Mutational Mtc6p attenuates autophagy and improves secretory expression of heterologous proteins in Kluyveromyces marxianus

Abstract

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.Electronic supplementary materialThe online version of this article (10.1186/s12934-018-0993-9) contains supplementary material, which is available to authorized users.

Characterization and modulation of endoplasmic reticulum stress response target genes in Kluyveromyces marxianus to improve secretory expressions of heterologous proteins

“…Genes encoding RuCelA and AnFaeA were integrated into the INU1 loci of a T1 strain to obtain LHP1021 and LHP643, respectively. The T1 strain was derived from FIM-1ΔU that improved the yield of heterologous proteins by attenuating autophagy [ 41 ]. In LHP1021 and LHP643, RuCelA and AnFaeA were expressed by a strong INU1 promoter and their secretions were directed by an alpha factor signal peptide from S. cerevisiae .…”

“…FIM-1ΔU strain was used as a wild-type strain for RNAseq [ 48 ]. T1 strain was used for expressions of lignocellulolytic enzymes [ 41 ].…”

“…Three CRISPR vectors, LHZ296, LHZ301 and LHZ531, were constructed and used as backbones to build CRISPR plasmids (Additional files 8 , 9 , 10 ). pUKD-N122-AUC contains two Sap I sites for the insertion of a target sequence in front of gRNA [ 41 ]. The ARS7 fragment in pUKD-N122-AUC was replaced by a pKD1 fragment [ 59 ], to obtain LHZ296.…”

A protocol of rapid laboratory evolution by genome shuffling in Kluyveromyces marxianus

“…In order to improve the efficiency of genome recombination, parent strains have different genetic backgrounds.The feruloyl esterase Est1E is used as a marker protein to measure the capacity of KM to express a heterologous protein. The expression level of Est1E is detected by a color-based assay [3] . The HML or HMR locus in the original parental strain is removed to produce stable haploid strain.…”

“…Both parental strains contain a deletion of URA3 to be compatible for a URA3 plasmid expressing the heterologous protein. The deletion is performed by the recombination with the aid of a CRISPR plasmid as described before [3] .…”