米曲霉磷酸甲羟戊酸激酶功能研究

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

摘要/Abstract

摘要：

磷酸甲羟戊酸激酶（PMK）是甲羟戊酸（MVA）途径的关键酶。在真菌中，MVA途径是麦角甾醇生物合成的上游，因此PMK也被称为Erg8。为了研究磷酸甲羟戊酸激酶在米曲霉（Aspergillus oryzae）麦角甾醇合成通路中的作用，对米曲霉AoErg8基因功能进行研究。采用生物信息学方法鉴定米曲霉中的该基因，通过系统发育树和酵母异源互补分析其是否保守，利用实时荧光定量PCR（RT-qPCR）检测其表达模式，同时通过荧光蛋白标记对其亚细胞定位进行分析，最后测定AoErg8基因过表达对米曲霉生长和麦角甾醇含量的影响。结果表明，AoErg8进化保守，其表达量在不同生长时间和不同非生物胁迫下均发生了改变；AoErg8能恢复酿酒酵母erg8突变体的温度敏感表型；AoErg8定位于细胞质中；米曲霉中AoErg8过表达导致麦角甾醇含量降低，并且影响米曲霉生长和孢子形成。因此，米曲霉AoErg8的功能相对保守，其过表达可以降低麦角甾醇含量并影响菌落生长和孢子形成。该研究进一步揭示丝状真菌米曲霉麦角甾醇生物合成和调控机理，为米曲霉或其他真菌脂质代谢的基因工程奠定基础。

关键词: 米曲霉, 磷酸甲羟戊酸激酶, 麦角甾醇, 亚细胞定位, 孢子形成

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

尚怡彤, 闫欢欢, 王丽红, 田学琴, 薛萍红, 罗涛, 胡志宏. 米曲霉磷酸甲羟戊酸激酶功能研究[J]. 生物技术通报, 2023, 39(12): 311-319.

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.

图/表 8

图1 MVA途径代谢通路甲羟戊酸途径由乙酰辅酶A在乙酰辅酶A乙酰基转移酶、3-羟基-3-甲基戊二酰辅酶A合酶、3-羟基-3-甲基戊二酰辅酶A还原酶、甲羟戊酸激酶、磷酸甲羟戊酸激酶、甲羟戊酸-5-焦磷酸脱羧酶的催化下生成异戊烯焦磷酸，在植物、动物、真菌中位于类异戊二烯物质、甾醇类等物质合成途径的上游，在生物体的生长与代谢活动中起着重要作用

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

表1 不同物种Erg8序列号

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

表2 供试引物

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

图2 不同物种磷酸甲羟戊酸激酶的系统发育分析

Fig. 2 Phylogenetic analysis of phosphomevalonate kinases from different species

图3 AoErg8在琼脂CD培养基上不同生长时间和不同非生物胁迫下的表达水平 A：生长24、48和72 h AoErg8的表达；B：不同温度下AoErg8的表达；C：不同盐胁迫条件下AoErg8的表达；D：不同浓度乙醇（EtOH）胁迫条件下AoErg8的表达。将野生型米曲霉孢子悬浮液接种在单独的CD琼脂培养基或补充有NaCl或乙醇的CD琼脂培养基上，并在30℃下孵育（温度应激除外）。为了测定不同生长时间的mRNA水平，在24、48和72 h获菌丝体；对于其他测试，在72 h收获菌丝体。使用24 h、30℃下和在0% NaCl/乙醇中的AoErg8表达水平作为相应的参考。数值代表3个独立实验的平均值±标准差。通过GraphPad的t检验进行统计分析，*：P<0.05，**：P <0.01，下同

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

图4 AoErg8可以恢复酿酒酵母erg8突变体的表型 A：野生型生长、erg8突变体（Y40835）、AoErg8转化子在30℃和37℃的YPD和YPG培养基上的生长；B：测量所有转化子中的麦角甾醇含量，将对照和转化子在液体YPG中在30℃下培养2 d，并收集酵母以测定麦角甾醇的含量。将AoErg8转化子的麦角甾醇含量与相应培养基中的pYes2.0/erg8突变体进行比较

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

图5 AoErg8过表达菌株的表型及麦角甾醇的含量 A：培养72 h后，CK（野生型米曲霉转化的pEX2B载体）、AoErg8过表达菌株在PDA培养基上的菌落形态；B：不同转基因菌株菌落的孢子数；C：不同转基因菌株的菌落直径；D：AoErg8过表达菌株的麦角甾醇含量。对照和转化子在30℃的DPY中培养3 d，并收集以测定麦角甾醇的含量。将每个实验组与相应的对照组进行比较

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

图6 AoErg8的亚细胞定位 A：用AoErg8-DsRed转化米曲霉3.042 ΔpyrG菌丝体；从左到右：DIC、DsRed荧光图像以及DsRed和DIC的合并图像；B：AoErg8-DsRed与细胞质的共定位；用AoErg8-DsRed和pt-pEX1-GFP载体共转化米曲霉3.042 ΔpyrG的菌丝体；从左到右：DIC，DsRed、GFP的荧光图像，以及DsRed、GFP和DIC的合并图像

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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