Systemic intracellular analysis for balancing complex biosynthesis in a transcriptionally deregulated Escherichia coli l-Methionine producer.

l-Methionine (l-Met) has gained remarkable interest due to its multifaceted and versatile applications in the fields of nutrition, pharmaceuticals and clinical practice. In this study, the fluxes of the challenging l-Met biosynthesis in the producer strain Escherichia coli (E. coli) DM2853 were fine-tuned to enable improved l-Met production. The potential bottlenecks identified in sulfur assimilation and l-Met synthesis downstream of O-succinyl-l-homoserine (OSHS) were addressed by overexpressing glutaredoxin 1 (grxA), thiosulfate sulfurtransferase (pspE) and O-succinylhomoserine lyase (metB). Although deemed as a straightforward target for improving glucose-to-Met conversion, the yields remained at approximately 12%-13% (g/g). Instead, intracellular l-Met pools increased by up to four-fold with accelerated kinetics. Overexpression of the Met exporter ygaZH may serve as a proper valve for releasing the rising internal Met pressure. Interestingly, the export kinetics revealed maximum saturated export rates already at low growth rates. This scenario is particularly advantageous for large-scale fermentation when product formation is ideally uncoupled from biomass formation to achieve maximum performance within the technical limits of large-scale bioreactors.

A Two-Compartment Fermentation System to Quantify Strain-Specific Interactions in Microbial Co-Cultures.

To fulfil the growing interest in investigating microbial interactions in co-cultures, a novel two-compartment bioreactor system was developed, characterised, and implemented. The system allowed for the exchange of amino acids and peptides via a polyethersulfone membrane that retained biomass. Further system characterisation revealed a Bodenstein number of 18, which hints at backmixing. Together with other physical settings, the existence of unwanted inner-compartment substrate gradients could be ruled out. Furthermore, the study of Damkoehler numbers indicated that a proper metabolite supply between compartments was enabled. Implementing the two-compartment system (2cs) for growing Streptococcus thermophilus and Lactobacillus delbrueckii subs. bulgaricus, which are microorganisms commonly used in yogurt starter cultures, revealed only a small variance between the one-compartment and two-compartment approaches. The 2cs enabled the quantification of the strain-specific production and consumption rates of amino acids in an interacting S. thermophilus-L. bulgaricus co-culture. Therefore, comparisons between mono- and co-culture performance could be achieved. Both species produce and release amino acids. Only alanine was produced de novo from glucose through potential transaminase activity by L. bulgaricus and consumed by S. thermophilus. Arginine availability in peptides was limited to S. thermophilus' growth, indicating active biosynthesis and dependency on the proteolytic activity of L. bulgaricus. The application of the 2cs not only opens the door for the quantification of exchange fluxes between microbes but also enables continuous production modes, for example, for targeted evolution studies.

Polyphosphate Kinases Phosphorylate Thiamine Phosphates.

Polyphosphate kinases (PPKs) catalyze the reversible transfer of the γ-phosphate moiety of ATP (or of another nucleoside triphosphate) to a growing chain of polyphosphate (polyP). In this study we describe that PPKs of various sources are additionally able to phosphorylate thiamine diphosphate (ThP2) to produce thiamine triphosphate (ThP3) and even thiamine tetraphosphate (ThP4) in vitro. Furthermore, all tested PPK2s, but not PPK1s, were able to phosphorylate thiamine monophosphate (ThP1) to ThP2 and ThP3 although at low efficiency. The predicted masses and identities of the mono- and oligo-phosphorylated thiamine metabolites were identified by high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). Moreover, the biological activity of ThP2, that was synthesized by phosphorylation of ThP1 with polyP and PPK, as a cofactor of ThP2-dependent enzymes (here transketolase TktA from Escherichia coli) was confirmed in a coupled enzyme assay. In conclusion, our study shows that PPKs are promiscuous enzymes that are presumably involved in the formation of a variety of phosphorylated metabolites in vivo.

Monitoring Intracellular Metabolite Dynamics in Saccharomyces cerevisiae during Industrially Relevant Famine Stimuli.

Carbon limitation is a common feeding strategy in bioprocesses to enable an efficient microbiological conversion of a substrate to a product. However, industrial settings inherently promote mixing insufficiencies, creating zones of famine conditions. Cells frequently traveling through such regions repeatedly experience substrate shortages and respond individually but often with a deteriorated production performance. A priori knowledge of the expected strain performance would enable targeted strain, process, and bioreactor engineering for minimizing performance loss. Today, computational fluid dynamics (CFD) coupled to data-driven kinetic models are a promising route for the in silico investigation of the impact of the dynamic environment in the large-scale bioreactor on microbial performance. However, profound wet-lab datasets are needed to cover relevant perturbations on realistic time scales. As a pioneering study, we quantified intracellular metabolome dynamics of Saccharomyces cerevisiae following an industrially relevant famine perturbation. Stimulus-response experiments were operated as chemostats with an intermittent feed and high-frequency sampling. Our results reveal that even mild glucose gradients in the range of 100 µmol·L-1 impose significant perturbations in adapted and non-adapted yeast cells, altering energy and redox homeostasis. Apparently, yeast sacrifices catabolic reduction charges for the sake of anabolic persistence under acute carbon starvation conditions. After repeated exposure to famine conditions, adapted cells show 2.7% increased maintenance demands.

Towards valorization of pectin-rich agro-industrial residues: Engineering of Saccharomyces cerevisiae for co-fermentation of d-galacturonic acid and glycerol.

Pectin-rich plant biomass residues represent underutilized feedstocks for industrial biotechnology. The conversion of the oxidized monomer d-galacturonic acid (d-GalUA) to highly reduced fermentation products such as alcohols is impossible due to the lack of electrons. The reduced compound glycerol has therefore been considered an optimal co-substrate, and a cell factory able to efficiently co-ferment these two carbon sources is in demand. Here, we inserted the fungal d-GalUA pathway in a strain of the yeast S. cerevisiae previously equipped with an NAD-dependent glycerol catabolic pathway. The constructed strain was able to consume d-GalUA with the highest reported maximum specific rate of 0.23 g gCDW-1 h-1 in synthetic minimal medium when glycerol was added. By means of a 13C isotope-labelling analysis, carbon from both substrates was shown to end up in pyruvate. The study delivers the proof of concept for a co-fermentation of the two 'respiratory' carbon sources to ethanol and demonstrates a fast and complete consumption of d-GalUA in crude sugar beet pulp hydrolysate under aerobic conditions. The future challenge will be to achieve co-fermentation under industrial, quasi-anaerobic conditions.

Compartment-specific 13C metabolic flux analysis reveals boosted NADPH availability coinciding with increased cell-specific productivity for IgG1 producing CHO cells after MTA treatment.

Increasing cell-specific productivities (CSPs) for the production of heterologous proteins in Chinese hamster ovary (CHO) cells is an omnipresent need in the biopharmaceutical industry. The novel additive 5'-deoxy-5'-(methylthio)adenosine (MTA), a chemical degradation product of S-(5'-adenosyl)-ʟ-methionine (SAM) and intermediate of polyamine biosynthesis, boosts the CSP of IgG1-producing CHO cells by 50%. Compartment-specific 13C flux analysis revealed a fundamental reprogramming of the central metabolism after MTA addition accompanied by cell-cycle arrest and increased cell volumes. Carbon fluxes into the pentose-phosphate pathway increased 22 fold in MTA-treated cells compared to that in non-MTA-treated reference cells. Most likely, cytosolic ATP inhibition of phosphofructokinase mediated the carbon detour. Mitochondrial shuttle activity of the α-ketoglurarate/malate antiporter (OGC) reversed, reducing cytosolic malate transport. In summary, NADPH supply in MTA-treated cells improved three fold compared to that in non-MTA-treated cells, which can be regarded as a major factor for explaining the boosted CSPs.

Compartment-specific metabolome labeling enables the identification of subcellular fluxes that may serve as promising metabolic engineering targets in CHO cells.

13C labeling data are used to calculate quantitative intracellular flux patterns reflecting in vivo conditions. Given that approaches for compartment-specific metabolomics exist, the benefits they offer compared to conventional non-compartmented 13C flux studies remain to be determined. Using compartment-specific labeling information of IgG1-producing Chinese hamster ovary cells, this study investigated differences of flux patterns exploiting and ignoring metabolic labeling data of cytosol and mitochondria. Although cellular analysis provided good estimates for the majority of intracellular fluxes, half of the mitochondrial transporters, and NADH and ATP balances, severe differences were found for some reactions. Accurate flux estimations of almost all iso-enzymes heavily depended on the sub-cellular labeling information. Furthermore, key discrepancies were found for the mitochondrial carriers vAGC1 (Aspartate/Glutamate antiporter), vDIC (Malate/H+ symporter), and vOGC (α-ketoglutarate/malate antiporter). Special emphasis is given to the flux of cytosolic malic enzyme (vME): it could not be estimated without the compartment-specific malate labeling information. Interesting enough, cytosolic malic enzyme is an important metabolic engineering target for improving cell-specific IgG1 productivity. Hence, compartment-specific 13C labeling analysis serves as prerequisite for related metabolic engineering studies.

Micro-aerobic production of isobutanol with engineered Pseudomonas putida.

Pseudomonas putida KT2440 is emerging as a promising microbial host for biotechnological industry due to its broad range of substrate affinity and resilience to physicochemical stresses. Its natural tolerance towards aromatics and solvents qualifies this versatile microbe as promising candidate to produce next generation biofuels such as isobutanol. In this study, we scaled-up the production of isobutanol with P. putida from shake flask to fed-batch cultivation in a 30 L bioreactor. The design of a two-stage bioprocess with separated growth and production resulted in 3.35 gisobutanol L-1. Flux analysis revealed that the NADPH expensive formation of isobutanol exceeded the cellular catabolic supply of NADPH finally causing growth retardation. Concomitantly, the cell counteracted to the redox imbalance by increased formation of 2-ketogluconic thereby providing electrons for the respiratory ATP generation. Thus, P. putida partially uncoupled ATP formation from the availability of NADH. The quantitative analysis of intracellular pyridine nucleotides NAD(P)+ and NAD(P)H revealed elevated catabolic and anabolic reducing power during aerobic production of isobutanol. Additionally, the installation of micro-aerobic conditions during production doubled the integral glucose-to-isobutanol conversion yield to 60 mgisobutanol gglucose -1 while preventing undesired carbon loss as 2-ketogluconic acid.

Identifying and Engineering Bottlenecks of Autotrophic Isobutanol Formation in Recombinant C. ljungdahlii by Systemic Analysis.

Clostridium ljungdahlii (C. ljungdahlii, CLJU) is natively endowed producing acetic acid, 2,3-butandiol, and ethanol consuming gas mixtures of CO2, CO, and H2 (syngas). Here, we present the syngas-based isobutanol formation using C. ljungdahlii harboring the recombinant amplification of the "Ehrlich" pathway that converts intracellular KIV to isobutanol. Autotrophic isobutanol production was studied analyzing two different strains in 3-L gassed and stirred bioreactors. Physiological characterization was thoroughly applied together with metabolic profiling and flux balance analysis. Thereof, KIV and pyruvate supply were identified as key "bottlenecking" precursors limiting preliminary isobutanol formation in CLJU[KAIA] to 0.02 g L-1. Additional blocking of valine synthesis in CLJU[KAIA]:ilvE increased isobutanol production by factor 6.5 finally reaching 0.13 g L-1. Future metabolic engineering should focus on debottlenecking NADPH availability, whereas NADH supply is already equilibrated in the current generation of strains.

Revisiting the Growth Modulon of Corynebacterium glutamicum Under Glucose Limited Chemostat Conditions.

Increasing the growth rate of the industrial host Corynebacterium glutamicum is a promising target to rise productivities of growth coupled product formation. As a prerequisite, detailed knowledge about the tight regulation network is necessary for identifying promising metabolic engineering goals. Here, we present comprehensive metabolic and transcriptional analysis of C. glutamicum ATCC 13032 growing under glucose limited chemostat conditions with µ = 0.2, 0.3, and 0.4 h-1. Intermediates of central metabolism mostly showed rising pool sizes with increasing growth. 13C-metabolic flux analysis (13C-MFA) underlined the fundamental role of central metabolism for the supply of precursors, redox, and energy equivalents. Global, growth-associated, concerted transcriptional patterns were not detected giving rise to the conclusion that glycolysis, pentose-phosphate pathway, and citric acid cycle are predominately metabolically controlled under glucose-limiting chemostat conditions. However, evidence is found that transcriptional regulation takes control over glycolysis once glucose-rich growth conditions are installed.

Streamlining the Analysis of Dynamic 13C-Labeling Patterns for the Metabolic Engineering of Corynebacterium glutamicum as l-Histidine Production Host.

Today's possibilities of genome editing easily create plentitudes of strain mutants that need to be experimentally qualified for configuring the next steps of strain engineering. The application of design-build-test-learn cycles requires the identification of distinct metabolic engineering targets as design inputs for subsequent optimization rounds. Here, we present the pool influx kinetics (PIK) approach that identifies promising metabolic engineering targets by pairwise comparison of up- and downstream 13C labeling dynamics with respect to a metabolite of interest. Showcasing the complex l-histidine production with engineered Corynebacterium glutamicuml-histidine-on-glucose yields could be improved to 8.6 ± 0.1 mol% by PIK analysis, starting from a base strain. Amplification of purA, purB, purH, and formyl recycling was identified as key targets only analyzing the signal transduction kinetics mirrored in the PIK values.

Characterization of Agrobacterium tumefaciens PPKs reveals the formation of oligophosphorylated products up to nucleoside nona-phosphates.

Agrobacterium tumefaciens synthesizes polyphosphate (polyP) in the form of one or two polyP granules per cell during growth. The A. tumefaciens genome codes for two polyphosphate kinase genes, ppk1AT and ppk2AT, of which only ppk1AT is essential for polyP granule formation in vivo. Biochemical characterization of the purified PPK1AT and PPK2AT proteins revealed a higher substrate specificity of PPK1AT (in particular for adenine nucleotides) than for PPK2AT. In contrast, PPK2AT accepted all nucleotides at comparable rates. Most interestingly, PPK2AT catalyzed also the formation of tetra-, penta-, hexa-, hepta-, and octa-phosphorylated nucleosides from guanine, cytosine, desoxy-thymidine, and uridine nucleotides and even nona-phosphorylated adenosine. Our data-in combination with in vivo results-suggest that PPK1AT is important for the formation of polyP whereas PPK2AT has the function to replenish nucleoside triphosphate pools during times of enhanced demand. The potential physiological function(s) of the detected oligophosphorylated nucleotides await clarification. KEY POINTS: •PPK1AT and PPK2AT have different substrate specificities, •PPK2AT is a subgroup 1 member of PPK2s, •PPK2AT catalyzes the formation of polyphosphorylated nucleosides.

Comparison of l-tyrosine containing dipeptides reveals maximum ATP availability for l-prolyl-l-tyrosine in CHO cells.

Increasing markets for biopharmaceuticals, including monoclonal antibodies, have triggered a permanent need for bioprocess optimization. Biochemical engineering approaches often include the optimization of basal and feed media to improve productivities of Chinese hamster ovary (CHO) cell cultures. Often, l-tyrosine is added as dipeptide to deal with its poor solubility at neutral pH. Showcasing IgG1 production with CHO cells, we investigated the supplementation of three l-tyrosine (TYR, Y) containing dipeptides: glycyl-l-tyrosine (GY), l-tyrosyl-l-valine (YV), and l-prolyl-l-tyrosine (PY). While GY and YV led to almost no phenotypic and metabolic differences compared to reference samples, PY significantly amplified TYR uptake thus maximizing related catabolic activity. Consequently, ATP formation was roughly four times higher upon PY application than in reference samples.

S-adenosylmethionine and methylthioadenosine boost cellular productivities of antibody forming Chinese hamster ovary cells.

The improvement of cell specific productivities for the formation of therapeutic proteins is an important step towards intensified production processes. Among others, the induction of the desired production phenotype via proper media additives is a feasible solution provided that said compounds adequately trigger metabolic and regulatory programs inside the cells. In this study, S-(5'-adenosyl)- l-methionine (SAM) and 5'-deoxy-5'-(methylthio)adenosine (MTA) were found to stimulate cell specific productivities up to approx. 50% while keeping viable cell densities transiently high and partially arresting the cell cycle in an anti-IL-8-producing CHO-DP12 cell line. Noteworthy, MTA turned out to be the chemical degradation product of the methyl group donor SAM and is consumed by the cells.

Electron availability in CO2 , CO and H2 mixtures constrains flux distribution, energy management and product formation in Clostridium ljungdahlii.

Acetogens such as Clostridium ljungdahlii can play a crucial role reducing the human CO2 footprint by converting industrial emissions containing CO2 , CO and H2 into valuable products such as organic acids or alcohols. The quantitative understanding of cellular metabolism is a prerequisite to exploit the bacterial endowments and to fine-tune the cells by applying metabolic engineering tools. Studying the three gas mixtures CO2  + H2 , CO and CO + CO2  + H2 (syngas) by continuously gassed batch cultivation experiments and applying flux balance analysis, we identified CO as the preferred carbon and electron source for growth and producing alcohols. However, the total yield of moles of carbon (mol-C) per electrons consumed was almost identical in all setups which underlines electron availability as the main factor influencing product formation. The Wood-Ljungdahl pathway (WLP) showed high flexibility by serving as the key NAD+ provider for CO2  + H2, whereas this function was strongly compensated by the transhydrogenase-like Nfn complex when CO was metabolized. Availability of reduced ferredoxin (Fdred ) can be considered as a key determinant of metabolic control. Oxidation of CO via carbon monoxide dehydrogenase (CODH) is the main route of Fdred formation when CO is used as substrate, whereas Fdred is mainly regenerated via the methyl branch of WLP and the Nfn complex utilizing CO2  + H2 . Consequently, doubled growth rates, highest ATP formation rates and highest amounts of reduced products (ethanol, 2,3-butanediol) were observed when CO was the sole carbon and electron source.

A universal polyphosphate kinase: PPK2c of Ralstonia eutropha accepts purine and pyrimidine nucleotides including uridine diphosphate.

Polyphosphosphate kinases (PPKs) catalyse the reversible transfer of the γ-phosphate group of a nucleoside-triphosphate to a growing chain of polyphosphate. Most known PPKs are specific for ATP, but some can also use GTP as a phosphate donor. In this study, we describe the properties of a PPK2-type PPK of the ß-proteobacterium Ralstonia eutropha. The purified enzyme (PPK2c) is highly unspecific and accepts purine nucleotides as well as the pyridine nucleotides including UTP as substrates. The presence of a polyP primer is not necessary for activity. The corresponding nucleoside diphosphates and microscopically detectable polyphosphate granules were identified as reaction products. PPK2c also catalyses the formation of ATP, GTP, CTP, dTTP and UTP from the corresponding nucleoside diphosphates, if polyP is present as a phosphate donor. Remarkably, the nucleoside-tetraphosphates AT(4)P, GT(4)P, CT(4)P, dTT(4)P and UT(4)P were also detected in substantial amounts. The low nucleotide specificity of PPK2c predestines this enzyme in combination with polyP to become a powerful tool for the regeneration of ATP and other nucleotides in biotechnological applications. As an example, PPK2c and polyP were used to replace ATP and to fuel the hexokinase-catalysed phosphorylation of glucose with only catalytic amounts of ADP. KEY POINTS: • PPK2c of R. eutropha can be used for regeneration of any NTP or dNTP. • PPK2c is highly unspecific and accepts all purine and pyrimidine nucleotides. • PPK2c forms polyphosphate granules in vitro from any NTP.

HILIC-Enabled 13C Metabolomics Strategies: Comparing Quantitative Precision and Spectral Accuracy of QTOF High- and QQQ Low-Resolution Mass Spectrometry.

Dynamic 13C-tracer-based flux analyses of in vivo reaction networks still require a continuous development of advanced quantification methods applying state-of-the-art mass spectrometry platforms. Utilizing alkaline HILIC chromatography, we adapt strategies for a systematic quantification study in non- and 13C-labeled multicomponent endogenous Corynebacterium glutamicum extracts by LC-QTOF high resolution (HRMS) and LC-QQQ tandem mass spectrometry (MS/MS). Without prior derivatization, a representative cross-section of 17 central carbon and anabolic key intermediates were analyzed with high selectivity and sensitivity under optimized ESI-MS settings. In column detection limits for the absolute quantification range were between 6.8-304.7 (QQQ) and 28.7-881.5 fmol (QTOF) with comparable linearities (3-5 orders of magnitude) and enhanced precision using QQQ-MRM detection. Tailor-made preparations of uniformly (U)13C-labeled cultivation extracts for isotope dilution mass spectrometry enabled the accurate quantification in complex sample matrices and extended linearities without effect on method parameters. Furthermore, evaluation of metabolite-specific m+1-to-m+0 ratios (ISR1:0) in non-labeled extracts exhibited sufficient methodical spectral accuracies with mean deviations of 3.89 ± 3.54% (QTOF) and 4.01 ± 3.01% (QQQ). Based on the excellent HILIC performance, conformity analysis of time-resolved isotopic enrichments in 13C-tracer experiments revealed sufficient spectral accuracy for QQQ-SIM detection. However, only QTOF-HRMS ensures determination of the full isotopologue space in complex matrices without mass interferences.

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.

From nutritional wealth to autophagy: In vivo metabolic dynamics in the cytosol, mitochondrion and shuttles of IgG producing CHO cells.

To fulfil the optimization needs of current biopharmaceutical processes the knowledge how to improve cell specific productivities is of outmost importance. This requires a detailed understanding of cellular metabolism on a subcellular level inside compartments such as cytosol and mitochondrion. Using IgG1 producing Chinese hamster ovary (CHO) cells, a pioneering protocol for compartment-specific metabolome analysis was applied. Various production-like growth conditions ranging from ample glucose and amino acid supply via moderate to severe nitrogen limitation were investigated in batch cultures. The combined application of quantitative metabolite pool analysis, 13C tracer studies and non-stationary flux calculations revealed that Pyr/H+ symport (MPC1/2) bore the bulk of the mitochondrial transport under ample nutrient supply. Glutamine limitation induced the concerted adaptation of the bidirectional Mal/aKG (OGC) and the Mal/HPO42- antiporter (DIC), even installing completely reversed shuttle fluxes. As a result, NADPH and ATP formation were adjusted to cellular needs unraveling the key role of cytosolic malic enzyme for NADPH production. Highest cell specific IgG1 productivities were closely correlated to a strong mitochondrial malate export according to the anabolic demands. The requirement to install proper NADPH supply for optimizing the production of monoclonal antibodies is clearly outlined. Interestingly, it was observed that mitochondrial citric acid cycle activity was always maintained enabling constant cytosolic adenylate energy charges at physiological levels, even under autophagy conditions.

Perfusion cultures require optimum respiratory ATP supply to maximize cell-specific and volumetric productivities.

Perfusion processes are an emerging alternative to common fed-batch processes in the growing biopharmaceutical industry. However, the challenge of maintaining high cell-specific productivities remains. In this study, glucose limitation was applied to two perfusion steady states and compared with a third steady state without any detectable limitation. The metabolic phenotype was enhanced under glucose limitation with a decrease of 30% in glucose uptake and 75% in lactate formation. Cell-specific productivities were substantially improved by 50%. Remarkably, the productivities showed a strong correlation to respiratory adenosine triphosphate (ATP) supply. As less reduced nicotinamide adenine dinucleotide (NADH) remained in the cytosol, the ATP generation from oxidative phosphorylation was increased by almost 30%. Consequently, the efficiency of carbon metabolism and the resulting respiratory ATP supply was crucial for maintaining the highly productive cellular state. This study highlights that glucose limitation can be used for process intensification in perfusion cultures as ATP generation via respiration is significantly increased, leading to elevated productivities.