Combining metabolic engineering and multiplexed screening methods for 3-Hydroxypropionic acid production in Pichia pastoris
Fina Romero, Albert (Universitat Autònoma de Barcelona. Departament d'Enginyeria Química, Biològica i Ambiental)
Heux, Stephanie (Université de Toulouse. Toulouse Biotechnology Institute)
Albiol i Sala, Joan (Universitat Autònoma de Barcelona. Departament d'Enginyeria Química, Biològica i Ambiental)
Ferrer, Pau (Universitat Autònoma de Barcelona. Departament d'Enginyeria Química, Biològica i Ambiental)

Data:	2022
Resum:	Production of 3-hydroxypropionic acid (3-HP) in Pichia pastoris (syn. Komagataella phaffii) via the malonyl-CoA pathway has been recently demonstrated using glycerol as a carbon source, but the reported metrics were not commercially relevant. The flux through the heterologous pathway from malonyl-CoA to 3-HP was hypothesized as the main bottleneck. In the present study, different metabolic engineering approaches have been combined to improve the productivity of the original 3-HP producing strains. To do so, an additional copy of the gene encoding for the potential rate-limiting step of the pathway, i. e. , the C-terminal domain of the malonyl-CoA reductase, was introduced. In addition, a variant of the endogenous acetyl-CoA carboxylase (ACC1S1132A) was overexpressed with the aim to increase the delivery of malonyl-CoA. Furthermore, the genes encoding for the pyruvate decarboxylase, aldehyde dehydrogenase and acetyl-CoA synthase, respectively, were overexpressed to enhance conversion of pyruvate into cytosolic acetyl-CoA, and the main gene responsible for the production of the by-product D-arabitol was deleted. Three different screening conditions were used to classify the performance of the different strains: 24-deep-well plates batch cultures, small-scale cultures in falcon tubes using FeedBeads® (i. e. , slow release of glycerol over time), and mini bioreactor batch cultures. The best two strains from the FeedBeads® screening, PpHP8 and PpHP18, were tested in bioreactor fed-batch cultures using a pre-fixed exponentially increasing feeding rate. The strain PpHP18 produced up to 37. 05 g L−1 of 3-HP at 0. 712 g L−1 h−1 with a final product yield on glycerol of 0. 194 Cmol−1 in fed-batch cultures. Remarkably, PpHP18 did not rank among the 2-top producer strains in small scale batch cultivations in deep-well plates and mini bioreactors, highlighting the importance of multiplexed screening conditions for adequate assessment of metabolic engineering strategies. These results represent a 50% increase in the product yield and final concentration, as well as over 30% increase in volumetric productivity compared to the previously obtained metrics for P. pastoris. Overall, the combination of glycerol as carbon source and a metabolically engineered P. pastoris strain resulted in the highest 3-HP concentration and productivity reported so far in yeast.
Ajuts:	Agencia Estatal de Investigación PID2019-104666GB-I00 Agència de Gestió d'Ajuts Universitaris i de Recerca 2017/SGR-1462 Ministerio de Ciencia e Innovación FPU17/05434
Nota:	Altres ajuts: Short-Term Fellowship (8853) from the European Molecular Biology Organization (EMBO)
Drets:	Aquest document està subjecte a una llicència d'ús Creative Commons. Es permet la reproducció total o parcial, la distribució, la comunicació pública de l'obra i la creació d'obres derivades, fins i tot amb finalitats comercials, sempre i quan es reconegui l'autoria de l'obra original.
Llengua:	Anglès
Document:	Article ; recerca ; Versió publicada
Matèria:	3-hydroxypropionic acid ; Pichia pastoris ; Glycerol ; Malonyl-CoA ; Acetyl-CoA ; Metabolic engineering
Publicat a:	Frontiers in Bioengineering and Biotechnology, Vol. 10 (July 2022) , art. 942304, ISSN 2296-4185

Dades de recerca relacionades amb l'article: https://ddd.uab.cat/record/267200
DOI: 10.3389/fbioe.2022.942304
PMID: 35935509

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Supplementary Material 5 p, 107.8 KB

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