Dataset Information

Heterologous expression of glucose oxidase in the yeast Kluyveromyces marxianus.

ABSTRACT:

Background

In spite of its advantageous physiological properties for bioprocess applications, the use of the yeast Kluyveromyces marxianus as a host for heterologous protein production has been very limited, in constrast to its close relative Kluyveromyces lactis. In the present work, the model protein glucose oxidase (GOX) from Aspergillus niger was cloned into K. marxianus CBS 6556 and into K. lactis CBS 2359 using three different expression systems. We aimed at verifying how each expression system would affect protein expression, secretion/localization, post-translational modification, and biochemical properties.

Results

The highest GOX expression levels (1552 units of secreted protein per gram dry cell weight) were achieved using an episomal system, in which the INU1 promoter and terminator were used to drive heterologous gene expression, together with the INU1 prepro sequence, which was employed to drive secretion of the enzyme. In all cases, GOX was mainly secreted, remaining either in the periplasmic space or in the culture supernatant. Whereas the use of genetic elements from Saccharomyces cerevisiae to drive heterologous protein expression led to higher expression levels in K. lactis than in K. marxianus, the use of INU1 genetic elements clearly led to the opposite result. The biochemical characterization of GOX confirmed the correct expression of the protein and showed that K. marxianus has a tendency to hyperglycosylate the protein, in a similar way as already observed for other yeasts, although this tendency seems to be smaller than the one of e.g. K. lactis and S. cerevisiae. Hyperglycosylation of GOX does not seem to affect its affinity for the substrate, nor its activity.

Conclusions

Taken together, our results indicate that K. marxianus is indeed a good host for the expression of heterologous proteins, not only for its physiological properties, but also because it correctly secretes and folds these proteins.

SUBMITTER: Rocha SN

PROVIDER: S-EPMC2817671 | biostudies-literature | 2010 Jan

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Heterologous expression of glucose oxidase in the yeast Kluyveromyces marxianus.

Rocha Saul N SN Abrahão-Neto José J Cerdán María E ME González-Siso María I MI Gombert Andreas K AK

Microbial cell factories 20100121

<h4>Background</h4>In spite of its advantageous physiological properties for bioprocess applications, the use of the yeast Kluyveromyces marxianus as a host for heterologous protein production has been very limited, in constrast to its close relative Kluyveromyces lactis. In the present work, the model protein glucose oxidase (GOX) from Aspergillus niger was cloned into K. marxianus CBS 6556 and into K. lactis CBS 2359 using three different expression systems. We aimed at verifying how each expr ...[more]

PMID: 20092622

Similar Datasets

Project description:BACKGROUND: Kluyveromyces marxianus has recently become a species of interest for ethanol production since it can produce ethanol at high temperature and on a wide variety of substrates. However, the reason why this yeast can produce ethanol at high temperature is largely unknown. RESULTS: The ethanol fermentation capability of K. marxianus GX-UN120 at 40°? was found to be the same as that of Saccharomyces cerevisiae at 34°?. Zymogram analysis showed that alcohol dehydrogenase 1 (KmAdh1) was largely induced during ethanol production, KmAdh4 was constitutively expressed at a lower level and KmAdh2 and KmAdh3 were almost undetectable. The genes encoding the four alcohol dehydrogenases (ADHs) were cloned from strain GX-UN120. Each KmADH was expressed in Escherichia coli and each recombinant protein was digested with enterokinase to remove the fusion protein. The optimum pH of the purified recombinant KmAdh1 was 8.0 and that of KmAdh2, KmAdh3 and KmAdh4 was 7.0. The optimum temperatures of KmAdh1, KmAdh2, KmAdh3 and KmAdh4 were 50, 45, 55 and 45°C, respectively. The K(m) values of the recombinant KmAdh1 and KmAdh2 were 4.0 and 1.2 mM for acetaldehyde and 39.7 and 49.5 mM for ethanol, respectively. The V(max) values of the recombinant KmAdh1 and KmAdh2 were 114.9 and 21.6 ?mol min?¹ mg?¹ for acetaldehyde and 57.5 and 1.8 ?mol min?¹ mg?¹ for ethanol, respectively. KmAdh3 and KmAdh4 catalyze the oxidation reaction of ethanol to acetaldehyde but not the reduction reaction of acetaldehyde to ethanol, and the K(m) values of the recombinant KmAdh3 and KmAdh4 were 26.0 and 17.0 mM for ethanol, respectively. The V(max) values of the recombinant KmAdh3 and KmAdh4 were 12.8 and 56.2 ?mol min?¹ mg?¹ for ethanol, respectively. CONCLUSION: These data in this study collectively indicate that KmAdh1 is the primary ADH responsible for the production of ethanol from the reduction of acetaldehyde in K. marxianus. The relatively high optimum temperature of KmAdh1 may partially explain the ability of K. marxianus to produce ethanol at high temperature. Understanding the biochemical characteristics of KmAdhs will enhance our fundamental knowledge of the metabolism of ethanol fermentation in K. marxianus.

Project description: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.

Project description:BackgroundThe yeast Kluyveromyces marxianus features specific traits that render it attractive for industrial applications. These include production of ethanol which, together with thermotolerance and the ability to grow with a high specific growth rate on a wide range of substrates, could make it an alternative to Saccharomyces cerevisiae as an ethanol producer. However, its ability to co-ferment C5 and C6 sugars under oxygen-limited conditions is far from being fully characterized.ResultsIn the present study, K. marxianus CBS712 strain was cultivated in defined medium with glucose and xylose as carbon source. Ethanol fermentation and sugar consumption of CBS712 were investigated under different oxygen supplies (1.75%, 11.00% and 20.95% of O2) and different temperatures (30°C and 41°C). By decreasing oxygen supply, independently from the temperature, both biomass production as well as sugar utilization rate were progressively reduced. In all the tested conditions xylose consumption followed glucose exhaustion. Therefore, xylose metabolism was mainly affected by oxygen depletion. Loss in cell viability cannot explain the decrease in sugar consumption rates, as demonstrated by single cell analyses, while cofactor imbalance is commonly considered as the main cause of impairment of the xylose reductase (KmXR) - xylitol dehydrogenase (KmXDH) pathway. Remarkably, when these enzyme activities were assayed in vitro, a significant decrease was observed together with oxygen depletion, not ascribed to reduced transcription of the corresponding genes.ConclusionsIn the present study both oxygen supply and temperature were shown to be key parameters affecting the fermentation capability of sugars in the K. marxianus CBS712 strain. In particular, a direct correlation was observed between the decreased efficiency to consume xylose with the reduced specific activity of the two main enzymes (KmXR and KmXDH) involved in its catabolism. These data suggest that, in addition to the impairment of the oxidoreductive pathway being determined by the cofactor imbalance, post-transcriptional and/or post-translational regulation of the pathway enzymes contributes to the efficiency of xylose catabolism in micro-aerobic conditions. Overall, the presented work provides novel information on the fermentation capability of the CBS712 strain that is currently considered as the reference strain of the genus K. marxianus.

Dataset Information

Heterologous expression of glucose oxidase in the yeast Kluyveromyces marxianus.

Background

Results

Conclusions

Publications

Heterologous expression of glucose oxidase in the yeast Kluyveromyces marxianus.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets