Mutational Mtc6p attenuates autophagy and improves secretory expression of heterologous proteins in Kluyveromyces marxianus.
Mutational Mtc6p attenuates autophagy and improves secretory expression of heterologous proteins in Kluyveromyces marxianus.
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突变的 Mtc6p 会减弱马克斯克鲁维酵母的自噬并提高异源蛋白的分泌表达。
DOI：
10.1186/s12934-018-0993-9
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发表时间：
2018-09-14
影响因子：
6.4
通讯作者：
Lu H
中科院分区：
文献类型：
--
作者：
Liu Y;Mo WJ;Shi TF;Wang MZ;Zhou JG;Yu Y;Yew WS;Lu H
背景马氏克鲁维酵母是一种新兴的异源蛋白质生物合成细胞工厂，其应用具有多种用途的巨大优势。结果构建了以异源阿魏酸酯酶Est1E为报告基因的重组马氏克氏菌(FIM1/Est1E)。对该重组子进行UV-60Co-γ辐照诱变，筛选出一株突变体(命名为T1)，与亲本重组体相比，异源Est1E的产量提高了至少10倍。转录扰动分析表明，T1突变体的胞内囊泡转运增强，而自噬减弱。此外，全基因组测序结合CRISPR/Cas9介导的基因编辑发现了一个新的功能蛋白Mtc6p，该蛋白在T1突变体中通过删除其ORF 755位的一个胞嘧啶而在Tyr251处过早终止。我们发现，在FIM1中缺失MTC6的C755导致Est1E的产量比FIM1增加了4.86倍，而自噬水平下降了47%；相反，当在T1突变体中恢复MTC6的C755时，Est1E的产量比T1下降了66%，而自噬水平增加了124%。此外，在含有弱化自噬(即FIM1mtc6C755Δ和t1)或阻断自噬(即FIM1atg1Δ和t1atg1Δ)的重组体中，另外三种异源蛋白的产量也得到了提高，特别是异源甘露聚糖酶Man330、β-1，4-内切聚糖酶XynCDBFV和常规EGFP。结论Mtc6p参与了自噬的调节；减弱或阻断自噬可以显著提高马氏链霉菌中目标蛋白的产量，这种调节可以通过关注Mtc6p靶标的过早突变来实现。
BackgroundThe yeast Kluyveromyces marxianus is an emerging cell factory for heterologous protein biosynthesis and its use holds tremendous advantages for multiple applications. However, which genes influence the productivity of desired proteins in K. marxianus has so far been investigated by very few studies.ResultsIn this study, we constructed a K. marxianus recombinant (FIM1/Est1E), which expressed the heterologous ruminal feruloyl esterase Est1E as reporter. UV-60Co-γ irradiation mutagenesis was performed on this recombinant, and one mutant (be termed as T1) was screened and reported, in which the productivity of heterologous Est1E was increased by at least tenfold compared to the parental FIM1/Est1E recombinant. Transcriptional perturbance was profiled and presented that the intracellular vesicle trafficking was enhanced while autophagy be weakened in the T1 mutant. Moreover, whole-genome sequencing combined with CRISPR/Cas9 mediated gene-editing identified a novel functional protein Mtc6p, which was prematurely terminated at Tyr251 by deletion of a single cytosine at 755 loci of its ORF in the T1 mutant. We found that deleting C755 of MTC6 in FIM1 led to 4.86-fold increase in the production of Est1E compared to FIM1, while the autophagy level decreased by 47%; on the contrary, when reinstating C755 of MTC6 in the T1 mutant, the production of Est1E decreased by 66% compared to T1, while the autophagy level increased by 124%. Additionally, in the recombinant with attenuated autophagy (i.e., FIM1 mtc6C755Δ and T1) or interdicted autophagy (i.e., FIM1 atg1Δ and T1 atg1Δ), the productivity of three other heterologous proteins was also increased, specifically the heterologous mannase Man330, the β-1,4-endoxylanase XynCDBFV or the conventional EGFP.ConclusionsOur results demonstrated that Mtc6p was involved in regulating autophagy; attenuating or interdicting autophagy would dramatically improve the yields of desired proteins in K. marxianus, and this modulation could be achieved by focusing on the premature mutation of Mtc6p target.
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影响因子：
6.4
作者：
Aw R;Polizzi KM
通讯作者：
Polizzi KM
DOI：
10.1083/jcb.201802003
发表时间：
2018-07-02
期刊：
The Journal of cell biology
影响因子：
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作者：
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通讯作者：
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影响因子：
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作者：
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通讯作者：
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影响因子：
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作者：
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通讯作者：
Lu, Hong
影响因子：
6.4
作者：
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通讯作者：
Gombert AK