Dataset Information

Yeast-based production and in situ purification of acetaldehyde.

ABSTRACT: Acetaldehyde is a platform chemical with a production volume of more than 1 Mt/a, but is chiefly synthesized from petrochemical feedstocks. We propose the fermentative conversion of glucose towards acetaldehyde via genetically modified S. cerevisiae. This allows for ethanol-free bioactaldehyde production. Exploiting the high volatility of the product, in situ gas stripping in an aerated reactor is inevitable and crucial due to the respiratory toxicity effects of the acetaldehyde overproduction. We devise a lab-scale setup for the recovery of the product from the off-gas. Water was chosen as a suitable solvent and the Henry coefficient of acetaldehyde in water was validated experimentally. Based on an experimentally verified capture efficiency of 75%, an acetaldehyde production rate of over 100 mg/g/h was reached in 200 mL lab-scale fermentations.

SUBMITTER: Mengers HG

PROVIDER: S-EPMC8948146 | biostudies-literature | 2022 Apr

REPOSITORIES: biostudies-literature

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Yeast-based production and in situ purification of acetaldehyde.

Mengers Hendrik G HG Westarp William Graf von WGV Brücker Daniela D Jupke Andreas A Blank Lars M LM

Bioprocess and biosystems engineering 20220208 4

Acetaldehyde is a platform chemical with a production volume of more than 1 Mt/a, but is chiefly synthesized from petrochemical feedstocks. We propose the fermentative conversion of glucose towards acetaldehyde via genetically modified S. cerevisiae. This allows for ethanol-free bioactaldehyde production. Exploiting the high volatility of the product, in situ gas stripping in an aerated reactor is inevitable and crucial due to the respiratory toxicity effects of the acetaldehyde overproduction. ...[more]

PMID: 35137261

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Project description:The commonly used host for industrial production of recombinant proteins Pichia pastoris, has been used in this work to produce the rabies virus glycoprotein (RABV-G). To allow a constitutive expression and the secretion of the expressed recombinant RABV-G, the PichiaPink™ commercialized expression vectors were modified to contain the constitutive GAP promoter and the α secretion signal sequences. Recombinant PichiaPink™ strains co-expressing the RABV-G and the protein chaperone PDI, have been then generated and screened for the best producer clone. The influence of seven carbon sources on the expression of the RABV-G, has been studied under different culture conditions in shake flask culture. An incubation temperature of 30°C under an agitation rate of 250 rpm in a filling volume of 10:1 flask/culture volume ratio were the optimal conditions for the RABV-G production in shake flask for all screened carbon sources. A bioreactor Fed batch culture has been then carried using glycerol and glucose as they were good carbon sources for cell growth and RABV-G production in shake flask scale. Cells were grown on glycerol during the batch phase then fed with glycerol or glucose defined solutions, a final RABV-G concentration of 2.7 µg/l was obtained with a specific product yield (YP/X) of 0.032 and 0.06 µg/g(DCW) respectively. The use of semi-defined feeding solution enhanced the production and the YP/X to 12.9 µg/l and 0.135 µg/g(DCW) respectively. However, the high cell density favored by these carbon sources resulted in oxygen limitation which influenced the glycosylation pattern of the secreted RABV-G. Alternatively, the use of sucrose as substrate for RABV-G production in large scale culture, resulted in less biomass production and a YP/X of 0.310 µg/g(DCW) was obtained. A cation exchange chromatography was then used for RABV-G purification as one step method. The purified protein was correctly folded and glycosylated and able to adopt trimeric conformation. The knowledges gained through this work offer a valuable insight into the bioprocess design of RABV-G production in Pichia pastoris to obtain a correctly folded protein which can be used during an immunization proposal for subunit Rabies vaccine development.

Project description:MotivationX-ray crystallography-based protein structure determination, which accounts for majority of solved structures, is characterized by relatively low success rates. One solution is to build tools which support selection of targets that are more likely to crystallize. Several in silico methods that predict propensity of diffraction-quality crystallization from protein chains were developed. We show that the quality of their predictions drops when applied to more recent crystallization trails, which calls for new solutions. We propose a novel approach that alleviates drawbacks of the existing methods by using a recent dataset and improved protocol to annotate progress along the crystallization process, by predicting the success of the entire process and steps which result in the failed attempts, and by utilizing a compact and comprehensive set of sequence-derived inputs to generate accurate predictions.ResultsThe proposed PPCpred (predictor of protein Production, Purification and Crystallization) predict propensity for production of diffraction-quality crystals, production of crystals, purification and production of the protein material. PPCpred utilizes comprehensive set of inputs based on energy and hydrophobicity indices, composition of certain amino acid types, predicted disorder, secondary structure and solvent accessibility, and content of certain buried and exposed residues. Our method significantly outperforms alignment-based predictions and several modern crystallization propensity predictors. Receiver operating characteristic (ROC) curves show that PPCpred is particularly useful for users who desire high true positive (TP) rates, i.e. low rate of mispredictions for solvable chains. Our model reveals several intuitive factors that influence the success of individual steps and the entire crystallization process, including the content of Cys, buried His and Ser, hydrophobic/hydrophilic segments and the number of predicted disordered segments.Availabilityhttp://biomine.ece.ualberta.ca/PPCpred/.Contactlkurgan@ece.ualberta.ca.

Dataset Information

Yeast-based production and in situ purification of acetaldehyde.

Publications

Yeast-based production and in situ purification of acetaldehyde.

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