Mechanistic platform knowledge of concomitant sugar uptake in Escherichia coli and analysis thereof

Eines der am häufigsten angewendeten Expressionssysteme bei rekombinanter Protein-produktion in Escherichia coli ist das T7-basierte pET-Expressionssystem. Kürzlich stellten wir im Rahmen einer Studie eine neue Induktionsstrategie basierend auf Laktose vor, in der sich zeigte, dass die Laktose-Aufnahme streng von der verfügbaren Menge an Glukose abhängig ist. Darauf basierend entwickelten wir ein mechanistisches Modell zur Bestimmung der Korrelation von gleichzeitiger Zucker-Aufnahme und zeigten, dass die Bestimmung dieser Korrelation die Möglichkeit bietet, Produktivität und Produktort über die Höhe der Laktose-Aufnahme zu steuern. In dieser Studie untermauern wir die Anwendung der entdeckten Korrelation für gleichzeitige Glukose- und Laktose-Aufnahme als Plattform-Wissen für BL21(DE3) Zellen mit pET-Plasmiden: Zum ersten Mal zeigen wir, dass die Korrelation für gleichzeitige Zuckeraufnahme den gleichen Trend aufweist, in E. coli BL21(DE3) Zellen, die verschiedene Produkte exprimieren. Die absolut Werte der Laktose-Aufnahme sind jedoch unterschiedlich. Außerdem entwickelten wir eine Strategie für schnelle und verlässliche Bestimmung der Korrelation. Diese Studie kann allen Wissenschaftlern, die mit rekombinanter Proteinproduktion in E. coli Stämmen arbeiten, behilflich sein, da einerseits mechanistisches Plattform-Wissen für E. coli Stämme mit pET-Plasmiden präsentiert wird und andererseits eine Strategie zur schnellen Bestimmung der mechanistischen Korrelation vorgestellt wird.When producing recombinant proteins Escherichia coli strain BL21(DE3) in combination with the T7-based pET-expression system is often the method of choice. Recently, we proposed a new induction strategy based on lactose, where the level of lactose uptake was shown to be strictly coupled to the abundance of glucose. Based thereon we developed a mechanistic model to determine the correlation of the concomitant sugar uptake and showed that establishment of the correlation can be used to gear productivity and product location through the level of lactose uptake. In this study we substantiated applicability of the established correlation for concomitant glucose and lactose uptake as platform knowledge for BL21(DE3) cells with pET-plasmids: we show for the first time that the correlation of concomitant sugar uptake exhibits the same trend in E. coli BL21(DE3) cells expressing different products, only absolute values diverge. Furthermore, we developed a strategy for fast and accurate determination of this correlation. This study will help all scientists working with recombinant E. coli strains, as not only mechanistic platform knowledge for E. coli strains carrying the pET expression system is revealed, but also a strategy for fast determination of the mechanistic correlation is offered.6

E. coli HMS174(DE3) is a sustainable alternative to BL21(DE3)

Abstract Background Escherichia coli is one of the most widely used hosts for recombinant protein production in academia and industry. Strain BL21(DE3) is frequently employed due to its advantageous feature of lacking proteases which avoids degradation of target protein. Usually it is used in combination with the T7-pET system where induction is performed by one point addition of IPTG. We recently published a few studies regarding lactose induction in BL21(DE3) strains. BL21(DE3) can only take up the glucose-part of the disaccharide when fed with lactose. However, initially additional glucose has to be supplied as otherwise the ATP-related lactose uptake barely happens. Yet, as lactose is an inexpensive compound compared to glucose and IPTG, a new induction strategy by a lactose-only feed during induction seems attractive. Thus, we investigated this idea in the galactose metabolizing strain HMS174(DE3). Results We show that strain HMS174(DE3) can be cultivated on lactose as sole carbon source during induction. We demonstrate that strain HMS174(DE3) exhibits higher product and biomass yields compared to BL21(DE3) when cultivated in a lactose fed-batch. More importantly, HMS174(DE3) cultivated on lactose even expresses more product than BL21(DE3) in a standard IPTG induced glucose fed-batch at the same growth rate. Finally, we demonstrate that productivity in HMS174(DE3) lactose-fed batch cultivations can easily be influenced by the specific lactose uptake rate (qs,lac). This is shown for two model proteins, one expressed in soluble form and one as inclusion body. Conclusions As strain HMS174(DE3) expresses even slightly higher amounts of target protein in a lactose fed-batch than BL21(DE3) in a standard cultivation, it seems a striking alternative for recombinant protein production. Especially for large scale production of industrial enzymes cheap substrates are essential. Besides cost factors, the strategy allows straight forward adjustment of specific product titers by variation of the lactose feed rate

Valorisation of cheese whey as substrate and inducer for recombinant protein production in E. coli HMS174(DE3)

Every year worldwide around 190 million tons of cheese whey are generated resulting in a huge environmental burden. We recently published a study where we showed that E. coli strain HMS174(DE3) can be cultivated using only lactose as C-source and inducer. Motivated by the results we investigated using a concentrated whey feed instead of the lactose feed. Spray drying whey and dissolving the powder allowed preparation of a 40-fold concentrated whey containing 91% lactose and 81% protein of the original whey. Cultivations using the concentrated whey feed instead of a defined lactose feed revealed 39% higher growth rates, 24% higher biomass yields and even higher specific product titers for the model enzymes, flavanone 3-hydroxylase and chalcone 3- hydroxylase. Our strategy simultaneously provides a cheap substrate for large-scale production of technical enzymes and an excellent opportunity for cheese whey valorization, reducing the biological burden resulting from whey wastewaters.Fonds zur Förderung der Wissenschaftlichen Forschung

Molecular and Enzymatic Characterization of Flavonoid 3′-Hydroxylase of Malus × domestica

Malus × domestica (apple) accumulates particularly high amounts of dihydrochalcones in various tissues, with phloridzin (phloretin 2′-O-glucoside) being prevalent, although small amounts of 3-hydroxyphloretin and 3-hydroxyphloridzin are also constitutively present. The latter was shown to correlate with increased disease resistance of transgenic M. × domestica plants. Two types of enzymes could be involved in 3-hydroxylation of dihydrochalcones: polyphenol oxidases or the flavonoid 3′-hydroxylase (F3′H), which catalyzes B-ring hydroxylation of flavonoids. We isolated two F3′H cDNA clones from apple leaves and tested recombinant Malus F3′Hs for their substrate specificity. From the two isolated cDNA clones, only F3′HII encoded a functionally active enzyme. In the F3′HI sequence, we identified two putatively relevant amino acids that were exchanged in comparison to that of a previously published F3′HI. Site directed mutagenesis, which exchanged an isoleucine into methionine in position 211 restored the functional activity, which is probably because it is located in an area involved in interaction with the substrate. In contrast to high activity with various flavonoid substrates, the recombinant enzymes did not accept phloretin under assay conditions, making an involvement in the dihydrochalcone biosynthesis unlikely