Recombinant Lipase from Gibberella zeae Exhibits Broad Substrate Specificity: A Comparative Study on Emulsified and Monomolecular Substrate.

Using the classical emulsified system and the monomolecular film technique, the substrate specificity of recombinant Gibberella zeae lipase (rGZEL) that originates from Gibberella zeae was characterized in detail. Under the emulsified reaction system, both phospholipase and glycolipid hydrolytic activities were observed, except for the predominant lipase activity. The optimum conditions for different activity exhibition were also determined. Compared with its lipase activity, a little higher ratio of glycolipid hydrolytic activity (0.06) than phospholipase activity (0.02) was found. rGZEL preferred medium chain-length triglycerides, while lower activity was found for the longer-chain triglyceride. Using the monomolecular film technique, we found that the preference order of rGZEL to different phospholipids was 1,2-diacyl-sn-glycero-3-phospho-l-serine (PS) > 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (PG) > 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) > l-α-phosphatidylinositol (PI) > cardiolipin (CL) > 3-sn-phosphatidic acid sodium salt (PA) > l-α-phosphatidylethanolamine (PE), while no hydrolytic activity was detected for sphingomyelin (SM). Moreover, rGZEL showed higher galactolipase activity on 1,2-distearoyimonoglactosylglyceride (MGDG). A kinetic study on the stereo- and regioselectivity of rGZEL was also performed by using three pairs of pseudodiglyceride enantiomers (DDGs). rGZEL presented higher preference for distal DDG enantiomers than adjacent ester groups, however, no hydrolytic activity to the sn-2 position of diglyceride analogs was found. Furthermore, rGZEL preferred the R configuration of DDG enantiomers. Molecular docking results were in concordance with in vitro tests.

Improving the catalytic potential and substrate tolerance of Gibberella intermedia nitrilase by whole-cell immobilization.

Comparative studies of immobilized and free cells of Gibberella intermedia CA3-1 in bioconversion of 3-cyanopyridine to nicotinic acid were performed. Entrapping method was chosen based on the advantages in enzymatic activity recovery, mechanical strength and preparation procedure. Four entrapment matrices were investigated and sodium alginate was screened to be the most suitable material. Maximal nitrilase activity of alginate immobilized cells was obtained under conditions of 2 % alginate, 0.6 % CaCl2, 0.4 g cell/g alginate, 1.8 mm bead size. The immobilized cells showed excellent substrate tolerance even when the 3-cyanopyridine concentration was 700 mM. The half-lives of immobilized cells at 30, 40 and 50 °C were 315, 117.5 and 10.9 h, respectively, correspondingly 1.4, 1.6 and 1.7-fold compared with that of the free cells. Efficient reusability of immobilized cells up to 28 batches was achieved and 205.7 g/(g dcw) nicotinic acid was obtained with 80.55 % enzyme activity preserved.

Isolation and characterization of Gibberella intermedia CA3-1, a novel and versatile nitrilase-producing fungus.

Nitrilase-mediated biocatalysis has attracted substantial attention for its application in carboxylic acid production in recent years. In the present study, the fungus CA3-1 was isolated and identified as Gibberella intermedia based on its morphology, its 18S ribosomal DNA (rDNA), and internal transcribed spacer (ITS) sequences. The enzymatic properties of G. intermedia resting cells were determined, and the optimum activity was achieved at 40 °C with pH 7.6. The half-lives of the nitrilase at 30, 40, and 50 °C were 231.1, 72.9, and 6.4 h, respectively. This Gibberella nitrilase showed a wide substrate spectrum with high specificity for heterocyclic and aliphatic nitriles. It remained extremely active in 5% propanol. The presence of Ag(+), Hg(2+), and excess substrate inhibited the nitrilase activity, whereas Fe(2+), Mn(2+), and Li(+) improved enzyme activity. 3-Cyanopyridine (50 mM) was hydrolyzed into nicotinic acid within 30 min, whereas only <5% of nicotinamide was detected. The results show that this fungal nitrilase is a promising candidate for commercial application in nicotinic acid production.

Functional analyses of two acetyl coenzyme A synthetases in the ascomycete Gibberella zeae.

Acetyl coenzyme A (acetyl-CoA) is a crucial metabolite for energy metabolism and biosynthetic pathways and is produced in various cellular compartments with spatial and temporal precision. Our previous study on ATP citrate lyase (ACL) in Gibberella zeae revealed that ACL-dependent acetyl-CoA production is important for histone acetylation, especially in sexual development, but is not involved in lipid synthesis. In this study, we deleted additional acetyl-CoA synthetic genes, the acetyl-CoA synthetases (ACS genes ACS1 and ACS2), to identify alternative acetyl-CoA production mechanisms for ACL. The ACS1 deletion resulted in a defect in sexual development that was mainly due to a reduction in 1-palmitoyl-2-oleoyl-3-linoleoyl-rac-glycerol production, which is required for perithecium development and maturation. Another ACS coding gene, ACS2, has accessorial functions for ACS1 and has compensatory functions for ACL as a nuclear acetyl-CoA producer. This study showed that acetate is readily generated during the entire life cycle of G. zeae and has a pivotal role in fungal metabolism. Because ACSs are components of the pyruvate-acetaldehyde-acetate pathway, this fermentation process might have crucial roles in various physiological processes for filamentous fungi.

New PCR assays for the identification of Fusarium verticillioides, Fusarium subglutinans, and other species of the Gibberella fujikuroi complex.

Fusarium verticillioides and Fusarium subglutinans are important fungal pathogens of maize and other cereals worldwide. In this study, we developed PCR-based protocols for the identification of these pathogens targeting the gaoB gene, which codes for galactose oxidase. The designed primers recognized isolates of F. verticillioides and F. subglutinans that were obtained from maize seeds from several producing regions of Brazil but did not recognize other Fusarium spp. or other fungal genera that were either obtained from fungal collections or isolated from maize seeds. A multiplex PCR protocol was established to simultaneously detect the genomic DNA from F. verticillioides and F. subglutinans. This protocol could detect the DNA from these fungi growing in artificially or naturally infected maize seeds. Another multiplex reaction with a pair of primers developed in this work combined with a pre-existing pair of primers has allowed identifying F. subglutinans, F. konzum, and F. thapsinum. In addition, the identification of F. nygamai was also possible using a combination of two PCR reactions described in this work, and another described in the literature.

ATP citrate lyase is required for normal sexual and asexual development in Gibberella zeae.

Adenosine triphosphate (ATP) citrate lyase (ACL) is a key enzyme in the production of cytosolic acetyl-CoA, which is crucial for de novo lipid synthesis and histone acetylation in mammalian cells. In this study, we characterized the mechanistic roles of ACL in the homothallic ascomycete fungus Gibberella zeae, which causes Fusarium head blight in major cereal crops. Deletion of ACL in the fungus resulted in a complete loss of self and female fertility as well as a reduction in asexual reproduction, virulence, and trichothecene production. When the wild-type strain was spermatized with the ACL deletion mutants, they produced viable ascospores, however ascospore delimitation was not properly regulated. Although lipid synthesis was not affected by ACL deletion, histone acetylation was dramatically reduced in the ACL deletion mutants during sexual development, suggesting that the defects in sexual reproduction were caused by the reduction in histone acetylation. This study is the first report demonstrating a link between sexual development and ACL-mediated histone acetylation in fungi.

Histidine kinase two-component response regulator proteins regulate reproductive development, virulence, and stress responses of the fungal cereal pathogens Cochliobolus heterostrophus and Gibberella zeae.

Histidine kinase (HK) phosphorelay signaling is a major mechanism by which fungi sense their environment. The maize pathogen Cochliobolus heterostrophus has 21 HK genes, 4 candidate response regulator (RR) genes (SSK1, SKN7, RIM15, REC1), and 1 gene (HPT1) encoding a histidine phosphotransfer domain protein. Because most HKs are expected to signal through RRs, these were chosen for deletion. Except for pigment and slight growth alterations for rim15 mutants, no measurable altered phenotypes were detected in rim15 or rec1 mutants. Ssk1p is required for virulence and affects fertility and proper timing of sexual development of heterothallic C. heterostrophus. Pseudothecia from crosses involving ssk1 mutants ooze masses of single ascospores, and tetrads cannot be found. Wild-type pseudothecia do not ooze. Ssk1p represses asexual spore proliferation during the sexual phase, and lack of it dampens asexual spore proliferation during vegetative growth, compared to that of the wild type. ssk1 mutants are heavily pigmented. Mutants lacking Skn7p do not display any of the above phenotypes; however, both ssk1 and skn7 mutants are hypersensitive to oxidative and osmotic stresses and ssk1 skn7 mutants are more exaggerated in their spore-type balance phenotype and more sensitive to stress than single mutants. ssk1 mutant phenotypes largely overlap hog1 mutant phenotypes, and in both types of mutant, the Hog1 target gene, MST1, is not induced. ssk1 and hog1 mutants were examined in the homothallic cereal pathogen Gibberella zeae, and pathogenic and reproductive phases of development regulated by Ssk1 and Hog1 were found to mirror, but also vary from, those of C. heterostrophus.

Genome mining for the discovery of new nitrilases in filamentous fungi.

PURPOSE OF WORK: our aim is to describe new fungal nitrilases whose sequences were published but whose catalytic properties were unknown. We adapted for expression in E. coli three of the genes and confirmed that the enzymes acted on organic nitriles. The genome mining approach was used to search for nitrilases in filamentous fungi. Synthetic genes encoding nitrilases in Aspergillus niger, Gibberella moniliformis and Neurospora crassa were expressed in Escherichia coli. This is the first heterologous expression of fungal enzymes of this type. The recombinant enzyme derived from G. moniliformis was an aromatic nitrilase with an activity of 390 U l(-1) culture with benzonitrile as substrate. This was much less than the activities of the recombinant enzymes derived from A. niger and N. crassa that had activities of 2500 and 2700 U l(-1) culture, respectively, with phenylacetonitrile as substrate.

A polyketide macrolactone synthase from the filamentous fungus Gibberella zeae.

Resorcylic acid lactones represent a unique class of fungal polyketides and display a wide range of biological activities, such as nanomolar inhibitors of Hsp90 and MAP kinase. The biosynthesis of these compounds is proposed to involve two fungal polyketide synthases (PKS) that function collaboratively to yield a 14-membered macrolactone with a resorcylate core. We report here the reconstitution of Gibberella zeae PKS13, which is the nonreducing PKS associated with zearalenone biosynthesis. Using a small molecule mimic of the natural hexaketide starter unit, we reconstituted the entire repertoire of PKS13 activities in vitro, including starter-unit selection, iterative condensation, regioselective C2-C7 cyclization, and macrolactone formation. PKS13 synthesized both natural 14-membered and previously uncharacterized 16-membered resorcylic acid lactones, indicating relaxed control in both iterative elongation and macrocyclization. PKS13 exhibited broad starter-unit specificities toward fatty acyl-CoAs ranging in sizes between C6 and C16 and displayed the highest activity toward decanoyl-CoA. PKS13 was shown to be active in Escherichia coli and synthesized numerous alkyl pyrones and alkyl resorcylic esters without exogenously supplied precursors. We demonstrated that PKS13 can interact with E. coli fatty acid biosynthetic machinery and can be primed with fatty-acyl ACPp at low-micromolar concentrations. PKS13 synthesized new polyketides in E. coli when the culture was supplemented with synthetic precursors, showcasing its utility in precursor-directed biosynthesis. PKS13 is therefore a highly versatile polyketide macrolactone synthase that is useful in the engineered biosynthesis of polyketides, including resorcylic acid lactones that are not found in nature.

Engineered biosynthesis of bacterial aromatic polyketides in Escherichia coli.

Bacterial aromatic polyketides are important therapeutic compounds including front line antibiotics and anticancer drugs. It is one of the last remaining major classes of natural products of which the biosynthesis has not been reconstituted in the genetically superior host Escherichia coli. Here, we demonstrate the engineered biosynthesis of bacterial aromatic polyketides in E. coli by using a dissected and reassembled fungal polyketide synthase (PKS). The minimal PKS of the megasynthase PKS4 from Gibberella fujikuroi was extracted by using two approaches. The first approach yielded a stand-alone Ketosynthase (KS)_malonyl-CoA:ACP transferase (MAT) didomain and an acyl-carrier protein (ACP) domain, whereas the second approach yielded a compact PKS (PKS_WJ) that consists of KS, MAT, and ACP on a single polypeptide. Both minimal PKSs produced nonfungal polyketides cyclized via different regioselectivity, whereas the fungal-specific C2-C7 cyclization mode was not observed. The kinetic properties of the two minimal PKSs were characterized to confirm both PKSs can synthesize polyketides with similar efficiency as the parent PKS4 megasynthase. Both minimal PKSs interacted effectively with exogenous polyketide cyclases as demonstrated by the synthesis of predominantly PK8 3 or NonaSEK4 6 in the presence of a C9-C14 or a C7-C12 cyclase, respectively. When PKS_WJ and downstream tailoring enzymes were expressed in E. coli, the expected nonaketide anthraquinone SEK26 was recovered in good titer. High-cell density fermentation was performed to demonstrate the scale-up potential of the in vivo platform for the biosynthesis of bacterial polyketides. Using engineered fungal PKSs can therefore be a general approach toward the heterologous biosynthesis of bacterial aromatic polyketides in E. coli.

Functional analyses of two syntaxin-like SNARE genes, GzSYN1 and GzSYN2, in the ascomycete Gibberella zeae.

We identified two syntaxin-like SNARE genes, named GzSYN1 and GzSYN2, from the plant pathogenic ascomycete Gibberella zeae, and characterized the functions and cellular localization of these genes. The GzSYN1 deletion mutant (Deltagzsyn1) had 71% reduced hyphal growth compared to the wild-type strain, but produced perithecia with normal ascospores. Deltagzsyn2 had the same hyphal growth rate as the wild-type, but completely lost both self and female fertility. When Deltagzsyn2 was spermatized for Deltamat1-1 or Deltamat1-2 strains, it retained its male fertility, but the ascus shape was abnormal and ascospore delimitation was delayed. The Deltagzsyn1 and Deltagzsyn2 virulence on barley was reduced by 67% and 75%, respectively, compared to the wild-type. The GFP::GzSYN1 fusion protein was localized in vesicles, vacuoles, plasma membranes, and septa, whereas GFP::GzSYN2 was found only in plasma membranes and septa. These results suggest that syntaxins have key roles in fungal development and virulence in G. zeae.

GzSNF1 is required for normal sexual and asexual development in the ascomycete Gibberella zeae.

The sucrose nonfermenting 1 (SNF1) protein kinase of yeast plays a central role in the transcription of glucose-repressible genes in response to glucose starvation. In this study, we deleted an ortholog of SNF1 from Gibberella zeae to characterize its functions by using a gene replacement strategy. The mycelial growth of deletion mutants (DeltaGzSNF1) was reduced by 21 to 74% on diverse carbon sources. The virulence of DeltaGzSNF1 mutants on barley decreased, and the expression of genes encoding cell-wall-degrading enzymes was reduced. The most distinct phenotypic changes were in sexual and asexual development. DeltaGzSNF1 mutants produced 30% fewer perithecia, which matured more slowly, and asci that contained one to eight abnormally shaped ascospores. Mutants in which only the GzSNF1 catalytic domain was deleted had the same phenotype changes as the DeltaGzSNF1 strains, but the phenotype was less extreme in the mutants with the regulatory domain deleted. In outcrosses between the DeltaGzSNF1 mutants, each perithecium contained approximately 70% of the abnormal ascospores, and approximately 50% of the asci showed unexpected segregation patterns in a single locus tested. The asexual spores of the DeltaGzSNF1 mutants were shorter and had fewer septa than those of the wild-type strain. The germination and nucleation of both ascospores and conidia were delayed in DeltaGzSNF1 mutants in comparison with those of the wild-type strain. GzSNF1 expression and localization depended on the developmental stage of the fungus. These results suggest that GzSNF1 is critical for normal sexual and asexual development in addition to virulence and the utilization of alternative carbon sources.

Roles of the glyoxylate and methylcitrate cycles in sexual development and virulence in the cereal pathogen Gibberella zeae.

The glyoxylate and methylcitrate cycles are involved in the metabolism of two- or three-carbon compounds in fungi. To elucidate the role(s) of these pathways in Gibberella zeae, which causes head blight in cereal crops, we focused on the functions of G. zeae orthologs (GzICL1 and GzMCL1) of the genes that encode isocitrate lyase (ICL) and methylisocitrate lyase (MCL), respectively, key enzymes in each cycle. The deletion of GzICL1 (DeltaGzICL1) caused defects in growth on acetate and in perithecium (sexual fruiting body) formation but not in virulence on barley and wheat, indicating that GzICL1 acts as the ICL of the glyoxylate cycle and is essential for self-fertility in G. zeae. In contrast, the DeltaGzMCL1 strains failed to grow on propionate but exhibited no major changes in other traits, suggesting that GzMCL1 is required for the methylcitrate cycle in G. zeae. Interestingly, double deletion of both GzICL1 and GzMCL1 caused significantly reduced virulence on host plants, indicating that both GzICL1 and GzMCL1 have redundant functions for plant infection in G. zeae. Thus, both GzICL1 and GzMCL1 may play important roles in determining major mycological and pathological traits of G. zeae by participating in different metabolic pathways for the use of fatty acids.

A thioesterase from an iterative fungal polyketide synthase shows macrocyclization and cross coupling activity and may play a role in controlling iterative cycling through product offloading.

Zearalenone, a fungal macrocyclic polyketide, is a member of the resorcylic acid lactone family. Herein, we characterize in vitro the thioesterase from PKS13 in zearalenone biosynthesis (Zea TE). The excised Zea TE catalyzes macrocyclization of a linear thioester-activated model of zearalenone. Zea TE also catalyzes the cross coupling of a benzoyl thioester with alcohols and amines. Kinetic characterization of the cross coupling is consistent with a ping-pong bi-bi mechanism, confirming an acyl-enzyme intermediate. Finally, the substrate specificity of the Zea TE indicates the TE may help control iterative cycling on PKS13 by rapidly offloading the final resorcylate-containing product.

Gibberella zeae chitin synthase genes, GzCHS5 and GzCHS7, are required for hyphal growth, perithecia formation, and pathogenicity.

Gibberella zeae causes Fusarium head blight of cereal crops, and sexual spores of the fungus play an important role as primary inocula. We isolated a restriction enzyme-mediated integration (REMI) transformant, ZH431, of G. zeae with defects in perithecia formation and virulence. Integration of the REMI vector resulted in disruption of GzCHS7 gene, which encodes a putative class VII chitin synthase with high similarity to Fusarium oxysporum ChsVb. A second chitin synthase, GzCHS5, is adjacently located in a head-to-head configuration with GzCHS7, and its deduced protein sequence showed similarity with a class V chitin synthase in F. oxysporum. Neither DeltaGzChs5 nor DeltaGzChs7 mutants produced perithecia or caused disease on barley heads. Microscopic observation revealed that both mutants formed balloon-shaped hyphae and intrahyphal hyphae and that cell wall rigidity of the mutants was weaker than that of the wild-type strain. Transcription profiles of GzCHS5 and GzCHS7 were not altered in DeltaGzChs7 and DeltaGzChs5, respectively, suggesting that transcription regulations of the genes are independent of each other. Our results demonstrate that GzCHS5 and GzCHS7 are indispensable for perithecia formation and pathogenicity as well as normal septa formation and hyphal growth in G. zeae.

A novel protease-resistant alpha-galactosidase with high hydrolytic activity from Gibberella sp. F75: gene cloning, expression, and enzymatic characterization.

A novel alpha-galactosidase gene (aga-F75) from Gibberella sp. F75 was cloned and expressed in Escherichia coli. The gene codes for a protein of 744 amino acids with a 24-residue putative signal peptide and a calculated molecular mass of 82.94 kDa. The native structure of the recombinant Aga-F75 was estimated to be a trimer or tetramer. The deduced amino acid sequence showed highest identity (69%) with an alpha-galactosidase from Hypocrea jecorina (Trichoderma reesei), a member of the glycoside hydrolase family 36. Purified recombinant Aga-F75 was optimally active at 60 degrees C and pH 4.0 and was stable at pH 3.0-12.0. The enzyme exhibited broad substrate specificity and substantial resistance to neutral and alkaline proteases. The enzyme K (m) values using pNPG, melibiose, stachyose, and raffinose as substrates were 1.06, 1.75, 54.26, and 8.23 mM, respectively. Compared with the commercial alpha-galactosidase (Aga-A) from Aspergillus niger var. AETL and a protease-resistant alpha-galactosidase (Aga-F78) from Rhizopus sp. F78, Aga-F75 released 1.4- and 4.9-fold more galactose from soybean meal alone, respectively, and 292.5- and 8.6-fold more galactose from soybean meal in the presence of trypsin, respectively. The pH and thermal stability and hydrolytic activity of Aga-F75 make it potentially useful in the food and feed industries.

Kinetics and regioselectivity of three GH62 α-L-arabinofuranosidases from plant pathogenic fungi.

BACKGOUND: Xylan is the second most abundant plant cell wall polysaccharide after cellulose with α-L-arabinofuranose (L-Araf) as one of the major side substituents. Capacity to degrade xylan is characteristic of many plant pathogens; and corresponding enzymes that debranch arabinoxylan provide tools to tailor xylan functionality or permit its full hydrolysis. METHOD: Three GH62_2 family α-arabinofuranosidases (Abfs) from plant pathogenic fungi, NhaAbf62A from Nectria haematococca, SreAbf62A from Sporisorium reilianum and GzeAbf62A from Gibberella zeae, were recombinantly produced in Escherichia coli. Their biochemical properties and substrate specificities were characterized in detail. Particularly with 1H NMR, the regioselectivity and debranching preference of the three Abfs were directly compared. RESULTS: The activities of selected Abfs towards arabinoxylan were all optimal at pH 6.5. Their preferred substrates were wheat arabinoxylan, followed by soluble oat spelt xylan. The Abfs displayed selectivity towards either α-(1 → 2) or α-(1 → 3)-L- Araf mono-substituents in arabinoxylan. Specifically, SreAbf62A and GzeAbf62A removed m-α-(1 → 3)-L-Araf and m-α-(1 → 2)-L-Araf substituents with a similar rates, whereas NhaAbf62A released m-α-(1 → 3)-L-Araf 1.9 times faster than m-α-(1 → 2)-L-Araf. MAJOR CONCLUSIONS: Building upon the known selectivity of GH62 family α-arabinofuranosidases towards L-Araf mono-substituents in xylans, the current study uncovers enzyme-dependent preferences towards m-α-(1 → 3)-L-Araf and m-α-(1 → 2)-L-Araf substitutions. Comparative sequence-structure analyses of Abfs identified an arginine residue in the xylose binding +2R subsite that was correlated to the observed enzyme-dependent L-Araf debranching preferences. GENERAL SIGNIFICANCE: This study expands the limited pool of characterized GH62 Abfs particularly those from plant pathogenic fungi, and provides biochemical details and methodology to evaluate regioselectivity within this glycoside hydrolase family.

Exploring the influence of phospholipid monolayer conformation and environmental conditions on the interfacial binding of Gibberella Zeae lipase.

The involvement of different parameters on Gibberella zeae lipase (GZEL) membrane binding were characterized by using monomolecular film technology and circular dichroism spectroscopy. Among four kinds of phospholipid monolayers, 1,2­dimyristoyl­sn­glycero­3­phosphoethanolamine have the highest maximum insertion pressure (MIP) value. Comparing the GZEL adsorption to phosphatidylcholine monolayers with different acyl chains in sn-1 and sn-2 positions, the higher MIP values were found for 1,2­dilauroyl­sn­glycero­3­phosphocholine. Significantly improvement between 1,2­dioleoyl­sn­glycero­3­phosphocholine and 1,2­distearoyl­sn­glycero­3­phosphocholine suggested that the presence of fatty acid unsaturation may affect protein adsorption by changing the chemical structure in each phospholipid. The MIP value was shown higher (48.6 mN m-1) at pH 5 and pH 6 (47.5 ±â€¯1.9 mN m-1) but decreased significantly (34.2 mN m-1) at pH 9. This may indicate that the proportion of helices in the protein decreases with the alteration of the catalytic center, thus affecting the binding of the protein to its substrate. The MIP values obviously decreased with increasing salt ion concentration, suggesting that excessive salt ion concentration may destabilize the secondary and tertiary structures of the protein, thereby affecting the characteristics of its adsorption at the interfaces. Present studies improve our understanding on the protein-membrane interaction of this enzyme.

Redirecting the cyclization steps of fungal polyketide synthase.

Regiospecific cyclizations of the nascent poly-beta-ketone backbones dictate the structures of polyketide natural products. The fungal iterative megasynthases use terminal thioesterase/claisen cyclase (TE/CLC) domains to direct the fate of the polyketide chains. In this work, we present two strategies toward redirecting the cyclization steps of fungal PKSs using the Gibberella fujikuroi PKS4. First, inactivation or removal of the TE/CLC domain resulted in the synthesis of the new polyketide SMA93 2. Complementation of the mutant PKS4 with a standalone TE/CLC domain restored the regioselective cyclization steps of PKS4 and led to the synthesis of SMA76 1, demonstrating that cyclization enzymes can interact with the megasynthase in trans. This led to the second approach in which various dissociated, bacterial tailoring enzymes were added to the megasynthase in trans. Addition of the act KR led to the synthesis of mutactin 3, while the addition of first ring and second ring cyclases yielded anthraquinone compounds DMAC 5 and SEK26 6. The cooperative activities of fungal and bacterial PKS components are especially important and enable synthesis of polyketides utilizing enzymes from two distinct families of PKSs.

Crystallization and preliminary crystallographic analysis of Gibberella zeae extracellular lipase.

Fusarium head blight, one of the most destructive crop diseases, is mainly caused by Fusarium graminearum (known in its sexual stage as Gibberella zeae). F. graminearum secretes various extracellular enzymes that have been hypothesized to be involved in host infection. One of the extracellular enzymes secreted by this organism is the G. zeae extracellular lipase (GZEL), which is encoded by the FGL1 gene. In order to solve the crystal structure of GZEL and to gain a better understanding of the biological functions of the protein and of possible inhibitory mechanisms of lipase inhibitors, recombinant GZEL was crystallized at 291 K using PEG 3350 as a precipitant. A data set was collected to 2.8 A resolution from a single flash-cooled crystal (100 K). The crystal belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 78.4, b = 91.0, c = 195.8 A, alpha = beta = gamma = 90 degrees . The presence of four molecules was assumed per asymmetric unit, which gave a Matthews coefficient of 2.6 A(3) Da(-1).