嗜热毁丝霉木糖代谢调控机制研究

doi:10.13304/j.nykjdb.2023.0151

摘要/Abstract

摘要：

为提高发酵菌株的木糖利用速率，以嗜热毁丝霉为体系，利用遗传学手段及转录组学技术，探究丝状真菌嗜热毁丝霉木糖代谢的调控机制。结果表明，在嗜热毁丝霉中，转录因子XlnR能够正向调控木糖代谢和转运以及半纤维酶的表达，是木糖条件下孢子萌发的必需因子。在木糖条件下，突变体ΔxlnR孢子丧失了萌发的能力，并且菌丝木糖利用速率显著降低，而过表达xlnR 使得木糖利用速率提升14.2%。转录组数据表明，敲除xlnR 导致木聚糖水解酶基因、木糖转运蛋白及代谢相关基因的转录水平显著降低。全局调控因子Cre-1是木糖及葡萄糖代谢的抑制因子，敲除cre-1后显著提升嗜热毁丝霉木糖和葡萄糖利用速率；同时，Cre-1可直接抑制xlnR及自身的表达。通过过表达木糖代谢激活因子XlnR编码基因xlnR，同时敲除抑制因子Cre-1编码基因cre-1，嗜热毁丝霉木糖利用速率提高100%。以上研究结果为生物质高效利用底盘细胞的创建提供了良好的出发菌株。

关键词: 嗜热毁丝霉, 木糖代谢, 转录调控, Cre-1, XlnR

Abstract:

In order to improve xylose utilization efficiency of fermentation strains， the regulation mechanism of xylose metabolism in filamentous fungi Myceliophthora thermophila was investigated using genetic methods and transcriptomic techniques. The results showed that the transcription factor XlnR could positively regulate xylose metabolism and transport， as well as hemicellulase gene expression in M. thermophila， and was required for conidia germination under xylose conditions. Under xylose condition， the conidia of mutant ΔxlnR lost the ability to germinate， and the xylose utilization efficiency of mycelia was significantly decreased， while the xylose utilization efficiency was increased by 14.2% due to xlnR overexpression. Transcriptomic analysis showed that knockout of xlnR resulted in lower transcription levels of the genes involved in xylanolytic enzymes， xylose transporter， and xylose catabolic pathway. The global regulatory factor Cre-1 was an inhibitor of xylose and glucose metabolism. Disruption of cre-1 could significantly improve the utilization efficiency of xylose and glucose in M. thermophila. Meanwhile， the Cre-1 could directly inhibit the expression of xlnR and itself. The overexpression of xlnR and knockout of cre-1 made the xylose metabolic utilization efficiency increased by 100%. Above results provided a good starting strain for the establishment of the cell chassis for the efficient utilization of biomass.

Key words: Myceliophthora thermophila, xylose metabolism, transcription regulation, Cre-1, XlnR

中图分类号:

S182

陈美欣, 顾淑莹, 刘佳, 刘浩, 李金根, 田朝光. 嗜热毁丝霉木糖代谢调控机制研究[J]. 中国农业科技导报, 2024, 26(12): 77-87.

Meixin CHEN, Shuying GU, Jia LIU, Hao LIU, Jingen LI, Chaoguang TIAN. Research on Regulation Mechanism of Xylose Metabolism in Myceliophthora thermophila[J]. Journal of Agricultural Science and Technology, 2024, 26(12): 77-87.

图/表 9

表1 扩增目的片段所用引物

Table 1 Primers used to amplify the target fragments

引物名称 Prime name	引物序列 Prime sequence（5’-3’）	备注 Note
cre-1-up-F/R	GAGGACTTGACGCTGC/GTCGTTAGACTCTTTCA	cre-1 5’
cre-1-down-F/R	GTGCCCCTGATAATAGC/CGACGGAATTGAGGATG	cre-1 3’
xlnR-up-F/R	CACAGTACACGAGGACTTG/CAATATCAGTTAACGTCG	xlnR 5’
xlnR-down-F/R	GAGTTCTTCTGATAATGAC/GAGGATCAGGACTGG	xlnR 3’
neo-F/R	CTACGACGTTAACTG/CAGAAGAACTCGTC	neo
bar-F/R	CAAAGATATTGAAG/GATCACTAGTACAGG	bar
OE xlnR-F/R	GTTCTTCAGAATGTTGT/GATCCCTACAGCGCCAGAC	xlnR
U6p-cre-1-R	GAGTTGCTCCAACACGAGGAAAGAAAGAAAAGAAG	U6p-cre-1-sgRNA
cre-1-gRNA-F	TGTTGGAGCAACTCCGCTTGTTTTAGAGCTAGAAATAG	U6p-cre-1-sgRNA
U6p-xlnR-R	TAGCTGGTTGCACTGATCGCGAGGAAAGAAAG	U6p-xlnR-sgRNA
xlnR-gRNA-F	AGTGCAACCAGCTAGTTTTAGAGCTAGAAATAGC	U6p-xlnR-sgRNA
U6-F/gRNA-R	GTGGAGTGAAGTTCGGAA/AAAAAGCACCGACTC	sgRNA
cas-F/R	TCCGAGGTTCGACATCAG/GCTCCCTCTAAACAAGTG	cas9
cre-1-emsa-F/R	CCAAAATCGGATCTGGTTCCGCGTGGATCCCAGCAGAAGCAGCAAGAACAACA/GATGCGGCCCTAGTGGTGGTGGTGGTGGTGCTCGAGCATGGCGGCGTGCTGATGATG	cre-1-EMSA
xlnR-emsa-F/R	CCAAAATCGGATCTGGTTCCGCGTGGATCCGCTCGGACTGGCCCTCAACCGA/CTAGTGGTGGTGGTGGTGGTGCTCGTGAATTCTCGGTCTTCTGGC	xlnR-EMSA

表 2 转化体验证引物

Table 2 Verification primer of mutation

引物名称

Prime name

引物序列

Primer sequence （5’-3’）

备注

Note

Orf-cre-1-F/R

CGATACTCGACCAAGAA/GAACCACTCTGGCTGCT

∆cre-1

Orf-xlnR-F/R

GCTCCCCATCGCCG/GGCCAGAAGACCGAG

∆xlnR

Orf-OExlnR-F/R

GCGTAACCCCACCAAC/GCTGATCTGACCAGT

OexlnR

表3 探针扩增引物

Table 3 Probe amplification primer

引物名称

Prime name

引物序列

Primer sequence （5’-3’）

备注

Note

cre-1-P-F/R

CCGTAGGTACCTGG/ GTTTTAAATGACGC

cre-1探针cre-1 probe

xlnR-P-F/R

GGCATCCCTTGGTC/ GCTCCGCCGGTGCTAA

xlnR探针xlnR probe

图1 野生型嗜热毁丝霉和突变体ΔxlnR 的表型A：接种突变体ΔxlnR孢子后木糖利用；B：接种突变体ΔxlnR孢子后阿拉伯糖利用；C：接种突变体ΔxlnR孢子后葡萄糖利用；D：突变体ΔxlnR 在木三糖、木二糖、木糖和葡萄糖条件下的生长表型；E：突变体ΔxlnR 在木糖、阿拉伯糖、葡萄糖固体平板上的表型

Fig. 1 Phenotypic analysis of M. thermophila strains WT and ΔxlnRA： Xylose utilization after the inoculation of the conidia from strains WT and ΔxlnR； B： Arabinose utilization after the inoculation of the conidia from strains WT and ΔxlnR； C： Glucose utilization after the inoculation of the conidia from strains WT and ΔxlnR； D： Growth phenotypes of ΔxlnR mutant on xylotriose， xylobiose， xylose and glucose； E： Growth phenotypes of ΔxlnR mutant on the agar plate with xylose， arabinose or glucose as the carbon source

图2 嗜热毁丝霉野生型菌株和突变体ΔxlnR菌丝的木糖利用

Fig. 2 Xylose utilization of the mycelia from M. thermophila strains WT and ΔxlnR

图3 嗜热毁丝霉突变体Δcre-1表型分析A：嗜热毁丝霉野生型菌株和突变体Δcre-1的木糖利用；B：嗜热毁丝霉野生型菌株和突变体Δcre-1的葡萄糖利用

Fig. 3 Phenotypic analysis of M. thermophila strain Δcre-1A： Xylose utilization of M. thermophila strains WT and Δcre-1； B： Glucose utilization of M. thermophila strains WT and Δcre-1

图4 嗜热毁丝霉野生型菌株和突变体Δcre-1、ΔxlnR在木糖诱导条件下的转录组分析A：嗜热毁丝霉野生型菌株和突变体Δcre-1、ΔxlnR在木糖诱导条件下的木糖代谢途径及磷酸戊糖途径相关基因表达量聚类分析；B：糖转运蛋白基因表达水平聚类分析；C：木聚糖酶基因及β-木糖苷酶基因表达水平聚类分析。xr—木糖还原酶基因；xdh—木酮糖还原酶基因；xks—木酮糖激酶基因；rki—核糖5-磷酸异构酶基因；rpe—磷酸核酮糖3-差向异构酶基因；tkl—转酮醇酶基因；tal—转醛酶基因

Fig. 4 Transcriptome analysis of M. thermophila strains WT， Δcre-1， and ΔxlnR mutation induced by xyloseA： Heatmap analysis of expression levels of genes involved in xylose catabolism and pentose phosphate pathway in strains WT， ΔxlnR， and Δcre-1 under xylose condition； B； Heatmap analysis of expression levels of putative sugar transporter genes； C： Heatmap analysis of expression levels of genes. xr—Xylose reductase gene； xdh—Xylitol dehydrogenase gene； xks—Xylulose kinase gene； rki—Ribose 5-phosphate isomerase gene； rpe—Ribulose phosphate 3-epimerase gene； tkl—Transketolase gene； tal—Transaldolase gene

图5 嗜热毁丝霉中Cre-1对转录激活因子XlnR的调控功能分析A：EMSA分析；B：在木糖条件下，基因xlnR 在野生型菌株和突变体Δcre-1 的表达水平；C：转录因子Cre-1和XlnR之间，以及二者对木糖代谢的调控关系

Fig. 5 Analysis of regulatory function of Cre-1 on transcriptional activator XlnR in M. thermophilaA： Analysis of EMSA； B： Expression level of xlnR in the wild-type strain and mutant Δcre-1 under xylose condition； C： Relationship between transcription factor Cre-1 and XlnR， and their regulation in xylose metabolism

图6 突变体OE_xlnR 和OE_xlnRΔcre-1 的木糖利用分析

Fig. 6 Xylose utilization of the mutants OE_xlnR and OE_xlnRΔcre-1

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