of 3 and 6 microM, respectively. Both substrates also inhibited ent-6alpha,7alpha-dihydroxy[14C4]kaurenoic acid metabolism by SG139-P450-1. Conversely, [¹⁴C₄]GA14 synthesis from [¹⁴C₄]GA12-aldehyde was inhibited by ent-[2H]kauradienoic acid and fujenal with I50 values of 10 and 30 microM, respectively. These results demonstrate that kaurenolides and seco-ring B kaurenoids are formed by the P450-1 monooxygenase (GA14 synthase) of G. fujikuroi and are thus side products that probably result from stabilization of radical intermediates involved in GA14 synthesis.

Malonek, S., Rojas, M.C., Hedden, P., Gaskin, P., Hopkins, P., and Tudzynski, B. (2005). Functional characterization of two cytochrome P450 monooxygenase genes, P450-1 and P450-4, of the gibberellic acid gene cluster in Fusarium proliferatum (Gibberella fujikuroi MP-D). Appl. Env. Microbiol. 71: 1462-1472.
Gibberella fujikuroi is a species complex with at least nine different biological species, termed mating populations (MPs) A to I, known to produce many different secondary metabolites. So far, gibberellin (GA) production is restricted to Fusarium fujikuroi (G. fujikuroi MP-C), although at least five other MPs contain all biosynthetic genes. Here, we analyze the GA gene cluster and GA pathway in the closest related species, F. proliferatum (MP-D) and demonstrate that the GA genes share a high degree of sequence homology with the corresponding genes of MP-C. The GA production capacity was restored after integration of the entire GA gene cluster from MP-C, indicating the existence of an active regulation system in F. proliferatum. The results further indicate that one reason for the loss of GA production is the accumulation of several mutations in the coding and 5'-non-coding regions of the ent-kaurene oxidase gene, P450-4.

Tudzynski, B. (2005) Gibberellin biosynthesis in fungi: genes, enzymes, evolution, and I impact on biotechnology. Appl Microbiol Biotechnol. 66: 597-611.
Gibberellins (GAs) constitute a large family of tetracyclic diterpenoid carboxylic acids, some members of which function as growth hormones in higher plants. As well as being phytohormones, GAs are also present in some fungi and bacteria. In recent years, GA biosynthetic genes from Fusarium fujikuroi and Arabidopsis thaliana have been cloned and well characterised. Although higher plants and the fungus both produce structurally identical GAs, there are important differences indicating that GA biosynthetic pathways have evolved independently in higher plants and fungi. The fact that horizontal gene transfer of GA genes from the plant to the fungus can be excluded, and that GA genes are obviously missing in closely related Fusarium species, raises the question of the origin of fungal GA biosynthetic genes. Besides characterisation of F. fujikuroi GA pathway genes, much progress has been made in the molecular analysis of regulatory mechanisms, especially the nitrogen metabolite repression controlling fungal GA biosynthesis. Basic research in this field has been shown to have an impact on biotechnology. Cloning of genes, construction of knock-out mutants, gene amplification, and regulation studies at the molecular level are powerful tools for improvement of production strains. Besides increased yields of the final product, GA3, it is now possible to produce intermediates of the GA biosynthetic pathway, such as ent-kaurene, ent-kaurenoic acid, and GA14, in high amounts using different knock-out mutants. This review concentrates mainly on the fungal biosynthetic pathway, the genes and enzymes involved, the regulation network, the biotechnological relevance of recent studies, and on evolutionary aspects of GA biosynthetic genes.

Malonek, S., Bömke, C., Bornberg-Bauer, E., Rojas, M.C., Hedden, P., Hopkins, P., and Tudzynski, B. (2005) Distribution of gibberellin biosynthetic genes and gibberellin production in the Gibberella fujikuroi species complex. Phytochem. 66: 1296-1311.
Gibberella fujikuroi is a monophyletic complex of at least nine sexually fertile biological species (mating populations, MP-A - MP-I) and more than 30 anamorphs in the genus Fusarium. They produce a variety of secondary metabolites, such as fumonisins, fusaproliferin, moniliformin, beauvericin, fusaric acid, and gibberellins (GAs), a group of plant hormones. In this study, we examined for the first time all nine sexually fertile species (MPs) and additional anamorphs within and outside the G. fujikuroi species complex for the presence of GA biosynthetic genes. So far, the ability to produce GAs was described only for Fusarium fujikuroi (G. fujikuroi MP-C), which contains seven clustered genes in the genome all participating in GA biosynthesis. We show that six other MPs (MPs B, D, E, F, G, and I) and most of the anamorphs within the species complex also consist of the entire GA gene cluster, whereas two MPs, F. verticillioides (MP-A), and F. circinatum (MP-H), contain only parts of the cluster. Despite the presence of the entire gene cluster in most of the species within the G. fujikuroi species complex, expression of GA biosynthetic genes and GA production were detected only in F. fujikuroi (MP-C). We used two new molecular marker genes, P450-4 from the GA gene cluster, and cpr, encoding the highly conserved NADPH cytochrome P450 reductase to study phylogenetic relationships within the G. fujikuroi species complex. The molecular phylogenetic studies for both genes have revealed good concordance to phylogenetic trees inferred from other genes. Furthermore, we discuss the role and evolutionary origin of the GA biosynthetic gene cluster.

[back]