Itaconate Production from Crude Substrates with U. maydis: Scale-up of an Industrially Relevant Bioprocess.

BACKGROUND: Industrial by-products accrue in most agricultural or food-related production processes, but additional value chains have already been established for many of them. Crude glycerol has a 60% lower market value than commercial glucose, as large quantities are produced in the biodiesel industry, but its valorisation is still underutilized. Due to its high carbon content and the natural ability of many microorganisms to metabolise it, microbial upcycling is a suitable option for this waste product. RESULTS: In this work, the use of crude glycerol for the production of the value-added compound itaconate is demonstrated using the smut fungus Ustilago maydis. Starting with a highly engineered strain, itaconate production from an industrial glycerol waste stream was quickly established on a small scale, and the resulting yields were already competitive with processes using commercial sugars. Adaptive laboratory evolution resulted in an evolved strain with a 72% increased growth rate on glycerol. In the subsequent development and optimisation of a fed-batch process on a 1.5-2 L scale, the use of molasses, a side stream of sugar beet processing, eliminated the need for other expensive media components such as nitrogen or vitamins for biomass growth. The optimised process was scaled up to 150 L, achieving an overall titre of 72 g L- 1, a yield of 0.34 g g- 1, and a productivity of 0.54 g L- 1 h- 1. CONCLUSIONS: Pilot-scale itaconate production from the complementary waste streams molasses and glycerol has been successfully established. In addition to achieving competitive performance indicators, the proposed dual feedstock strategy offers lower process costs and carbon footprint for the production of bio-based itaconate.

Beyond rational-biosensor-guided isolation of 100 independently evolved bacterial strain variants and comparative analysis of their genomes.

BACKGROUND: In contrast to modern rational metabolic engineering, classical strain development strongly relies on random mutagenesis and screening for the desired production phenotype. Nowadays, with the availability of biosensor-based FACS screening strategies, these random approaches are coming back into fashion. In this study, we employ this technology in combination with comparative genome analyses to identify novel mutations contributing to product formation in the genome of a Corynebacterium glutamicum L-histidine producer. Since all known genetic targets contributing to L-histidine production have been already rationally engineered in this strain, identification of novel beneficial mutations can be regarded as challenging, as they might not be intuitively linkable to L-histidine biosynthesis. RESULTS: In order to identify 100 improved strain variants that had each arisen independently, we performed > 600 chemical mutagenesis experiments, > 200 biosensor-based FACS screenings, isolated > 50,000 variants with increased fluorescence, and characterized > 4500 variants with regard to biomass formation and L-histidine production. Based on comparative genome analyses of these 100 variants accumulating 10-80% more L-histidine, we discovered several beneficial mutations. Combination of selected genetic modifications allowed for the construction of a strain variant characterized by a doubled L-histidine titer (29 mM) and product yield (0.13 C-mol C-mol-1) in comparison to the starting variant. CONCLUSIONS: This study may serve as a blueprint for the identification of novel beneficial mutations in microbial producers in a more systematic manner. This way, also previously unexplored genes or genes with previously unknown contribution to the respective production phenotype can be identified. We believe that this technology has a great potential to push industrial production strains towards maximum performance.

Development of an itaconic acid production process with Ustilaginaceae on alternative feedstocks.

BACKGROUND: Currently, Aspergillus terreus is used for the industrial production of itaconic acid. Although, alternative feedstock use in fermentations is crucial for cost-efficient and sustainable itaconic acid production, their utilisation with A. terreus most often requires expensive pretreatment. Ustilaginacea are robust alternatives for itaconic acid production, evading the challenges, including the pretreatment of crude feedstocks regarding reduction of manganese concentration, that A. terreus poses. RESULTS: In this study, five different Ustilago strains were screened for their growth and production of itaconic acid on defined media. The most promising strains were then used to find a suitable alternative feedstock, based on the local food industry. U. cynodontis ITA Max pH, a highly engineered production strain, was selected to determine the biologically available nitrogen concentration in thick juice and molasses. Based on these findings, thick juice was chosen as feedstock to ensure the necessary nitrogen limitation for itaconic acid production. U. cynodontis ITA Max pH was further characterised regarding osmotolerance and product inhibition and a successful scale-up to a 2 L stirred tank reactor was accomplished. A titer of 106.4 gitaconic acid/L with a theoretical yield of 0.50 gitaconic acid/gsucrose and a space-time yield of 0.72 gitaconic acid/L/h was reached. CONCLUSIONS: This study demonstrates the utilisation of alternative feedstocks to produce ITA with Ustilaginaceae, without drawbacks in either titer or yield, compared to glucose fermentations.

Characterization and engineering of branched short-chain dicarboxylate metabolism in Pseudomonas reveals resistance to fungal 2-hydroxyparaconate.

In recent years branched short-chain dicarboxylates (BSCD) such as itaconic acid gained increasing interest in both medicine and biotechnology. Their use as building blocks for plastics urges for developing microbial upcycling strategies to provide sustainable end-of-life solutions. Furthermore, many BSCD exhibit anti-bacterial properties or exert immunomodulatory effects in macrophages, indicating a medical relevance for this group of molecules. For both of these applications, a detailed understanding of the microbial metabolism of these compounds is essential. In this study, the metabolic pathway of BSCD degradation from Pseudomonas aeruginosa PAO1 was studied in detail by heterologously transferring it to Pseudomonas putida. Heterologous expression of the PA0878-0886 itaconate metabolism gene cluster enabled P. putida KT2440 to metabolize itaconate, (S)- and (R)-methylsuccinate, (S)-citramalate, and mesaconate. The functions of the so far uncharacterized genes PA0879 and PA0881 were revealed and proven to extend the substrate range of the core degradation pathway. Furthermore, the uncharacterized gene PA0880 was discovered to encode a 2-hydroxyparaconate (2-HP) lactonase that catalyzes the cleavage of the itaconate derivative 2-HP to itatartarate. Interestingly, 2-HP was found to inhibit growth of the engineered P. putida on itaconate. All in all, this study extends the substrate range of P. putida to include BSCD for bio-upcycling of high-performance polymers, and also identifies 2-HP as promising candidate for anti-microbial applications.

Experimental kS estimation: A comparison of methods for Corynebacterium glutamicum from lab to microfluidic scale.

Knowledge about the specific affinity of whole cells toward a substrate, commonly referred to as kS , is a crucial parameter for characterizing growth within bioreactors. State-of-the-art methodologies measure either uptake or consumption rates at different initial substrate concentrations. Alternatively, cell dry weight or respiratory data like online oxygen and carbon dioxide transfer rates can be used to estimate kS . In this work, a recently developed substrate-limited microfluidic single-cell cultivation (sl-MSCC) method is applied for the estimation of kS values under defined environmental conditions. This method is benchmarked with two alternative microtiter plate methods, namely high-frequency biomass measurement (HFB) and substrate-limited respiratory activity monitoring (sl-RA). As a model system, the substrate affinity kS of Corynebacterium glutamicum ATCC 13032 regarding glucose was investigated assuming a Monod-type growth response. A kS of <70.7 mg/L (with 95% probability) with HFB, 8.55 ± 1.38 mg/L with sl-RA, and 2.66 ± 0.99 mg/L with sl-MSCC was obtained. Whereas HFB and sl-RA are suitable for a fast initial kS estimation, sl-MSCC allows an affinity estimation by determining tD at concentrations less or equal to the kS value. Thus, sl-MSCC lays the foundation for strain-specific kS estimations under defined environmental conditions with additional insights into cell-to-cell heterogeneity.

Robotic workflows for automated long-term adaptive laboratory evolution: improving ethanol utilization by Corynebacterium glutamicum.

BACKGROUND: Adaptive laboratory evolution (ALE) is known as a powerful tool for untargeted engineering of microbial strains and genomics research. It is particularly well suited for the adaptation of microorganisms to new environmental conditions, such as alternative substrate sources. Since the probability of generating beneficial mutations increases with the frequency of DNA replication, ALE experiments are ideally free of constraints on the required duration of cell proliferation. RESULTS: Here, we present an extended robotic workflow for performing long-term evolution experiments based on fully automated repetitive batch cultures (rbALE) in a well-controlled microbioreactor environment. Using a microtiter plate recycling approach, the number of batches and thus cell generations is technically unlimited. By applying the validated workflow in three parallel rbALE runs, ethanol utilization by Corynebacterium glutamicum ATCC 13032 (WT) was significantly improved. The evolved mutant strain WT_EtOH-Evo showed a specific ethanol uptake rate of 8.45 ± 0.12 mmolEtOH gCDW-1 h-1 and a growth rate of 0.15 ± 0.01 h-1 in lab-scale bioreactors. Genome sequencing of this strain revealed a striking single nucleotide variation (SNV) upstream of the ald gene (NCgl2698, cg3096) encoding acetaldehyde dehydrogenase (ALDH). The mutated basepair was previously predicted to be part of the binding site for the global transcriptional regulator GlxR, and re-engineering demonstrated that the identified SNV is key for enhanced ethanol assimilation. Decreased binding of GlxR leads to increased synthesis of the rate-limiting enzyme ALDH, which was confirmed by proteomics measurements. CONCLUSIONS: The established rbALE technology is generally applicable to any microbial strain and selection pressure that fits the small-scale cultivation format. In addition, our specific results will enable improved production processes with C. glutamicum from ethanol, which is of particular interest for acetyl-CoA-derived products.

Discovery of novel amino acid production traits by evolution of synthetic co-cultures.

BACKGROUND: Amino acid production features of Corynebacterium glutamicum were extensively studied in the last two decades. Many metabolic pathways, regulatory and transport principles are known, but purely rational approaches often provide only limited progress in production optimization. We recently generated stable synthetic co-cultures, termed Communities of Niche-optimized Strains (CoNoS), that rely on cross-feeding of amino acids for growth. This setup has the potential to evolve strains with improved production by selection of faster growing communities. RESULTS: Here we performed adaptive laboratory evolution (ALE) with a CoNoS to identify mutations that are relevant for amino acid production both in mono- and co-cultures. During ALE with the CoNoS composed of strains auxotrophic for either L-leucine or L-arginine, we obtained a 23% growth rate increase. Via whole-genome sequencing and reverse engineering, we identified several mutations involved in amino acid transport that are beneficial for CoNoS growth. The L-leucine auxotrophic strain carried an expression-promoting mutation in the promoter region of brnQ (cg2537), encoding a branched-chain amino acid transporter in combination with mutations in the genes for the Na+/H+-antiporter Mrp1 (cg0326-cg0321). This suggested an unexpected link of Mrp1 to L-leucine transport. The L-arginine auxotrophic partner evolved expression-promoting mutations near the transcriptional start site of the yet uncharacterized operon argTUV (cg1504-02). By mutation studies and ITC, we characterized ArgTUV as the only L-arginine uptake system of C. glutamicum with an affinity of KD = 30 nM. Finally, deletion of argTUV in an L-arginine producer strain resulted in a faster and 24% higher L-arginine production in comparison to the parental strain. CONCLUSION: Our work demonstrates the power of the CoNoS-approach for evolution-guided identification of non-obvious production traits, which can also advance amino acid production in monocultures. Further rounds of evolution with import-optimized strains can potentially reveal beneficial mutations also in metabolic pathway enzymes. The approach can easily be extended to all kinds of metabolite cross-feeding pairings of different organisms or different strains of the same organism, thereby enabling the identification of relevant transport systems and other favorable mutations.

Communities of Niche-optimized Strains (CoNoS) - Design and creation of stable, genome-reduced co-cultures.

Current bioprocesses for production of value-added compounds are mainly based on pure cultures that are composed of rationally engineered strains of model organisms with versatile metabolic capacities. However, in the comparably well-defined environment of a bioreactor, metabolic flexibility provided by various highly abundant biosynthetic enzymes is much less required and results in suboptimal use of carbon and energy sources for compound production. In nature, non-model organisms have frequently evolved in communities where genome-reduced, auxotrophic strains cross-feed each other, suggesting that there must be a significant advantage compared to growth without cooperation. To prove this, we started to create and study synthetic communities of niche-optimized strains (CoNoS) that consists of two strains of the same species Corynebacterium glutamicum that are mutually dependent on one amino acid. We used both the wild-type and the genome-reduced C1* chassis for introducing selected amino acid auxotrophies, each based on complete deletion of all required biosynthetic genes. The best candidate strains were used to establish several stably growing CoNoS that were further characterized and optimized by metabolic modelling, microfluidic experiments and rational metabolic engineering to improve amino acid production and exchange. Finally, the engineered CoNoS consisting of an l-leucine and l-arginine auxotroph showed a specific growth rate equivalent to 83% of the wild type in monoculture, making it the fastest co-culture of two auxotrophic C. glutamicum strains to date. Overall, our results are a first promising step towards establishing improved biobased production of value-added compounds using the CoNoS approach.

Metabolic fluxes for nutritional flexibility of Mycobacterium tuberculosis.

The co-catabolism of multiple host-derived carbon substrates is required by Mycobacterium tuberculosis (Mtb) to successfully sustain a tuberculosis infection. However, the metabolic plasticity of this pathogen and the complexity of the metabolic networks present a major obstacle in identifying those nodes most amenable to therapeutic interventions. It is therefore critical that we define the metabolic phenotypes of Mtb in different conditions. We applied metabolic flux analysis using stable isotopes and lipid fingerprinting to investigate the metabolic network of Mtb growing slowly in our steady-state chemostat system. We demonstrate that Mtb efficiently co-metabolises either cholesterol or glycerol, in combination with two-carbon generating substrates without any compartmentalisation of metabolism. We discovered that partitioning of flux between the TCA cycle and the glyoxylate shunt combined with a reversible methyl citrate cycle is the critical metabolic nodes which underlie the nutritional flexibility of Mtb. These findings provide novel insights into the metabolic architecture that affords adaptability of bacteria to divergent carbon substrates and expand our fundamental knowledge about the methyl citrate cycle and the glyoxylate shunt.

Hot isopropanol quenching procedure for automated microtiter plate scale 13C-labeling experiments.

BACKGROUND: Currently, the generation of genetic diversity for microbial cell factories outpaces the screening of strain variants with omics-based phenotyping methods. Especially isotopic labeling experiments, which constitute techniques aimed at elucidating cellular phenotypes and supporting rational strain design by growing microorganisms on substrates enriched with heavy isotopes, suffer from comparably low throughput and the high cost of labeled substrates. RESULTS: We present a miniaturized, parallelized, and automated approach to 13C-isotopic labeling experiments by establishing and validating a hot isopropanol quenching method on a robotic platform coupled with a microbioreactor cultivation system. This allows for the first time to conduct automated labeling experiments at a microtiter plate scale in up to 48 parallel batches. A further innovation enabled by the automated quenching method is the analysis of free amino acids instead of proteinogenic ones on said microliter scale. Capitalizing on the latter point and as a proof of concept, we present an isotopically instationary labeling experiment in Corynebacterium glutamicum ATCC 13032, generating dynamic labeling data of free amino acids in the process. CONCLUSIONS: Our results show that a robotic liquid handler is sufficiently fast to generate informative isotopically transient labeling data. Furthermore, the amount of biomass obtained from a sub-milliliter cultivation in a microbioreactor is adequate for the detection of labeling patterns of free amino acids. Combining the innovations presented in this study, isotopically stationary and instationary automated labeling experiments can be conducted, thus fulfilling the prerequisites for 13C-metabolic flux analyses in high-throughput.

Biosensor-based growth-coupling and spatial separation as an evolution strategy to improve small molecule production of Corynebacterium glutamicum.

Evolutionary engineering is a powerful method to improve the performance of microbial cell factories, but can typically not be applied to enhance the production of chemicals due to the lack of an appropriate selection regime. We report here on a new strategy based on transcription factor-based biosensors, which directly couple production to growth. The growth of Corynebacterium glutamicum was coupled to the intracellular concentration of branched-chain amino acids, by integrating a synthetic circuit based on the Lrp biosensor upstream of two growth-regulating genes, pfkA and hisD. Modelling and experimental data highlight spatial separation as key strategy to limit the selection of 'cheater' strains that escaped the evolutionary pressure. This approach facilitated the isolation of strains featuring specific causal mutations enhancing amino acid production. We envision that this strategy can be applied with the plethora of known biosensors in various microbes, unlocking evolution as a feasible strategy to improve production of chemicals.

Pseudomonas putida KT2440 endures temporary oxygen limitations.

The obligate aerobic nature of Pseudomonas putida, one of the most prominent whole-cell biocatalysts emerging for industrial bioprocesses, questions its ability to be cultivated in large-scale bioreactors, which exhibit zones of low dissolved oxygen tension. P. putida KT2440 was repeatedly subjected to temporary oxygen limitations in scale-down approaches to assess the effect on growth and an exemplary production of rhamnolipids. At those conditions, the growth and production of P. putida KT2440 were decelerated compared to well-aerated reference cultivations, but remarkably, final biomass and rhamnolipid titers were similar. The robust growth behavior was confirmed across different cultivation systems, media compositions, and laboratories, even when P. putida KT2440 was repeatedly exposed to dual carbon and oxygen starvation. Quantification of the nucleotides ATP, ADP, and AMP revealed a decrease of intracellular ATP concentrations with increasing duration of oxygen starvation, which can, however, be restored when re-supplied with oxygen. Only small changes in the proteome were detected when cells encountered oscillations in dissolved oxygen tensions. Concluding, P. putida KT2440 appears to be able to cope with repeated oxygen limitations as they occur in large-scale bioreactors, affirming its outstanding suitability as a whole-cell biocatalyst for industrial-scale bioprocesses.

Metabolic and process engineering for microbial production of protocatechuate with Corynebacterium glutamicum.

3,4-Dihydroxybenzoate (protocatechuate, PCA) is a phenolic compound naturally found in edible vegetables and medicinal herbs. PCA is of high interest in the chemical industry and has wide potential for pharmaceutical applications. We designed and constructed a novel Corynebacterium glutamicum strain to enable the efficient utilization of d-xylose for microbial production of PCA. Shake flask cultivation of the engineered strain showed a maximum PCA titer of 62.1 ± 12.1 mM (9.6 ± 1.9 g L-1 ) from d-xylose as the primary carbon and energy source. The corresponding yield was 0.33 C-mol PCA per C-mol d-xylose, which corresponds to 38% of the maximum theoretical yield. Under growth-decoupled bioreactor conditions, a comparable PCA titer and a total amount of 16.5 ± 1.1 g PCA could be achieved when d-glucose and d-xylose were combined as orthogonal carbon substrates for biocatalyst provision and product synthesis, respectively. Downstream processing of PCA was realized via electrochemically induced crystallization by taking advantage of the pH-dependent properties of PCA. This resulted in a maximum final purity of 95.4%. The established PCA production process represents a highly sustainable approach, which will serve as a blueprint for the bio-based production of other hydroxybenzoic acids from alternative sugar feedstocks.

Robotic integration enables autonomous operation of laboratory scale stirred tank bioreactors with model-driven process analysis.

Given its geometric similarity to large-scale production plants and the excellent possibilities for precise process control and monitoring, the classic stirred tank bioreactor (STR) still represents the gold standard for bioprocess development at a laboratory scale. However, compared to microbioreactor technologies, bioreactors often suffer from a low degree of process automation and deriving key performance indicators (KPIs) such as specific rates or yields often requires manual sampling and sample processing. A widely used parallelized STR setup was automated by connecting it to a liquid handling system and controlling it with a custom-made process control system. This allowed for the setup of a flexible modular platform enabling autonomous operation of the bioreactors without any operator present. Multiple unit operations like automated inoculation, sampling, sample processing and analysis, and decision making, for example for automated induction of protein production were implemented to achieve such functionality. The data gained during application studies was used for fitting of bioprocess models to derive relevant KPIs being in good agreement with literature. By combining the capabilities of STRs with the flexibility of liquid handling systems, this platform technology can be applied to a multitude of different bioprocess development pipelines at laboratory scale.

Separation of 13C- and 15N-Isotopologues of Amino Acids with a Primary Amine without Mass Resolution by Means of O-Phthalaldehyde Derivatization and Collision Induced Dissociation.

Computational and experimental advances of recent years have culminated in establishing 13C-Metabolic Flux Analysis (13C-MFA) as a routine methodology to unravel the fluxome. As the acronym suggests, 13C-MFA has relied on the relative abundance of 13C-isotopes in metabolites for flux inference, most commonly measured by mass spectrometry. In this manuscript we expand the scope of labeling measurements to the case of simultaneous 13C- and 15N-labeling of amino acids. Analytically, the separation of isotopologues of this metabolite class can only be achieved at resolving power beyond 65,000. In this manuscript we harvest an overlooked property of the collision induced dissociation of amino acid adducts to discern 13C- and 15N- isotopologues of amino acids with a primary amine without separating them in the m/z domain.

Identification of modifications procuring growth on xylose in recombinant Saccharomyces cerevisiae strains carrying the Weimberg pathway.

The most prevalent xylose-assimilating pathways in recombinant Saccharomyces cerevisiae, i.e. the xylose isomerase (XI) and the xylose reductase/xylitol dehydrogenase (XR/XDH) pathways, channel the carbon flux through the pentose phosphate pathway and further into glycolysis. In contrast, the oxidative and non-phosphorylative bacterial Weimberg pathway channels the xylose carbon through five steps into the metabolic node α-ketoglutarate (αKG) that can be utilized for growth or diverted into production of various metabolites. In the present study, steps preventing the establishment of a functional Weimberg pathway in S. cerevisiae were identified. Using an original design where a S. cerevisiae strain was expressing the essential four genes of the Caulobacter crescentus pathway (xylB, xylD, xylX, xylA) together with a deletion of FRA2 gene to upregulate the iron-sulfur metabolism, it was shown that the C. crescentus αKG semialdehyde dehydrogenase, XylA was not functional in S. cerevisiae. When replaced by the recently described analog from Corynebacterium glutamicum, KsaD, significantly higher in vitro activity was observed but the strain did not grow on xylose. Adaptive laboratory evolution (ALE) on a xylose/glucose medium on this strain led to a loss of XylB, the first step of the Weimberg pathway, suggesting that ALE favored minimizing the inhibiting xylonate accumulation by restricting the upper part of the pathway. Therefore three additional gene copies of the lower Weimberg pathway (XylD, XylX and KsaD) were introduced. The resulting S. cerevisiae strain (ΔΔfra2, xylB, 4x (xylD-xylX-ksaD)) was able to generate biomass from xylose and Weimberg pathway intermediates were detected. To our knowledge this is the first report of a functional complete Weimberg pathway expressed in fungi. When optimized this pathway has the potential to channel xylose towards value-added specialty chemicals such as dicarboxylic acids and diols.

Multiscale dynamic modeling and simulation of a biorefinery.

A biorefinery comprises a variety of process steps to synthesize products from sustainable natural resources. Dynamic plant-wide simulation enhances the process understanding, leads to improved cost efficiency and enables model-based operation and control. It is thereby important for an increased competitiveness to conventional processes. To this end, we developed a Modelica library with replaceable building blocks that allow dynamic modeling of an entire biorefinery. For the microbial conversion step, we built on the dynamic flux balance analysis (DFBA) approach to formulate process models for the simulation of cellular metabolism under changing environmental conditions. The resulting system of differential-algebraic equations with embedded optimization criteria (DAEO) is solved by a tailor-made toolbox. In summary, our modeling framework comprises three major pillars: A Modelica library of dynamic unit operations, an easy-to-use interface to formulate DFBA process models and a DAEO toolbox that allows simulation with standard environments based on the Modelica modeling language. A biorefinery model for dynamic simulation of the OrganoCat pretreatment process and microbial conversion of the resulting feedstock by Corynebacterium glutamicum serves as case study to demonstrate its practical relevance.

Modulation of the central carbon metabolism of Corynebacterium glutamicum improves malonyl-CoA availability and increases plant polyphenol synthesis.

In recent years microorganisms have been engineered towards synthesizing interesting plant polyphenols such as flavonoids and stilbenes from glucose. Currently, the low endogenous supply of malonyl-CoA, indispensable for plant polyphenol synthesis, impedes high product titers. Usually, limited malonyl-CoA availability during plant polyphenol production is avoided by supplementing fatty acid synthesis-inhibiting antibiotics such as cerulenin, which are known to increase the intracellular malonyl-CoA pool as a side effect. Motivated by the goal of microbial polyphenol synthesis being independent of such expensive additives, we used rational metabolic engineering approaches to modulate regulation of fatty acid synthesis and flux into the tricarboxylic acid cycle (TCA cycle) in Corynebacterium glutamicum strains capable of flavonoid and stilbene synthesis. Initial experiments showed that sole overexpression of genes coding for the native malonyl-CoA-forming acetyl-CoA carboxylase is not sufficient for increasing polyphenol production in C. glutamicum. Hence, the intracellular acetyl-CoA availability was also increased by reducing the flux into the TCA cycle through reduction of citrate synthase activity. In defined cultivation medium, the constructed C. glutamicum strains accumulated 24 mg·L -1 (0.088 mM) naringenin or 112 mg·L -1 (0.49 mM) resveratrol from glucose without supplementation of phenylpropanoid precursor molecules or any inhibitors of fatty acid synthesis.

A FRET-based biosensor for the quantification of glucose in culture supernatants of mL scale microbial cultivations.

BACKGROUND: In most microbial cultivations D-glucose is the main carbon and energy source. However, quantification of D-glucose especially in small scale is still challenging. Therefore, we developed a FRET-based glucose biosensor, which can be applied in microbioreactor-based cultivations. This sensor consists of a glucose binding protein sandwiched between two fluorescent proteins, constituting a FRET pair. Upon D-glucose binding the sensor undergoes a conformational change which is translated into a FRET-ratio change. RESULTS: The selected sensor shows an apparent Kd below 1.5 mM D-glucose and a very high sensitivity of up to 70% FRET-ratio change between the unbound and the glucose-saturated state. The soluble sensor was successfully applied online to monitor the glucose concentration in an Escherichia coli culture. Additionally, this sensor was utilized in an at-line process for a Corynebacterium glutamicum culture as an example for a process with cell-specific background (e.g. autofluorescence) and medium-induced quenching. Immobilization of the sensor via HaloTag® enabled purification and covalent immobilization in one step and increased the stability during application, significantly. CONCLUSION: A FRET-based glucose sensor was used to quantify D-glucose consumption in microtiter plate based cultivations. To the best of our knowledge, this is the first method reported for online quantification of D-glucose in microtiter plate based cultivations. In comparison to D-glucose analysis via an enzymatic assay and HPLC, the sensor performed equally well, but enabled much faster measurements, which allowed to speed up microbial strain development significantly.

FeedER: a feedback-regulated enzyme-based slow-release system for fed-batch cultivation in microtiter plates.

With the advent of modern genetic engineering methods, microcultivation systems have become increasingly important tools for accelerated strain phenotyping and bioprocess engineering. While these systems offer sophisticated capabilities to screen batch processes, they lack the ability to realize fed-batch processes, which are used more frequently in industrial bioprocessing. In this study, a novel approach to realize a feedback-regulated enzyme-based slow-release system (FeedER), allowing exponential fed-batch for microscale cultivations, was realized by extending our existing Mini Pilot Plant technology with a customized process control system. By continuously comparing the experimental growth rates with predefined set points, the automated dosage of Amyloglucosidase enzyme for the cleavage of dextrin polymers into D-glucose monomers is triggered. As a prerequisite for stable fed-batch operation, a constant pH is maintained by automated addition of ammonium hydroxide. We show the successful application of FeedER to study fed-batch growth of different industrial model organisms including Corynebacterium glutamicum, Pichia pastoris, and Escherichia coli. Moreover, the comparative analysis of a C. glutamicum GFP producer strain, cultivated under microscale batch and fed-batch conditions, revealed two times higher product yields under slow growing fed-batch operation. In summary, FeedER enables to run 48 parallel fed-batch experiments in an automated and miniaturized manner, and thereby accelerates industrial bioprocess development at the screening stage.