Production and secretion dynamics of prokaryotic Penicillin G acylase in Pichia pastoris

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F61388971%3A_____%2F20%3A00534894" target="_blank" >RIV/61388971:_____/20:00534894 - isvavai.cz</a>

Nalezeny alternativní kódy

RIV/00216208:11310/20:10411686 RIV/60461373:22330/20:43920754

Výsledek na webu

<a href="https://link.springer.com/article/10.1007/s00253-020-10669-x" target="_blank" >https://link.springer.com/article/10.1007/s00253-020-10669-x</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1007/s00253-020-10669-x" target="_blank" >10.1007/s00253-020-10669-x</a>

Jazyk výsledku

angličtina

Název v původním jazyce

Production and secretion dynamics of prokaryotic Penicillin G acylase in Pichia pastoris

Popis výsledku v původním jazyce

To take full advantage of recombinant Pichia pastoris (Komagataella phaffii) as a production system for heterologous proteins, the complex protein secretory process should be understood and optimised by circumventing bottlenecks. Typically, little or no attention has been paid to the fate of newly synthesised protein inside the cell, or its passage through the secretory pathway, and only the secreted product is measured. However, the system's productivity (i.e. specific production rate q(p)), includes productivity of secreted (q(p,extra)) plus intracellularly accumulated (q(p,intra)) protein. In bioreactor cultivations with P. pastoris producing penicillin G acylase, we studied the dynamics of product formation, i.e. both the specific product secretion (q(p,extra)) and product retention (q(p,intra)) as functions of time, as well as the kinetics, i.e. productivity in relation to specific growth rate (mu). Within the time course, we distinguished (I) an initial phase with constant productivities, where the majority of product accumulated inside the cells, and q(p,extra), which depended on mu in a bell-shaped manner, (II) a transition phase, in which intracellular product accumulation reached a maximum and productivities (intracellular, extracellular, overall) were changing, (III) a new phase with constant productivities, where secretion prevailed over intracellular accumulation, q(p,extra) was linearly related to mu and was up to three times higher than in initial phase (I), while q(p,intra) decreased 4-6-fold. We show that stress caused by heterologous protein production induces cellular imbalance leading to a secretory bottleneck that ultimately reaches equilibrium. This understanding may help to develop cultivation strategies for improving protein secretion from P. pastoris.

Název v anglickém jazyce

Production and secretion dynamics of prokaryotic Penicillin G acylase in Pichia pastoris

Popis výsledku anglicky

To take full advantage of recombinant Pichia pastoris (Komagataella phaffii) as a production system for heterologous proteins, the complex protein secretory process should be understood and optimised by circumventing bottlenecks. Typically, little or no attention has been paid to the fate of newly synthesised protein inside the cell, or its passage through the secretory pathway, and only the secreted product is measured. However, the system's productivity (i.e. specific production rate q(p)), includes productivity of secreted (q(p,extra)) plus intracellularly accumulated (q(p,intra)) protein. In bioreactor cultivations with P. pastoris producing penicillin G acylase, we studied the dynamics of product formation, i.e. both the specific product secretion (q(p,extra)) and product retention (q(p,intra)) as functions of time, as well as the kinetics, i.e. productivity in relation to specific growth rate (mu). Within the time course, we distinguished (I) an initial phase with constant productivities, where the majority of product accumulated inside the cells, and q(p,extra), which depended on mu in a bell-shaped manner, (II) a transition phase, in which intracellular product accumulation reached a maximum and productivities (intracellular, extracellular, overall) were changing, (III) a new phase with constant productivities, where secretion prevailed over intracellular accumulation, q(p,extra) was linearly related to mu and was up to three times higher than in initial phase (I), while q(p,intra) decreased 4-6-fold. We show that stress caused by heterologous protein production induces cellular imbalance leading to a secretory bottleneck that ultimately reaches equilibrium. This understanding may help to develop cultivation strategies for improving protein secretion from P. pastoris.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

—

OECD FORD obor

10606 - Microbiology

Projekt

—

Návaznosti

I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Rok uplatnění

2020

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Název periodika

Applied Microbiology and Biotechnology

ISSN

0175-7598

e-ISSN

—

Svazek periodika

104

Číslo periodika v rámci svazku

13

Stát vydavatele periodika

DE - Spolková republika Německo

Počet stran výsledku

14

Strana od-do

5787-5800

Kód UT WoS článku

000533813200001

EID výsledku v databázi Scopus

2-s2.0-85084981824