Scaling up E. coli from the lab to industrial conditions: Lessons learned to engineer robust processes and production hosts

For commercialization, strain and bioprocess developments need to be successfully transferred from the lab to industrial scale. Often, this step crucially decides about economic feasibility and survival of the approach. Accordingly, profound understanding of impact factors that hamper the successful scale-up is key, either to create novel microbial production platforms with enhanced robustness or to improve bioreactor design targeting minimized impact on cellular performance. Using an experimental scale-up simulator consisting of a stirred tank reactor (STR) and a plug flow reactor (PFR) Escherichia coli was exposed to typical large-scale mixing conditions in continuous experiments. Installing mixing times of about 110 seconds and simulating fluctuating availability of carbon and nitrogen sources, short-term responses revealed the repeated on/off switching of about 600 genes (Löffler et al.,Metab Eng 2016; Simen et al. Microbial Biotechnol 2017). Dynamics of gene expression and protein formation were modelled using an agent-based approach and simulating large-scale conditions (Nieß et al. Frontiers Microbiol., 2017). ATP balancing of gene expression and protein formation showed that maintenance demands increased by ~50%. Thereof, strategies for genome reduction were deduced. Large-scale simulation revealed the dominating role of the alarmone ppGpp which triggers the on/off-switching of the stringent response. Accordingly, a novel chassis was engineered such that intracellular ppGpp levels were no more affected thereby disconnecting the extracellular stimulus from the intracellular response, even under nitrogen or carbon limitation. Experimental studies outline the energetic advantages of stringent response deficient production hosts. Additionally, changes were implemented in central metabolism finally yielding E. coli HGT (high glucose throughput, Michalowski et al., Metab Eng 2017). The patent-filed strain offers about 10 fold risen glucose uptake rates (relative to maintenance demands under glucose limitation) under resting conditions which is beneficial for large-scale production processes

Compartment-specific metabolome analysis reveals the tight link between IgG1 formation and necessarily high mitochondrial shuttle activities in Chinese Hamster Ovary cells

Chinese hamster ovary (CHO) cells are the dominating host for the production of pharmaceutical proteins, in particular monoclonal antibodies (mABs). Although production titers improved more than 100 fold during the last 2 decades, similar enhancements of cell specific productivities are less pronounced. They demand for detailed subcellular studies to identify promising metabolic engineering targets. In this context, our study focused on compartment specific metabolome analysis to measure metabolic patterns in the cytosol and in the mitochondrion during cell cultivation. Thereof, in vivo shuttle activities were calculated and correlated with cell specific IgG1 formation rates. The compartment-specific metabolome and labelling analysis (13C) distinguishes between cytosol and mitochondrion. Metabolomics and instationary 13C metabolic flux analysis build on preliminary own studies of 13C analytics (Teleki et al., Anal Biochem 2015; Teleki et al. Metab Eng 2017) and compartment-specific metabolomics (Matuszczyk et al., Biotechnol J 2015; Pfitzenmaier et al., Biotechnol J 2016). Further development and optimization has been performed finally reaching the current status that allows monitoring compartment-specific flux distributions and shuttle activities during the course of cell cultivation. Studying multiple periods of an IgG1 production process the crucial role of the mitochondrion not only as a provider of ATP but also as an essential part of metabolism was unraveled. 13C flux analysis disclosed the time-variant activities of the mitochondrial shuttles that are tightly linked to mitochondrial and cytosolic metabolism. Clear evidence was found that mAB production strongly depends on sufficient NADPH supply provided by cytosolic malic enzyme activity and malate export from the mitochondrio

Tracking dipeptides at work-uptake and intracellular fate in CHO culture

Artículo científicoMarket demands for monoclonal antibodies (mAbs) are steadily increasing worldwide. As a result, production processes using Chinese hamster ovary cells (CHO) are in the focus of ongoing intensification studies for maximizing cell-specific and volumetric productivities. This includes the optimization of animal-derived component free (ADCF) cultivation media as part of good cell culture practice. Dipeptides are known to improve CHO culture performance. However, little or even conflicting assumptions exist about their putative import and functionality inside the cells. A set of well-known performance boosters and new dipeptide prospects was evaluated. The present study revealed that dipeptides are indeed imported in the cells, where they are decomposed to the amino acids building blocks. Subsequently, they are metabolized or, unexpectedly, secreted to the medium. Monoclonal antibody production boosting additives like l-alanine-l-glutamine (AQ) or glycyl-l-glutamine (GQ) can be assigned to fast or slow dipeptide uptake, respectively, thus pinpointing to the need to study dipeptide kinetics and to adjust their feeding individually for optimizing mAb production.Instituto Tecnológico de Costa Rica. Escuela de Químic

The Less the Better: How Suppressed Base Addition Boosts Production of Monoclonal Antibodies With Chinese Hamster Ovary Cells

Biopharmaceutical production processes strive for the optimization of economic efficiency. Among others, the maximization of volumetric productivity is a key criterion. Typical parameters such as partial pressure of CO2 (pCO2) and pH are known to influence the performance although reasons are not yet fully elucidated. In this study the effects of pCO2 and pH shifts on the phenotypic performance were linked to metabolic and energetic changes. Short peak performance of qmAb (23 pg/cell/day) was achieved by early pCO2 shifts up to 200 mbar but followed by declining intracellular ATP levels to 2.5 fmol/cell and 80% increase of qLac. On the contrary, steadily rising qmAb could be installed by slight pH down-shifts ensuring constant cell specific ATP production (qATP) of 27 pmol/cell/day and high intracellular ATP levels of about 4 fmol/cell. As a result, maximum productivity was achieved combining highest qmAb (20 pg/cell/day) with maximum cell density and no lactate formation. Our results indicate that the energy availability in form of intracellular ATP is crucial for maintaining antibody synthesis and reacts sensitive to pCO2 and pH-process parameters typically responsible for inhomogeneities after scaling up

Pseudomonas putida KT2440 is naturally endowed to withstand industrial-scale stress conditions

Pseudomonas putida is recognized as a very promising strain for industrial application due to its high redox capacity and frequently observed tolerance towards organic solvents. In this research, we studied the metabolic and transcriptional response of P. putida KT2440 exposed to large-scale heterogeneous mixing conditions in the form of repeated glucose shortage. Cellular responses were mimicked in an experimental setup comprising a stirred tank reactor and a connected plug flow reactor. We deciphered that a stringent response-like transcriptional regulation programme is frequently induced, which seems to be linked to the intracellular pool of 3-hydroxyalkanoates (3-HA) that are known to serve as precursors for polyhydroxyalkanoates (PHA). To be precise, P. putida is endowed with a survival strategy likely to access cellular PHA, amino acids and glycogen in few seconds under glucose starvation to obtain ATP from respiration, thereby replenishing the reduced ATP levels and the adenylate energy charge. Notably, cells only need 0.4% of glucose uptake to build those 3-HA-based energy buffers. Concomitantly, genes that are related to amino acid catabolism and β-oxidation are upregulated during the transient absence of glucose. Furthermore, we provide a detailed list of transcriptional short- and long-term responses that increase the cellular maintenance by about 17% under the industrial-like conditions tested.publishersversionpublishe

Continuous Adaptive Evolution of a Fast-Growing Corynebacterium glutamicum Strain Independent of Protocatechuate

Graf M, Haas T, Mueller F, et al. Continuous Adaptive Evolution of a Fast-Growing Corynebacterium glutamicum Strain Independent of Protocatechuate. Frontiers in Microbiology . 2019;10: 1648.Corynebacterium glutamicum is a commonly applied host for the industrial production of amino acids. While valued for its robustness, it is somewhat inferior to competing strains such as Escherichia coli because of the relatively low growth rate of 0.40 h(-1) in synthetic, industrial media. Accordingly, adaptive laboratory evolution (ALE) experiments were performed in continuous cultivation mode to select for a growth-improved host. To ensure industrial attractiveness, this ALE study aimed at a reduction of dependency on costly growth-boosting additives such as protocatechuate (RCA) or complex media supplements. Consequently, double selection pressures were installed consisting of a steady increase in growth rate demands and a parallel reduction of complex medium fractions. Selection yielded C. glutamicum EVO5 achieving 0.54 h(-1) and 1.03g(Glc) g(CDW)(-1) h(-1) in minimal medium without above mentioned supplements. Sequencing revealed 10 prominent mutations, three of them in key regulator genes

Identifying the Growth Modulon of Corynebacterium glutamicum

The growth rate (μ) of industrially relevant microbes, such as Corynebacterium glutamicum, is a fundamental property that indicates its production capacity. Therefore, understanding the mechanism underlying the growth rate is imperative for improving productivity and performance through metabolic engineering. Despite recent progress in the understanding of global regulatory interactions, knowledge of mechanisms directing cell growth remains fragmented and incomplete. The current study investigated RNA-Seq data of three growth rate transitions, induced by different pre-culture conditions, in order to identify transcriptomic changes corresponding to increasing growth rates. These transitions took place in minimal medium and ranged from 0.02 to 0.4 h-1 μ. This study enabled the identification of 447 genes as components of the growth modulon. Enrichment of genes within the growth modulon revealed 10 regulons exhibiting a significant effect over growth rate transition. In summary, central metabolism was observed to be regulated by a combination of metabolic and transcriptional activities orchestrating control over glycolysis, pentose phosphate pathway, and the tricarboxylic acid cycle. Additionally, major responses to changes in the growth rate were linked to iron uptake and carbon metabolism. In particular, genes encoding glycolytic enzymes and the glucose uptake system showed a positive correlation with the growth rate