Contribution of membrane lipid unsaturation to temperaturedependent changes of secreted protein in the filamentous fungus
Aspergillus oryzae
Ryo IWAMA
Department of Biotechnology, The University of Tokyo
Research objectiveFilamentous fungi are eukaryotic microorganisms characterized by a long
filamentous cell morphology. These fungi are widely used in industrial fermentation
applications because of their ability to secrete various degradative enzymes into theextracellular space. The high extracellular secretory capacity of filamentous fungi for various
degradative enzymes is due to active vesicular transport, through which biomembranecomponents and cell wall synthases are continuously transported to the mycelial tip.
Phospholipids are the main component of the biomembrane that forms the transport vesicles.
There are various types of phospholipids, depending on the hydrophilic head and hydrophobic
tail. During industrial use, filamentous fungi are exposed to various stresses, including heatstress generated by fungi. Environmental temperature significantly affects biomembrane
properties. However, the effects of changes in phospholipid acyl chains on protein secretionin filamentous fungi are unclear. This study aimed to clarify the relationship between
incubation temperature, biomembrane phospholipid composition, and protein secretion in
Aspergillus oryzae, an industrially important filamentous fungus.
Methods
Generation of gene disruptant strains: The regulation of acyl chains of phospholipids infilamentous fungi is unclear. In this study, I focused on Aspergillus nidulans, which has long
been used as a model organism for filamentous fungi, and A. oryzae. Candidate genedisruption strains will be generated from these strains.
Lipidome analysis: Phospholipids were extracted from cultured mycelia using the BUME
method1) and subjected to quantitative phospholipid analysis using liquid chromatographytandem mass spectrometry (LC/MS/MS).
Examination of extracellular protein secretion capacity To analyze the extracellular protein
secretory capacity, the size of the halo after the iodine-starch reaction using a plate medium
containing starch was quantified using ImageJ software.

Results
Regulation of phospholipid unsaturation in A. nidulans
Lipidome analysis was performed using phospholipids extracted from A. nidulans
cultured in minimal liquid medium. Phospholipids having 34 and 36 carbons on the twoconstituent acyl chains were detected. Focusing on phospholipids with the 36-carbon acylchain, the number of double bonds in the acyl chain ranged from zero to six. There were up to
three double bonds in the fatty acids comprising the phospholipid acyl chain, indicating that
phospholipids with two multiple double-bond fatty acids are universally present in filamentous
fungi. Previous studies have demonstrated that filamentous fungi show growth patterns inwhich conidia germinate after non-polar swelling of the conidia. I examined the phospholipid
composition during the life cycle and discovered a transient increase in highly unsaturated
phospholipids, with a maximum value for the time at which the conidia germinated.
Furthermore, lipidome analysis of mycelia generated on a solid medium revealed theabundance of highly unsaturated phospholipids in areas where many cells displayed
filamentous morphology.
In A. nidulans, sdeA and sdeB encode ∆9 desaturases that introduce the first doublebond to fatty acids, odeA encodes ∆12 desaturase that introduces the second, and odeB encodes∆15 desaturase that introduces the third. However, the effects of these genes on biomembrane
phospholipids have not been analyzed in A. nidulans. In this study, I generated single
disruptant strains of these genes and analyzed the composition of their phospholipid acylchains. This in vivo analysis revealed that the odeA- or odeB-disrupted strains did not produce
phospholipids with five or more double bonds or three or more double bonds, respectively.
Effects of genes regulating phospholipid unsaturation on protein secretion in A. oryzae
In A. oryzae, AO090005000456, AO090102000339, AO090026000799, andAO090103000283 are putative ∆9 desaturase genes, AO090001000224 is a putative ∆12
desaturase gene, and AO0900010000714 is a putative ∆15 desaturase gene. These weredesignated sdeA, sdeB, sdeC, sdeD, odeA, and odeB in A. oryzae, respectively. The sdeA- andodeA-disrupted strains showed severe growth retardation at 20°C, and significant growth
retardation at 25°C and 30°C (Figure panel A). In A. nidulans, the effects of gene disruption
differed between A. oryzae and A. nidulans; the sdeA-disrupted strain grew more poorly athigher temperatures and the odeA-disrupted strain grew to a certain extent at lowertemperatures. When A. oryzae and A. nidulans were cultured at 20°C, 30°C, and 37°C andanalyzed for biomembrane phospholipid composition, the number of double bonds in
phospholipids increased at low temperatures in these two strains. The two strains exhibited
similar changes in acyl chain composition in response to temperature changes.

Figure. Phenotypes of fatty acid desaturase deficient strains in A. oryzae. (A) Conidia(103) of the wild-type (WT), ∆sdeA, ∆sdeB, ∆sdeC, ∆sdeD, ∆odeA, and ∆odeB were inoculated onto CDmedium and incubated for 3.5 days at 20°C, 25°C, 30°C, or 35°C. The sizes of the colonies weremeasured. (B) Conidia (103) of these strains were inoculated onto starch-containing CD medium andincubated for 3.5 days at 25°C or 30°C. The sizes of the halos produced in the iodine-starch test weremeasured. Bars indicate the mean of five independent experiments and dots indicate raw data. Error bars
represent standard error.

These disruption strains were cultured in a starch-containing plate medium, and theability to secrete proteins was evaluated based on the size of the halo in the iodine-starch test.
The ∆sdeA strain incubated at 20°C showed a significantly smaller halo; on the other hand,∆odeA strain incubated at 30°C showed a significantly larger halo (Figure panel B). Becausethe ∆odeA strain exhibited growth retardation even at 30°C in liquid CD medium, thephospholipid composition of the ∆odeA strain has not yet been analyzed. In A. nidulans, odeA
significantly increased phospholipids with two oleic acids as acyl chains. Therefore, toevaluate the effect of oleic acid on protein excretion, the halo size of the wild-type strain wasmeasured in starch-containing CD medium with oleic acid. A slight increase in the halo wasobserved. However, the same effect was observed when other fatty acids were added.
Therefore, the cause of the larger halo in the ∆odeA strain was unclear.
Conclusion
In A. oryzae, the halo sizes at 30°C were significantly larger in the ∆odeA strain.
There were no significant differences in halo size between the wild-type and ∆sdeA strains at30°C. The ∆sdeA strain showed growth retardation at both temperatures, as did the ∆odeAstrain. Therefore, poor growth is not expected to affect halo size. In the future, it will benecessary to clarify how the secretion of other proteins is altered, and the mechanism
responsible for this change in the ∆odeA strain.