Mutational Mtc6p attenuates autophagy and improves secretory expression of heterologous proteins in Kluyveromyces marxianus.
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ABSTRACT: BACKGROUND:The 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. RESULTS:In this study, we constructed a K. marxianus recombinant (FIM1/Est1E), which expressed the heterologous ruminal feruloyl esterase Est1E as reporter. UV-60Co-? 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 mtc6C755? and T1) or interdicted autophagy (i.e., FIM1 atg1? and T1 atg1?), the productivity of three other heterologous proteins was also increased, specifically the heterologous mannase Man330, the ?-1,4-endoxylanase XynCDBFV or the conventional EGFP. CONCLUSIONS:Our 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.
SUBMITTER: Liu Y
PROVIDER: S-EPMC6138896 | biostudies-literature | 2018 Sep
REPOSITORIES: biostudies-literature
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