Anaerobic α-Amylase Production and Secretion with Fumarate as the Final Electron Acceptor in Saccharomyces cerevisiae

Liu, Zihe; Österlund, Tobias; Hou, Jin; Petranović, Dina; Nielsen, Jens

doi:10.1128/aem.03207-12

Cited by 25 publications

(11 citation statements)

References 44 publications

(42 reference statements)

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“…Figure 4 clearly shows the role of the stress in the improvement of product formation. The increase in the product formation under anaerobic condition was reported by Liu et al [23]. They found out higher amylase production in S. cerevisiae under anaerobic conditions than aerobic condition.…”

Section: Discussionsupporting

confidence: 58%

Two‐compartment processing as a tool to boost recombinant protein production

Jazini

Herwig

2013

Engineering in Life Sciences

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Pichia pastoris is used extensively as a production platform for many recombinant proteins. The dissolved oxygen (DO) is one of the most important factors influencing protein production. The influence of the DO on productivity has not been studied independent from the feed rate. In this work, various DO levels were investigated independent from the feed rate. The model system was recombinant P. pastoris under the control of methanol‐induced alcohol oxidase promoter, which expressed HRP as the target protein. No significant effect was observed in terms of titer and specific productivity, which is a confirmation of the fact that the DO in a one‐compartment system cannot boost productivity for the model system under study. Hence, a two‐compartment system (a single reactor coupled with a plug flow reactor) was designed and implemented in order to apply oxygen‐related stress in the plug flow reactor and allow the cells to be recovered in the main reactor. Doing so, more than two‐fold increase in the titer and productivity and three‐fold increase in protein‐specific activity were achieved. Hence, partial application of oxygen‐related stress in the two‐compartment system was proposed as a process technology to enhance protein production.

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Section: Discussionsupporting

confidence: 58%

Two‐compartment processing as a tool to boost recombinant protein production

Jazini

Herwig

2013

Engineering in Life Sciences

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“…Ero1 is a flavoprotein and its reoxidation, following disulfide bond catalysis, can be driven by oxidized FAD (22, 23). Furthermore, studies of oxidative folding under anaerobic conditions suggest that fumarate is the terminal electron acceptor and Osm1 generates free oxidized FAD via the reduction of fumarate (24). Consistent with this model, we found that the ER-form of Osm1 (Met32Ala) but not the mitochondrial-form (Met1Ala) supported growth under anaerobic conditions (Fig.…”

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confidence: 99%

Targeting and plasticity of mitochondrial proteins revealed by proximity-specific ribosome profiling

Williams¹,

Jan²,

Weissman³

2014

Science

348

383

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Nearly all mitochondrial proteins are nuclear-encoded and are targeted to their mitochondrial destination from the cytosol. Here, we used proximity-specific ribosome profiling to comprehensively measure translation at the mitochondrial surface in yeast. Most inner membrane proteins were co-translationally targeted to mitochondria, reminiscent of proteins entering the endoplasmic reticulum (ER). Comparison between mitochondrial and ER localization demonstrated that the vast majority of proteins were targeted to a specific organelle. A prominent exception was the fumarate reductase Osm1, known to reside in mitochondria. We identified a conserved ER isoform of Osm1, which contributes to the oxidative protein folding capacity of the organelle. This dual localization was enabled by alternative translation initiation sites encoding distinct targeting signals. These findings highlight the exquisite in vivo specificity of organellar targeting mechanisms.

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“…In our case, this may be related to the improved cell performance showed by the hap1 expressing the recombinant hemoglobin compared with the hap1 control. It has recently been demonstrated that anaerobic metabolism has a positive effect enhancing protein production by reducing cellular stress levels caused by the aerobic respiration as shown with other recombinant proteins, such as α‐amylase (Liu et al, ). This might be also the case for the reported K. lactis protein production, where HAP1 deletion improved the production and secretion of recombinant inulinase (Yu et al, ), although unfortunately there is no physiological data to confirm this hypothesis.…”

Section: Resultsmentioning

confidence: 98%

Engineering the oxygen sensing regulation results in an enhanced recombinant human hemoglobin production by Saccharomyces cerevisiae

Martínez

Liu

Petranović

et al. 2014

Biotech & Bioengineering

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Efficient production of appropriate oxygen carriers for transfusions (blood substitutes or artificial blood) has been pursued for many decades, and to date several strategies have been used, from synthetic polymers to cell-free hemoglobin carriers. The recent advances in the field of metabolic engineering also allowed the generation of different genetically modified organisms for the production of recombinant human hemoglobin. Several studies have showed very promising results using the bacterium Escherichia coli as a production platform, reporting hemoglobin titers above 5% of the total cell protein content. However, there are still certain limitations regarding the protein stability and functionality of the recombinant hemoglobin produced in bacterial systems. In order to overcome these limitations, yeast systems have been proposed as the eukaryal alternative. We recently reported the generation of a set of plasmids to produce functional human hemoglobin in Saccharomyces cerevisiae, with final titers of active hemoglobin exceeding 4% of the total cell protein. In this study, we propose a strategy for further engineering S. cerevisiae by altering the oxygen sensing pathway by deleting the transcription factor HAP1, which resulted in an increase of the final recombinant active hemoglobin titer exceeding 7% of the total cellular protein.

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Anaerobic α-Amylase Production and Secretion with Fumarate as the Final Electron Acceptor in Saccharomyces cerevisiae

Cited by 25 publications

References 44 publications

Two‐compartment processing as a tool to boost recombinant protein production

Two‐compartment processing as a tool to boost recombinant protein production

Targeting and plasticity of mitochondrial proteins revealed by proximity-specific ribosome profiling

Engineering the oxygen sensing regulation results in an enhanced recombinant human hemoglobin production by Saccharomyces cerevisiae

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