Study on the Function of Phosphomevalonate Kinase in Aspergillus oryzae

doi:10.13560/j.cnki.biotech.bull.1985.2023-0702

Abstract

Abstract:

Phosphomevalonate kinase（PMK）is a key enzyme in the mevalonate（MVA）pathway. In fungi, the MVA pathway is the upstream of ergosterol biosynthesis and therefore PMK is also known as Erg8. In order to study the role of phosphomevalonate kinase in ergosterol synthesis pathway of Aspergillus oryzae, the function of AoErg8 gene in A. oryzae was preliminarily studied. Bioinformatics method was used to identify AoErg8. Phylogenetic tree and yeast allogeneic complementation were used to analyze whether this gene was conserved. Then the subcellular location was determined via fluorescence protein labelling and gene expression pattern were analyzed via RT-qPCR. Finally, the effects of overexpression of this gene on the growth and ergosterol content of A. oryzae were determined. AoErg8 was evolutionarily conservative, and its expression was different under different growth times and different abiotic stress. AoErg8 restored the temperature sensitive phenotype of Saccharomyces cerevisiae erg8 mutant. AoErg8 was located in the cytoplasm. The overexpression of AoErg8 led to the decrease of ergosterol content, and affected growth and spore formation. Therefore, the function of AoErg8 in A. oryzae was conservative and its overexpression reduceed the ergosterol content, also affected the growth of colonies and sporulation. This study further reveals the biosynthesis and regulatory mechanisms of ergosterol in this important filamentous fungus A. oryzae and lays the foundation for genetic engineering for lipid metabolism in A. oryzae or other fungi.

Key words: Aspergillus oryzae, phosphomevalonate kinase, ergosterol, subcellular localization, sporulation

SHANG Yi-tong, YAN Huan-huan, WANG Li-hong, TIAN Xue-qin, XUE Ping-hong, LUO Tao, HU Zhi-hong. Study on the Function of Phosphomevalonate Kinase in Aspergillus oryzae[J]. Biotechnology Bulletin, 2023, 39(12): 311-319.

Figures/Tables 8

Fig. 1 MVA pathway metabolic synthesis diagram MVA pathway is formed by acetyl-CoA under the catalysis of acetyl-CoA acetyltransferase, 3-hydroxy-3-methylglutaryl-CoA synthase, 3-hydroxy-3-methylglutaryl-CoA reductase, mevalonate kinase, phosphomevalonate kinase, mevalonate 5-pyrophosphate decarboxylase to generate isopentenyl-5-pyrophosphate. In plants, animals,and fungi, it is located in the upstream of isoprenoids and sterols, playing an important role in the growth and metabolic activities of organisms

Table 1 Erg8 numbers of different species

物种名 Species name	序列号GI No.
黄花蒿Artemisia annua	gi\|1387810006
人参Panax ginseng	gi\|555431957
黄花蒿Artemisia annua	gi\|1387770975
丹参Salvia miltiorrhiza	gi\|374639353
番茄Solanum lycopersicum	gi\|1480008164
海枣Phoenix dactylifera	gi\|672109111
短花药野生稻Oryza brachyantha	gi\|1002850285
玉米Zea mays	gi\|1126132679
卷柏Selaginella moellendorffii	gi\|1376963038
地钱Marchantia polymorpha	gi\|1376841057
小立碗藓Physcomitrium patens	gi\|1373905490
高卢蜜环菌Armillaria gallica	gi\|1243522655
米曲霉Aspergillus oryzae	gi\|391866384
酿酒酵母Saccharomyces cerevisiae	gi\|171479
植物乳杆菌Lactiplantibacillus plantarum	gi\|935445216
金黄色葡萄球菌Staphylococcus aureus	gi\|897312969
欧洲熊蜂Bombus terrestris	gi\|526117733
家蚕Bombyx mori	gi\|526117733
智人Homo sapiens	gi\|1294782
小鼠Mus musculus	gi\|32363399

Table 2 Primers used in this study

引物Primer	序列Sequence（5'-3'）
rH-F	GACAACATCCAGGGTATCACTAAGC
rH-R	GGTCTCCTCGTAGATCATGGCA
qRT-AoErg8-F	GGCTCTAGTAACTGCCCTAGTA
qRT-AoErg8-R	AGCCTGTGCCAAGTTATGAA
pEX2B-AoErg8-F	TTCACGTGCCCGTGCTTAAGATGTCTTATCCGCCATCCGGGAG
pEX2B-AoErg8-R	GAGGCCATGATATCCTTAAGAAGCCAACCGGCATATTGGTC
pYES2.0-AoErg8-F	CTATAGGGAATATTAAGCTTATGTCTTATCCGCCATCCGGG
pYES2.0-AoErg8-R	GATGGATATCTGCAGAATTCAAGCCAACCGGCATATTGGTC

Fig. 2 Phylogenetic analysis of phosphomevalonate kinases from different species

Fig.3 Expressions of AoErg8 on agar CD medium under different growth time and different abiotic stress A: Expression of AoErg8 at 24, 48, and 72 h of growth. B: Expression of AoErg8 at different temperatures. C: Expression of AoErg8 under different salt stress conditions.D: Expression of AoErg8 under different ethanol（EtOH）stress. In order to determine the level of mRNA at different growth times, Mycelium was harvested at 24, 48 and 72 h. For other tests, harvest Mycelium at 72 h. The expression levels of AoErg8 at 24 h, 30℃, and in 0% NaCl/ethanol were used as corresponding references. The numerical value represents the mean ± standard deviation of three independent experiments. Statistical analysis using GraphPad’s t-test, *: P<0.05, **: P <0.01, the same below

Fig. 4 AoErg8 may restore the phenotype of Saccharomyces cerevisiae erg8 mutant A: The growth of wild-type growth, erg8 mutant（Y40835）, and AoErg8 transformant on YPD and YPG media at 30℃ and 37℃. B: Measure the ergosterol content in all transformants, incubate the control and transformants in liquid YPG at 30℃ for 2 d, and collect yeast to determine the ergosterol content. Compare the ergosterol content of AoErg8 transformant with the pYES2.0/erg8 mutant in the corresponding culture medium

Fig. 5 Phenotype and ergosterol content of AoErg8 overexpressing strain A: After 72 h of cultivation, the colony morphology of CK（pEX2B vector transformed by wild-type A. oryzae）and AoErg8 overexpressing strains on PDA medium. B: Spore count of different transgenic strains. C: Colony diameter of different transgenic strains. D: Ergosterol content of AoErg8 overexpressing strains. The control and transformants were cultured in DPY at 30℃ for 3 d and collected for determination of ergosterol content. Compare each experimental group with the corresponding control group

Fig. 6 Subcellular localization of AoErg8 A: The mycelium of A. oryzae 3.042 ΔpyrG transformed with AoErg8-DsRed. Left to right: bright field（DIC）, fluorescent images of DsRed and merged image of DsRed and bright field. B: Co-localization of AoErg8 DsRed with cytoplasm. The A. oryzae 3.042 ΔpyrG mycelium co-transformed of AoErg8-DsRed and pt-pEX1-GFP carriers. From left to right: bright field（DIC）, fluorescent images of DsRed, GFP, and merged images of DsRed, GFP, and DIC

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