Effects of glycerol supply and specific growth rate on methanol-free production of CALB by P-pastoris: functional characterisation of a novel promoter

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F60461373%3A22330%2F17%3A43914615" target="_blank" >RIV/60461373:22330/17:43914615 - isvavai.cz</a>

Výsledek na webu

<a href="https://link.springer.com/article/10.1007%2Fs00253-017-8123-x" target="_blank" >https://link.springer.com/article/10.1007%2Fs00253-017-8123-x</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1007/s00253-017-8123-x" target="_blank" >10.1007/s00253-017-8123-x</a>

Jazyk výsledku

angličtina

Název v původním jazyce

Effects of glycerol supply and specific growth rate on methanol-free production of CALB by P-pastoris: functional characterisation of a novel promoter

Popis výsledku v původním jazyce

As Pichia pastoris (syn. Komagataella sp.) yeast can secrete pure recombinant proteins at high rates, it is a desirable production system. The function of a novel synthetic variant of the AOX1 promoter was characterised comprehensively using a strain secreting Candida antarctica lipase B (CALB) as a model. A new time-saving approach was introduced to determine, in only one experiment, the hitherto unknown relationship between specific product formation rate (q (p)) and specific growth rate (mu). Tight control of recombinant protein formation was possible in the absence of methanol, while using glycerol as a sole carbon/energy source. CALB was not synthesised during batch cultivation in excess glycerol (&gt; 10 g l(-1)) and at a growth rate close to mu (max) (0.15 h(-1)). Between 0.017 and 0.115 h(-1) in glycerol-limited fedbatch cultures, basal levels of q (p) &gt; 0.4 mg g(-1) h(-1) CALB were reached, independent of the mu at which the culture grew. At mu &gt; 0.04 h(-1), an elevated q (p) occurred temporarily during the first 20 h after changing to fedbatch mode and decreased thereafter to basal. In order to accelerate the determination of the q (p)(mu) relationship (kinetics of product formation), the entire mu range was covered in a single fedbatch experiment. By linearly increasing and decreasing glycerol addition rates, mu values were repeatedly shifted from 0.004 to 0.074 h(-1) and vice versa. Changes in q (p) were related to changes in mu. A rough estimation of mu range suitable for production was possible in a single fedbatch, thus significantly reducing the experimental input over previous approaches comprising several experiments.

Název v anglickém jazyce

Effects of glycerol supply and specific growth rate on methanol-free production of CALB by P-pastoris: functional characterisation of a novel promoter

Popis výsledku anglicky

As Pichia pastoris (syn. Komagataella sp.) yeast can secrete pure recombinant proteins at high rates, it is a desirable production system. The function of a novel synthetic variant of the AOX1 promoter was characterised comprehensively using a strain secreting Candida antarctica lipase B (CALB) as a model. A new time-saving approach was introduced to determine, in only one experiment, the hitherto unknown relationship between specific product formation rate (q (p)) and specific growth rate (mu). Tight control of recombinant protein formation was possible in the absence of methanol, while using glycerol as a sole carbon/energy source. CALB was not synthesised during batch cultivation in excess glycerol (&gt; 10 g l(-1)) and at a growth rate close to mu (max) (0.15 h(-1)). Between 0.017 and 0.115 h(-1) in glycerol-limited fedbatch cultures, basal levels of q (p) &gt; 0.4 mg g(-1) h(-1) CALB were reached, independent of the mu at which the culture grew. At mu &gt; 0.04 h(-1), an elevated q (p) occurred temporarily during the first 20 h after changing to fedbatch mode and decreased thereafter to basal. In order to accelerate the determination of the q (p)(mu) relationship (kinetics of product formation), the entire mu range was covered in a single fedbatch experiment. By linearly increasing and decreasing glycerol addition rates, mu values were repeatedly shifted from 0.004 to 0.074 h(-1) and vice versa. Changes in q (p) were related to changes in mu. A rough estimation of mu range suitable for production was possible in a single fedbatch, thus significantly reducing the experimental input over previous approaches comprising several experiments.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

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OECD FORD obor

10606 - Microbiology

Projekt

—

Návaznosti

I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Rok uplatnění

2017

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

101

Číslo periodika v rámci svazku

8

Stát vydavatele periodika

US - Spojené státy americké

Počet stran výsledku

14

Strana od-do

3163-3176

Kód UT WoS článku

000399167800012

EID výsledku v databázi Scopus

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