The effect of hypoxia on the lipidome of recombinant Pichia pastoris

Cultivation of recombinant Pichia pastoris (Komagataella sp.) under hypoxic conditions has a strong positive effect on specific productivity when the glycolytic GAP promoter is used for recombinant protein expression, mainly due to upregulation of glycolytic conditions. In addition, transcriptomic analyses of hypoxic P. pastoris pointed out important regulation of lipid metabolism and unfolded protein response (UPR). Notably, UPR that plays a role in the regulation of lipid metabolism, amino acid metabolism and protein secretion, was found to be upregulated under hypoxia. To improve our understanding of the interplay between lipid metabolism, UPR and protein secretion, the lipidome of a P. pastoris strain producing an antibody fragment was studied under hypoxic conditions. Furthermore, lipid composition analyses were combined with previously available transcriptomic datasets to further understand the impact of hypoxia on lipid metabolism. Chemostat cultures operated under glucose-limiting conditions under normoxic and hypoxic conditions were analyzed in terms of intra/extracellular product distribution and lipid composition. Integrated analysis of lipidome and transcriptome datasets allowed us to demonstrate an important remodeling of the lipid metabolism under limited oxygen availability. Additionally, cells with reduced amounts of ergosterol through fluconazole treatment were also included in the study to observe the impact on protein secretion and its lipid composition. Our results show that cells adjust their membrane composition in response to oxygen limitation mainly by changing their sterol and sphingolipid composition. Although fluconazole treatment results a different lipidome profile than hypoxia, both conditions result in higher recombinant protein secretion levels. The online version of this article (doi:10.1186/s12934-017-0699-4) contains supplementary material, which is available to authorized users

Systems metabolic engineering for recombinant protein production in Pichia pastoris

Departament responsable de la tesi: Departament d'Enginyeria Química, Biològica i Ambiental.El llevat metilotròfic Pichia pastoris (Komagataella sp.) és un dels sistemes d'expressió més atractius per a la producció de proteïna recombinant, mercat contínuament en expansió. El fort promotor del gen de l'alcohol oxidasa 1 (PAOX1), induït per metanol però reprimit per glucosa, glicerol o etanol, és un dels més emprats per aquest propòsit. No obstant, existeixen encara diversos colls d'ampolla fisiològics que limitant el procés.En aquest context, diferents estratègies han estat proposades i provades per tal de millorar la producció heteròloga de molts tipus diferents de proteïna. Les aproximacions més habituals inclouen increment en el nombre de còpies de gen heteròleg, enginyeria de promotors i modificació dels mecanismes de plegament i secreció. L'objectiu d'aquesta tesi ha estat el desenvolupament de noves estratègies per incrementar la producció de proteïna recombinant, emprant la lipasa de Rhizopus oryzae (Rol) com a proteïna model en el sistema d'expressió basat en el PAOX1.Primerament, els gens del factors de transcripció de PAOX1, MXR1 i MIT1, es van sobreexpressar constitutivament per tal de millorar la transcripció de ROL. Això es va confirmar degut a una millora en la capacitat assimilatòria de metanol i un increment en els nivells relatius de mRNA de ROL i varis gens relacionats amb el metabolisme del metanol, i.e. revertint l'efecte de titulació causat per la transcripció de múltiples cassettes d'expressió de ROL. Tot i aquestes millores, els nivells extracel·lulars d'activitat lipàsica no van augmentar de forma significativa en cultius en quimiòstat, apuntant a colls d'ampolla addicionals limitant la producció de Rol.En segon lloc, es van explorar possibles dianes d'enginyeria metabòlica en el metabolisme cel·lular de P. pastoris emprant el model metabòlic a escola genoma (GEM) consens iMT1026 v3.0. Aquest pas in silico va proporcionar diversos knock-outs prometedors que serien experimentalment testats fent servir el sistema d'edició genòmica CRISPR/Cas9. Les simulacions apuntaven a la disponibilitat de NADPH i una limitada aportació de determinats aminoàcids (serina i cisteïna) com a potencials factors limitants de la producció de Rol. Una reducció en el fitnes cel·lular que afecta a la viabilitat de les soques que es buscaven obtenir va impedir la verificació de la majoria dels knock-outs proposats.Finalment, donat que les nostres anàlisis i estudis prèviament publicats identificaven el NADPH com un cofactor important limitant la producció de proteïna recombinant, els nostres esforços es van dirigir a incrementar la seva disponibilitat a través d'estratègies de knock-in de gens. Específicament, vam sobreexpressar dos gens que codificaven per enzims redox, una NADH quinasa i una NADH oxidasa, amb l'objectiu de pertorbar directament l'equilibri redox de la cèl·lula. A més, es va comprovar l'efecte fisiològic d'aquests enzims fent servir diferents mescles co-substrat/metanol com a font de carboni. En resum, vam observar un increment en la producció de proteïna recombinant amb diferents graus de millora depenent de la font de carboni provada. També vam realitzar anàlisis transcriptòmiques i una avaluació in silico dels nostres resultats per tal de presentar una interpretació millor de l'estat fisiològic de la cèl·lula. Dins del nostre coneixement, aquest és el primer estudi dirigit a incrementar la generació de NADPH en un sistema d'expressió basat en PAOX1, en condicions de creixement en metanol.A grans trets, noves estratègies d'enginyeria de soques han estat proposades i provades durant l'execució d'aquest estudi. A més a més, s'han aplicat GEMs i aproximacions relacionades amb biologia de sistemes, demostrant que són eines potents i prometedores per al disseny racional d'organismes industrials.The methylotrophic yeast Pichia pastoris (Komagataella sp.) is one of the most attractive expression systems for heterologous protein production, which constitutes a continuously expanding market. The strong alcohol oxidase gene 1 promoter (PAOX1), induced by methanol but repressed by glucose, glycerol or ethanol, is one of the most used for this purpose. Nevertheless, there still exist several physiological bottlenecks limiting the process. In this context, several strategies have been proposed and tested in order to improve the heterologous production of many different types of proteins. Common approaches include increasing heterologous gene copy number, promoter engineering and modification of the folding and secretory mechanisms. The aim of this thesis has been the development of new strategies to increase recombinant protein yields, using the Rhizopus oryzae lipase (Rol) as model protein in a PAOX1-based expression system. Firstly, the PAOX1 transcription factor genes MXR1 and MIT1 were constitutively overexpressed aiming at improving ROL transcription. This was confirmed by an improved methanol assimilation capacity and an increase in relative mRNA levels of ROL and several genes related with methanol metabolism, i.e. reverting the titration effect caused by the transcription of multiple ROL expression cassettes. Despite such improvements, extracellular lipase activity levels did not increase significantly in chemostat cultures, pointing out to additional bottlenecks limiting Rol production. Second, possible metabolic engineering targets in P. pastoris' cell metabolism were explored using the consensus genome-scale metabolic model (GEM) iMT1026 v3.0. This in silico step provided several promising knock-outs which were going to be experimentally tested using the CRISPR/Cas9 genome editing system. The simulations pointed to NADPH availability and limited supply of some amino acids (serine and cysteine) as potential Rol production limiting factors. A reduction in cell fitness affecting the viability of the obtained strains impeded to verify most of the proposed knock-outs. Finally, since our in silico analyses and previously published studies identified NADPH as an important limiting cofactor in recombinant protein production, our efforts were geared towards increasing its availability through gene knock-in strategies. Specifically, we overexpressed two genes encoding redox enzymes, a NADH kinase and a NADH oxidase, with the aim to directly perturb the cell's redox balance. Further, we tested the physiological effect of these enzymes using different co-substrate/methanol mixtures as carbon source. In short, we observed an increase in recombinant protein production with different degrees of improvement depending on the carbon source(s) tested. We also performed a transcriptomic analysis and an in silico evaluation of our results in order to provide a better interpretation of the cell physiological state. To our knowledge, this is the first study aiming to increase NADPH generation in the PAOX1-based expression system, under methanol growth conditions. Overall, novel strain engineering strategies have been proposed and tested during the execution of this study. Furthermore, GEMs and related systems biology approaches were applied, proving to be promising powerful tools for rational engineering of industrial microorganisms

Deregulation of methanol metabolism reverts transcriptional limitations of recombinant Pichia pastoris (Komagataella spp) with multiple expression cassettes under control of the AOX1 promoter

The methanol-regulated alcohol oxidase promoter (P AOX1 ) of Pichia pastoris (syn. Komagataella spp.) is one of the strongest promoters for heterologous gene expression. Although increasing the gene dosage is a common strategy to improve recombinant protein productivities, P. pastoris strains harboring more than two copies of a Rhizopus oryzae lipase gene (ROL) have previously shown a decrease in cell growth, lipase production, and substrate consumption, as well as a significant transcriptional downregulation of methanol metabolism. This pointed to a potential titration effect of key transcriptional factors methanol expression regulator 1 (Mxr1) and methanol-induced transcription factor (Mit1) regulating methanol metabolism caused by the insertion of multiple expression vectors. To prove this hypothesis, a set of strains carrying one and four copies of ROL (1C and 4C, respectively) were engineered to coexpress one or two copies of MXR1*, coding for an Mxr1 variant insensitive to repression by 14-3-3 regulatory proteins, or one copy of MIT1. Small-scale cultures revealed that growth, Rol productivity, and methanol consumption were improved in the 4C-MXR1* and 4C-MIT1, strains growing on methanol as a sole carbon source, whereas only a slight increase in productivity was observed for re-engineered 1C strains. We further verified the improved performance of these strains in glycerol-/methanol-limited chemostat cultures

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Dataset from Redox engineering by ectopic overexpression of NADH kinase in recombinant Pichia pastoris (Komagataella phaffii) Study

Dataset corresponding to the NAD kinase redox engineering study in P.pastoris referenced below. Includes cultivation experimental data, droplet digital PCR data and computer simulation results in different files in Microsoft Excel 2013 Format

Eliminación del nitrógeno amoniacal en aguas residuales sanitarias

Premi "Principado de Asturias" atorgat per la Fundación Técnica Industrial del Consejo General de Colegios de Ingenieros Técnicos Industriales de EspañaLa presencia de NH4+ de origen orgánico en las corrientes de aguas residuales industriales es un problema que en la actualidad afecta a un número creciente de industrias. En este trabajo se ha comprobado el efecto del borboteo de aire atmosférico en el seno de un medio acuoso que simula la composición de las aguas residuales sanitarias, que son el problema. El objetivo de este tratamiento es aumentar la capacidad oxidante del medio para inhibir la reacción de descomposición de la urea, que es la que genera el NH4+ en el agua residual. El control y el seguimiento de los experimentos se ha realizado con un electrodo selectivo de amonio. El tratamiento ha tenido éxito en cuanto a la eliminación de amono, tal como se muestra en los apartados de resultados y conclusiones.Peer ReviewedAward-winningPostprint (published version

Eliminación del nitrógeno amoniacal en aguas residuales sanitarias

Premi "Principado de Asturias" atorgat per la Fundación Técnica Industrial del Consejo General de Colegios de Ingenieros Técnicos Industriales de EspañaLa presencia de NH4+ de origen orgánico en las corrientes de aguas residuales industriales es un problema que en la actualidad afecta a un número creciente de industrias. En este trabajo se ha comprobado el efecto del borboteo de aire atmosférico en el seno de un medio acuoso que simula la composición de las aguas residuales sanitarias, que son el problema. El objetivo de este tratamiento es aumentar la capacidad oxidante del medio para inhibir la reacción de descomposición de la urea, que es la que genera el NH4+ en el agua residual. El control y el seguimiento de los experimentos se ha realizado con un electrodo selectivo de amonio. El tratamiento ha tenido éxito en cuanto a la eliminación de amono, tal como se muestra en los apartados de resultados y conclusiones.Peer ReviewedAward-winnin

Eliminación del nitrógeno amoniacal en aguas residuales sanitarias

Premi "Principado de Asturias" atorgat per la Fundación Técnica Industrial del Consejo General de Colegios de Ingenieros Técnicos Industriales de EspañaLa presencia de NH4+ de origen orgánico en las corrientes de aguas residuales industriales es un problema que en la actualidad afecta a un número creciente de industrias. En este trabajo se ha comprobado el efecto del borboteo de aire atmosférico en el seno de un medio acuoso que simula la composición de las aguas residuales sanitarias, que son el problema. El objetivo de este tratamiento es aumentar la capacidad oxidante del medio para inhibir la reacción de descomposición de la urea, que es la que genera el NH4+ en el agua residual. El control y el seguimiento de los experimentos se ha realizado con un electrodo selectivo de amonio. El tratamiento ha tenido éxito en cuanto a la eliminación de amono, tal como se muestra en los apartados de resultados y conclusiones.Peer ReviewedAward-winnin

The effect of hypoxia on the lipidome of recombinant Pichia pastoris

Cultivation of recombinant Pichia pastoris (Komagataella sp.) under hypoxic conditions has a strong positive effect on specific productivity when the glycolytic GAP promoter is used for recombinant protein expression, mainly due to upregulation of glycolytic conditions. In addition, transcriptomic analyses of hypoxic P. pastoris pointed out important regulation of lipid metabolism and unfolded protein response (UPR). Notably, UPR that plays a role in the regulation of lipid metabolism, amino acid metabolism and protein secretion, was found to be upregulated under hypoxia. To improve our understanding of the interplay between lipid metabolism, UPR and protein secretion, the lipidome of a P. pastoris strain producing an antibody fragment was studied under hypoxic conditions. Furthermore, lipid composition analyses were combined with previously available transcriptomic datasets to further understand the impact of hypoxia on lipid metabolism. Chemostat cultures operated under glucose-limiting conditions under normoxic and hypoxic conditions were analyzed in terms of intra/extracellular product distribution and lipid composition. Integrated analysis of lipidome and transcriptome datasets allowed us to demonstrate an important remodeling of the lipid metabolism under limited oxygen availability. Additionally, cells with reduced amounts of ergosterol through fluconazole treatment were also included in the study to observe the impact on protein secretion and its lipid composition. Our results show that cells adjust their membrane composition in response to oxygen limitation mainly by changing their sterol and sphingolipid composition. Although fluconazole treatment results a different lipidome profile than hypoxia, both conditions result in higher recombinant protein secretion levels. The online version of this article (doi:10.1186/s12934-017-0699-4) contains supplementary material, which is available to authorized users