Lars M. Blank

Microbial hyaluronic acid production

AbstractsHyaluronic acid (HA) is a commercially valuable medical biopolymer increasingly produced through microbial fermentation. Viscosity limits product yield and the focus of research and development has been on improving the key quality parameters, purity and molecular weight. Traditional strain and process optimisation has yielded significant improvements, but appears to have reached a limit. Metabolic engineering is providing new opportunities and HA produced in a heterologous host is about to enter the market. In order to realise the full potential of metabolic engineering, however, greater understanding of the mechanisms underlying chain termination is required.

Chemical and biological single cell analysis

Single cells represent the minimal functional unit of life. A major goal of biology is to understand the mechanisms operating in this minimal unit. Nowadays, analysis of the single cell can be performed at unprecedented resolution using new lab-on-a-chip devices and advanced analytical methods. While cell handling and cultivation devices can be classified into finite volume reactors and flow systems, the analytical approaches differ in respect to invasive (i.e. chemical) and noninvasive (i.e. biological/living cell) analysis. Using these new and exciting technologies cell-to-cell differences, originating from regulatory circuits and distinct microenvironments, can now be explored. For example, it could be shown that the rates of transcription and translation are stochastic. Chemical and biological single cell analyses provide an unprecedented access to the understanding of cell-to-cell differences and basic biological concepts.

Metabolic response of Pseudomonas putida during redox biocatalysis in the presence of a second octanol phase

A key limitation of whole‐cell redox biocatalysis for the production of valuable, specifically functionalized products is substrate/product toxicity, which can potentially be overcome by using solvent‐tolerant micro‐organisms. To investigate the inter‐relationship of solvent tolerance and energy‐dependent biocatalysis, we established a model system for biocatalysis in the presence of toxic low logPow solvents: recombinant solvent‐tolerant Pseudomonas putida DOT‐T1E catalyzing the stereospecific epoxidation of styrene in an aqueous/octanol two‐liquid phase reaction medium. Using 13C tracer based metabolic flux analysis, we investigated the central carbon and energy metabolism and quantified the NAD(P)H regeneration rate in the presence of toxic solvents and during redox biocatalysis, which both drastically increased the energy demands of solvent‐tolerant P. putida. According to the driven by demand concept, the NAD(P)H regeneration rate was increased up to eightfold by two mechanisms: (a) an increase in glucose uptake rate without secretion of metabolic side products, and (b) reduced biomass formation. However, in the presence of octanol, only ∼ 1% of the maximally observed NAD(P)H regeneration rate could be exploited for styrene epoxidation, of which the rate was more than threefold lower compared with operation with a non‐toxic solvent. This points to a high energy and redox cofactor demand for cell maintenance, which limits redox biocatalysis in the presence of octanol. An estimated upper bound for the NAD(P)H regeneration rate available for biocatalysis suggests that cofactor availability does not limit redox biocatalysis under optimized conditions, for example, in the absence of toxic solvent, and illustrates the high metabolic capacity of solvent‐tolerant P. putida. This study shows that solvent‐tolerant P. putida have the remarkable ability to compensate for high energy demands by boosting their energy metabolism to levels up to an order of magnitude higher than those observed during unlimited growth.

Metabolic capacity estimation of Escherichia coli as a platform for redox biocatalysis: constraint‐based modeling and experimental verification

Whole‐cell redox biocatalysis relies on redox cofactor regeneration by the microbial host. Here, we applied flux balance analysis based on the Escherichia coli metabolic network to estimate maximal NADH regeneration rates. With this optimization criterion, simulations showed exclusive use of the pentose phosphate pathway at high rates of glucose catabolism, a flux distribution usually not found in wild‐type cells. In silico, genetic perturbations indicated a strong dependency of NADH yield and formation rate on the underlying metabolic network structure. The linear dependency of measured epoxidation activities of recombinant central carbon metabolism mutants on glucose uptake rates and the linear correlation between measured activities and simulated NADH regeneration rates imply intracellular NADH shortage. Quantitative comparison of computationally predicted NADH regeneration and experimental epoxidation rates indicated that the achievable biocatalytic activity is determined by metabolic and enzymatic limitations including non‐optimal flux distributions, high maintenance energy demands, energy spilling, byproduct formation, and uncoupling. The results are discussed in the context of cellular optimization of biotransformation processes and may guide a priori design of microbial cells as redox biocatalysts. Biotechnol. Bioeng. 2008;100: 1050–1065.

Quantitative Physiology of Pichia pastoris During Glucose-Limited High-Cell Density Fed-Batch Cultivation for Recombinant Protein Production

Pichia pastoris has become one of the major microorganisms for the production of proteins in recent years. This development was mainly driven by the readily available genetic tools and the ease of high‐cell density cultivations using methanol (or methanol/glycerol mixtures) as inducer and carbon source. To overcome the observed limitations of methanol use such as high heat development, cell lysis, and explosion hazard, we here revisited the possibility to produce proteins with P. pastoris using glucose as sole carbon source. Using a recombinant P. pastoris strain in glucose limited fed‐batch cultivations, very high‐cell densities were reached (more than 200 gCDW L−1) resulting in a recombinant protein titer of about 6.5 g L−1. To investigate the impact of recombinant protein production and high‐cell density fermentation on the metabolism of P. pastoris, we used 13C‐tracer‐based metabolic flux analysis in batch and fed‐batch experiments. At a controlled growth rate of 0.12 h−1 in fed‐batch experiments an increased TCA cycle flux of 1.1 mmol g−1 h−1 compared to 0.7 mmol g−1 h−1 for the recombinant and reference strains, respectively, suggest a limited but significant flux rerouting of carbon and energy resources. This change in flux is most likely causal to protein synthesis. In summary, the results highlight the potential of glucose as carbon and energy source, enabling high biomass concentrations and protein titers. The insights into the operation of metabolism during recombinant protein production might guide strain design and fermentation development. Biotechnol. Bioeng. 2010;107: 357–368.

Correlation between TCA cycle flux and glucose uptake rate during respiro-fermentative growth of Saccharomyces cerevisiae

Glucose repression of the tricarboxylic acid (TCA) cycle in Saccharomyces cerevisiae was investigated under different environmental conditions using (13)C-tracer experiments. Real-time quantification of the volatile metabolites ethanol and CO(2) allowed accurate carbon balancing. In all experiments with the wild-type, a strong correlation between the rates of growth and glucose uptake was observed, indicating a constant yield of biomass. In contrast, glycerol and acetate production rates were less dependent on the rate of glucose uptake, but were affected by environmental conditions. The glycerol production rate was highest during growth in high-osmolarity medium (2.9 mmol g(-1) h(-1)), while the highest acetate production rate of 2.1 mmol g(-1) h(-1) was observed in alkaline medium of pH 6.9. Under standard growth conditions (25 g glucose l(-1) , pH 5.0, 30 degrees C) S. cerevisiae had low fluxes through the pentose phosphate pathway and the TCA cycle. A significant increase in TCA cycle activity from 0.03 mmol g(-1) h(-1) to about 1.7 mmol g(-1) h(-1) was observed when S. cerevisiae grew more slowly as a result of environmental perturbations, including unfavourable pH values and sodium chloride stress. Compared to experiments with high glucose uptake rates, the ratio of CO(2) to ethanol increased more than 50 %, indicating an increase in flux through the TCA cycle. Although glycolysis and the ethanol production pathway still exhibited the highest fluxes, the net flux through the TCA cycle increased significantly with decreasing glucose uptake rates. Results from experiments with single gene deletion mutants partially impaired in glucose repression (hxk2, grr1) indicated that the rate of glucose uptake correlates with this increase in TCA cycle flux. These findings are discussed in the context of regulation of glucose repression.

Carbon Metabolism Limits Recombinant Protein Production in Pichia pastoris

The yeast Pichia pastoris enables efficient (high titer) recombinant protein production. As the molecular tools required are well established and gene specific optimizations of transcription and translation are becoming available, metabolism moves into focus as possible limiting factor of recombinant protein production in P. pastoris. To investigate the impact of recombinant protein production on metabolism systematically, we constructed strains that produced the model protein β‐aminopeptidase BapA of Sphingosinicella xenopeptidilytica at different production yields. The impact of low to high BapA production on cell physiology was quantified. The data suggest that P. pastoris compensates for the additional resources required for recombinant protein synthesis by reducing by‐product formation and by increasing energy generation via the TCA cycle. Notably, the activity of the TCA cycle was constant with a rate of 2.1 ± 0.1 mmol g  CDW−1  h−1 irrespective of significantly reduced growth rates in high BapA producing strains, suggesting an upper limit of TCA cycle activity. The reduced growth rate could partially be restored by providing all 20 proteinogenic amino acids in the fermentation medium. Under these conditions, the rate of BapA synthesis increased twofold. The successful supplementation of the growth medium by amino acids to unburden cellular metabolism during recombinant protein production suggests that the metabolic network is a valid target for future optimization of protein production by P. pastoris. Biotechnol. Bioeng. 2011; 108:1942–1953.

The polyhydroxyalkanoate metabolism controls carbon and energy spillage in Pseudomonas putida

The synthesis and degradation of polyhydroxyalkanoates (PHAs), the storage polymer of many bacteria, is linked to the operation of central carbon metabolism. To rationalize the impact of PHA accumulation on central carbon metabolism of the prototype bacterium Pseudomonas putida, we have revisited PHA production in quantitative physiology experiments in the wild-type strain vs. a PHA negative mutant growing under low nitrogen conditions. When octanoic acid was used as PHA precursor and as carbon and energy source, we have detected higher intracellular flux via acetyl-CoA in the mutant strain than in the wild type, which correlates with the stimulation of the TCA cycle and glyoxylate shunt observed on the transcriptional level. The mutant defective in carbon and energy storage spills the additional resources, releasing CO(2) instead of generating biomass. Hence, P. putida operates the metabolic network to optimally exploit available resources and channels excess carbon and energy to storage via PHA, without compromising growth. These findings demonstrate that the PHA metabolism plays a critical role in synchronizing global metabolism to availability of resources in PHA-producing microorganisms.

Response of Pseudomonas putida KT2440 to Increased NADH and ATP Demand

ABSTRACT Adenosine phosphate and NAD cofactors play a vital role in the operation of cell metabolism, and their levels and ratios are carefully regulated in tight ranges. Perturbations of the consumption of these metabolites might have a great impact on cell metabolism and physiology. Here, we investigated the impact of increased ATP hydrolysis and NADH oxidation rates on the metabolism of Pseudomonas putida KT2440 by titration of 2,4-dinitrophenol (DNP) and overproduction of a water-forming NADH oxidase, respectively. Both perturbations resulted in a reduction of the biomass yield and, as a consequence of the uncoupling of catabolic and anabolic activities, in an amplification of the net NADH regeneration rate. However, a stimulation of the specific carbon uptake rate was observed only when P. putida was challenged with very high 2,4-dinitrophenol concentrations and was comparatively unaffected by recombinant NADH oxidase activity. This behavior contrasts with the comparably sensitive performance described, for example, for Escherichia coli or Saccharomyces cerevisiae. The apparent robustness of P. putida metabolism indicates that it possesses a certain buffering capacity and a high flexibility to adapt to and counteract different stresses without showing a distinct phenotype. These findings are important, e.g., for the development of whole-cell redox biocatalytic processes that impose equivalent burdens on the cell metabolism: stoichiometric consumption of (reduced) redox cofactors and increased energy expenditures, due to the toxicity of the biocatalytic compounds.

Quantification of metabolic limitations during recombinant protein production in Escherichia coli.

Escherichia coli is one of the major microorganisms for recombinant protein production because it has been best characterized in terms of molecular genetics and physiology, and because of the availability of various expression vectors and strains. The synthesis of proteins is one of the most energy consuming processes in the cell, with the result that cellular energy supply may become critical. Indeed, the so called metabolic burden of recombinant protein synthesis was reported to cause alterations in the operation of the hosts central carbon metabolism. To quantify these alterations in E. coli metabolism in dependence of the rate of recombinant protein production, (13)C-tracer-based metabolic flux analysis in differently induced cultures was used. To avoid dilution of the (13)C-tracer signal by the culture history, the recombinant protein produced was used as a flux probe, i.e., as a read out of intracellular flux distributions. In detail, an increase in the generation rate rising from 36 mmol(ATP)g(CDW)(-1)h(-1) for the reference strain to 45 mmol(ATP)g(CDW)(-1)h(-1) for the highest yielding strain was observed during batch cultivation. Notably, the flux through the TCA cycle was rather constant at 2.5±0.1 mmol g(CDW)(-1)h(-1), hence was independent of the induced strength for gene expression. E. coli compensated for the additional energy demand of recombinant protein synthesis by reducing the biomass formation to almost 60%, resulting in excess NADPH. Speculative, this excess NADPH was converted to NADH via the soluble transhydrogenase and subsequently used for ATP generation in the electron transport chain. In this study, the metabolic burden was quantified by the biomass yield on ATP, which constantly decreased from 11.7g(CDW)mmol(ATP)(-1) for the reference strain to 4.9g(CDW)mmol(ATP)(-1) for the highest yielding strain. The insights into the operation of the metabolism of E. coli during recombinant protein production might guide the optimization of microbial hosts and fermentation conditions.