Monitoring of transcriptome and proteome profiles to investigate the cellular response of E. coli towards recombinant protein expression under defined chemostat conditions

bIn this study, we focus on production of heterologous ␣-amylase in the yeast Saccharomyces cerevisiae under anaerobic conditions. We compare the metabolic fluxes and transcriptional regulation under aerobic and anaerobic conditions, with the objective of identifying the final electron acceptor for protein folding under anaerobic conditions. We find that yeast produces more amylase under anaerobic conditions than under aerobic conditions, and we propose a model for electron transfer under anaerobic conditions. According to our model, during protein folding the electrons from the endoplasmic reticulum are transferred to fumarate as the final electron acceptor. This model is supported by findings that the addition of fumarate under anaerobic (but not aerobic) conditions improves cell growth, specifically in the ␣-amylase-producing strain, in which it is not used as a carbon source. Our results provide a model for the molecular mechanism of anaerobic protein secretion using fumarate as the final electron acceptor, which may allow for further engineering of yeast for improved protein secretion under anaerobic growth conditions.

mentioning

confidence: 99%

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

Liu

Österlund

Hou

et al. 2013

“…The availabilities of foldases, chaperones, redox-equivalents, glycans, trafficking vesicles, etc., are tuned in response to the expression level of the recombinant protein and the load with respect to the secretory pathway. Layers of quality control corresponding to protein folding and secretion are involved and must be well coordinated in order to avoid cellular stresses that may cause reductions of cell growth and protein secretion (41,42) or even apoptosis and cell death (43). No mutations were found in stress-related genes in M715, suggesting that the stress response may be regulated by a mechanism different from that seen with the M1052 strain.…”

Section: ])mentioning

confidence: 99%

Improved Production of a Heterologous Amylase in Saccharomyces cerevisiae by Inverse Metabolic Engineering

Liu

Österlund

et al. 2014

cThe increasing demand for industrial enzymes and biopharmaceutical proteins relies on robust production hosts with high protein yield and productivity. Being one of the best-studied model organisms and capable of performing posttranslational modifications, the yeast Saccharomyces cerevisiae is widely used as a cell factory for recombinant protein production. However, many recombinant proteins are produced at only 1% (or less) of the theoretical capacity due to the complexity of the secretory pathway, which has not been fully exploited. In this study, we applied the concept of inverse metabolic engineering to identify novel targets for improving protein secretion. Screening that combined UV-random mutagenesis and selection for growth on starch was performed to find mutant strains producing heterologous amylase 5-fold above the level produced by the reference strain. Genomic mutations that could be associated with higher amylase secretion were identified through whole-genome sequencing. Several single-point mutations, including an S196I point mutation in the VTA1 gene coding for a protein involved in vacuolar sorting, were evaluated by introducing these to the starting strain. By applying this modification alone, the amylase secretion could be improved by 35%. As a complement to the identification of genomic variants, transcriptome analysis was also performed in order to understand on a global level the transcriptional changes associated with the improved amylase production caused by UV mutagenesis.

“…Nevertheless, the combined optimized features employed in the new high-performance expression plasmids facilitated the production of 82.5 mg per g CDW Gfp in B. megaterium shakingflask cultivations, which is even more than described for fedbatch cultivations of E. coli (73.7 mg per g CDW ) (10). Thus, a competitive protein production system for the alternative bacterial host B. megaterium was developed.…”

Section: B Megaterium Cells Employing Pssbm78 (ϫ35mentioning

confidence: 99%

“…For example, Biedendieck et al (5) produced 5.2 mg per g cell dry weight (g CDW ) in a fed-batch cultivation employing the xylose-inducible protein production system in B. megaterium. In comparison, Dürrschmid et al (10) produced 73.7 mg green fluorescent protein (Gfp) per g CDW by using E. coli with a T7 RNA polymerase-dependent gene expression system in a comparable cultivation approach. Consequently, in this work, a directed systematic optimization of the xylose-inducible protein production and export system was carried out in order to reach the protein production efficiency of E. coli.…”

mentioning

confidence: 99%

High-Yield Intra- and Extracellular Protein Production Using Bacillus megaterium

Stammen

Müller

Korneli

et al. 2010

The Bacillus megaterium protein production system based on the inducible promoter of the xyl operon (P xylA ) was systematically optimized. Multiple changes in basic promoter elements, such as the ؊10 and ؊35 region and the ribosome-binding site, resulted in an 18-fold increase of protein production compared to the production of the previously established system. The production in shaking-flask culture of green fluorescent protein (Gfp) as a model product led to 82.5 mg per g cell dry weight (g CDW ) or 124 mg liter ؊1 . In fed-batch cultivation, the volumetric protein yield was increased 10-fold to 1.25 g liter ؊1 , corresponding to 36.8 mg protein per g CDW . Furthermore, novel signal peptides for Sec-dependent protein secretion were predicted in silico using the B. megaterium genome. Subsequently, leader peptides of Vpr, NprM, YngK, YocH, and a computationally designed artificial peptide were analyzed experimentally for their potential to facilitate the secretion of the heterologous model protein Thermobifida fusca hydrolase (Tfh). The best extracellular protein production, 5,000 to 6,200 U liter ؊1 (5.3 to 6.6 mg liter ؊1 ), was observed for strains where the Tfh export was facilitated by a codonoptimized leader peptide of YngK and by the signal peptide of YocH. Further increases in extracellular protein production were achieved when leader peptides were used in combination with the optimized expression system. In this case, the greatest extracellular enzyme amount of 7,200 U liter ؊1 , 7.7 mg liter ؊1 , was achieved by YocH leader peptide-mediated protein export. Nevertheless, the observed principal limitations in protein export might be related to components of the Sec-dependent protein transport system.