Strategies for strain improvement in Fusarium fujikuroi: overexpression and localization of key enzymes of the isoprenoid pathway and their impact on gibberellin biosynthesis

Abstract

The rice pathogen Fusarium fujikuroi is known to produce a wide range of secondary metabolites, such as the pigments bikaverin and fusarubins, the mycotoxins fusarins and fusaric acid, and the phytohormones gibberellic acids (GAs), which are applied as plant growth regulators in agri- and horticulture. The development of high-producing strains is a prerequisite for the efficient biotechnological production of GAs. In this work, we used different molecular approaches for strain improvement to directly affect expression of early isoprenoid genes as well as GA biosynthetic genes. Overexpression of the first GA pathway gene ggs2, encoding geranylgeranyl diphosphate synthase 2, or additional integration of ggs2 and cps/ks, the latter encoding the bifunctional ent-copalyldiphosphate synthase/ent-kaurene synthase, revealed an enhanced production level of 150%. However, overexpression of hmgR and fppS, encoding the key enzymes of the mevalonate pathway, hydroxymethylglutaryl coenzyme A reductase, and farnesyldiphosphate synthase, resulted in a reduced production level probably due to a negative feedback regulation of HmgR. Subsequent deletion of the transmembrane domains of HmgR and overexpression of the remaining catalytic domain led to an increased GA content (250%). Using green fluorescent protein and mCherry fusion constructs, we localized Cps/Ks in the cytosol, Ggs2 in small point-like structures, which are not the peroxisomes, and HmgR at the endoplasmatic reticulum. In summary, it was shown for the first time that amplification or truncation of key enzymes of the isoprenoid and GA pathway results in elevated production levels (2.5-fold). Fluorescence microscopy revealed localization of the key enzymes in different compartments.

The global regulator FfSge1 is required for expression of secondary metabolite gene clusters but not for pathogenicity in Fusarium fujikuroi

“…However, changes in an available acetyl‐CoA pool are not necessarily linked to an altered production rate of certain SMs due to the presence of other limiting factors. One such example is the negative feedback loop of the 3‐hydroxy‐3‐methyl‐glutaryl‐CoA reductase (HmgR) in the isoprenoid pathway: overexpression of HMGR resulted in decreased instead of elevated GA levels in F. fujikuroi (Albermann et al ., ). In addition, other global regulators, such as the velvet protein FfVel1 which negatively affects BIK biosynthesis, might overrule the activating effect of FfSge1 on this metabolite.…”

Spatial and temporal control of fungal natural product synthesis

“…Another biosynthetic pathway with enzymes localized to the cytosol is the diterpenoid phytohormone, gibberellin, produced by Fusarium fujikuroi (see section 3.2.3 for more details). In gibberellin biosynthesis, ent -copalyldiphosphate/ ent -kaurene synthase (Cps/Ks), a middle-pathway bifunctional enzyme that catalyzes conversion of geranylgeranyl diphosphate (GGDP) to ent -kaurene is found to localize to the cytosol 50 . It may be noteworthy that unlike the peroxisomes that can either house the initial or terminal biosynthetic step(s) of a pathway, the cytosol as of to date, is reported to only house intermediate steps of a biosynthetic pathway.…”

Metabolic engineering of the Stevia rebaudiana ent-kaurene biosynthetic pathway in recombinant Escherichia coli

“…A reductase (HmgR), in rice pathogen Fusarium fujikuroi (Albermann et al, 2013). Wang et al (2013) reported that the specific yield of ent-kaurene was enhanced by assembling and modulating the efflux pumps such as the AcrB and TolC components in E. coli.…”

“…Recently, Albermann et al (2013) have shown that GA production was elevated by overexpression of the GA pathway genes, geranylgeranyl diphosphate synthase 2 (ggs2) and bifunctional entcopalyldiphosphate synthase/ent-kaurene synthase (cps/ks), and truncation of the key enzyme of the mevalonate pathway, hydroxymethylglutaryl coenzyme. A reductase (HmgR), in rice pathogen Fusarium fujikuroi (Albermann et al, 2013).…”