Metabolic control analysis enabled the improvement of the L-cysteine production process with Escherichia coli.

L-cysteine is an amino acid with relevance to the pharmaceutical, food, feed, and cosmetic industry. The environmental and societal impact of its chemical production has led to the development of more sustainable fermentative L-cysteine production processes with engineered E. coli based on glucose and thiosulfate as sulphur source. Still, most of the published processes show low yields. For the identification of further metabolic engineering targets, engineered E. coli cells were withdrawn from a fed-batch production process, followed by in vivo metabolic control analysis (MCA) based on the data of short-term perturbation experiments, metabolomics (LC-MS), and thermodynamic flux analysis (TFA). In vivo MCA indicated that the activities of the L-cysteine synthases of the cells withdrawn from the production process might be limiting, and we hypothesised that the L-cysteine precursor O-acetylserine (OAS) might be exported from the cells faster than it took to transform OAS into L-cysteine. By increasing the expression of the L-cysteine synthases, either sulfocysteine synthase or L-cysteine synthase, which transform OAS into L-cysteine, an improvement of up to 70% in specific L-cysteine productivity and up to 47% in the final L-cysteine concentration was achieved in standardised fed-batch processes thereby increasing the yield on glucose by more than 85 to 9.2% (w/w). KEY POINTS: • Metabolic control analysis was applied to analyse L-cysteine production with E. coli • OAS export was faster than its transformation to L-cysteine • Overexpression of L-cysteine synthases improved L-cysteine productivity and yield.

3D bioprinting of microorganisms: principles and applications.

In recent years, the ability to create intricate, live tissues and organs has been made possible thanks to three-dimensional (3D) bioprinting. Although tissue engineering has received a lot of attention, there is growing interest in the use of 3D bioprinting for microorganisms. Microorganisms like bacteria, fungi, and algae, are essential to many industrial bioprocesses, such as bioremediation as well as the manufacture of chemicals, biomaterials, and pharmaceuticals. This review covers current developments in 3D bioprinting methods for microorganisms. We go over the bioink compositions designed to promote microbial viability and growth, taking into account factors like nutrient delivery, oxygen supply, and waste elimination. Additionally, we investigate the most important bioprinting techniques, including extrusion-based, inkjet, and laser-assisted approaches, as well as their suitability with various kinds of microorganisms. We also investigate the possible applications of 3D bioprinted microbes. These range from constructing synthetic microbial consortia for improved metabolic pathway combinations to designing spatially patterned microbial communities for enhanced bioremediation and bioprocessing. We also look at the potential for 3D bioprinting to advance microbial research, including the creation of defined microenvironments to observe microbial behavior. In conclusion, the 3D bioprinting of microorganisms marks a paradigm leap in microbial bioprocess engineering and has the potential to transform many application areas. The ability to design the spatial arrangement of various microorganisms in functional structures offers unprecedented possibilities and ultimately will drive innovation.

Simultaneous Formate and Syngas Conversion Boosts Growth and Product Formation by Clostridium ragsdalei.

Electrocatalytic CO2 reduction to CO and formate can be coupled to gas fermentation with anaerobic microorganisms. In combination with a competing hydrogen evolution reaction in the cathode in aqueous medium, the in situ, electrocatalytic produced syngas components can be converted by an acetogenic bacterium, such as Clostridium ragsdalei, into acetate, ethanol, and 2,3-butanediol. In order to study the simultaneous conversion of CO, CO2, and formate together with H2 with C. ragsdalei, fed-batch processes were conducted with continuous gassing using a fully controlled stirred tank bioreactor. Formate was added continuously, and various initial CO partial pressures (pCO0) were applied. C. ragsdalei utilized CO as the favored substrate for growth and product formation, but below a partial pressure of 30 mbar CO in the bioreactor, a simultaneous CO2/H2 conversion was observed. Formate supplementation enabled 20-50% higher growth rates independent of the partial pressure of CO and improved the acetate and 2,3-butanediol production. Finally, the reaction conditions were identified, allowing the parallel CO, CO2, formate, and H2 consumption with C. ragsdalei at a limiting CO partial pressure below 30 mbar, pH 5.5, n = 1200 min-1, and T = 32 °C. Thus, improved carbon and electron conversion is possible to establish efficient and sustainable processes with acetogenic bacteria, as shown in the example of C. ragsdalei.

Challenges and Advances in the Bioproduction of L-Cysteine.

L-cysteine is a proteogenic amino acid with many applications in the pharmaceutical, food, animal feed, and cosmetic industries. Due to safety and environmental issues in extracting L-cysteine from animal hair and feathers, the fermentative production of L-cysteine offers an attractive alternative using renewable feedstocks. Strategies to improve microbial production hosts like Pantoea ananatis, Corynebacterium glutamicum, Pseudomonas sp., and Escherichia coli are summarized. Concerning the metabolic engineering strategies, the overexpression of feedback inhibition-insensitive L-serine O-acetyltransferase and weakening the degradation of L-cysteine through the removal of L-cysteine desulfhydrases are crucial adjustments. The overexpression of L-cysteine exporters is vital to overcome the toxicity caused by intracellular accumulating L-cysteine. In addition, we compiled the process engineering aspects for the bioproduction of L-cysteine. Utilizing the energy-efficient sulfur assimilation pathway via thiosulfate, fermenting cheap carbon sources, designing scalable, fed-batch processes with individual feedings of carbon and sulfur sources, and implementing efficient purification techniques are essential for the fermentative production of L-cysteine on an industrial scale.

Impact of mixing insufficiencies on L-phenylalanine production with an Escherichia coli reporter strain in a novel two-compartment bioreactor.

BACKGROUND: The omnipresence of population heterogeneity in industrial bioprocesses originates from prevailing dynamic bioprocess conditions, which promote differences in the expression of cellular characteristics. Despite the awareness, the concrete consequences of this phenomenon remain poorly understood. RESULTS: Therefore, for the first time, a L-phenylalanine overproducing Escherichia coli quadruple reporter strain was established for monitoring of general stress response, growth behavior, oxygen limitation and product formation of single cells based on mTagBFP2, mEmerald, CyOFP1, and mCardinal2 expression measured by flow cytometry. This strain was applied for the fed-batch production of L-phenylalanine from glycerol and ammonia in a stirred-tank bioreactor at homogeneous conditions compared to the same process in a novel two-compartment bioreactor. This two-compartment bioreactor consists of a stirred-tank bioreactor with an initial volume of 0.9 L (homogeneous zone) with a coiled flow inverter with a fixed working volume of 0.45 L as a bypass (limitation zone) operated at a mean hydraulic residence time of 102 s. The product formation was similar in both bioreactor setups with maximum L-phenylalanine concentrations of 21.1 ± 0.6 g L-1 demonstrating the consistency of this study's microbial L-phenylalanine production. However, cell growth was vulnerable to repetitive exposure to the dynamically changing conditions in the two-compartment bioreactor with maximum biomass yields reduced by 21%. The functionality of reporter molecules was approved in the stirred-tank bioreactor cultivation, in which expressed fluorescence levels of all four markers were in accordance with respective process state variables. Additional evaluation of the distributions on single-cell level revealed the presence of population heterogeneity in both bioprocesses. Especially for the marker of the general stress response and the product formation, the corresponding histograms were characterized by bimodal shapes and broad distributions. These phenomena were pronounced particularly at the beginning and the end of the fed-batch process. CONCLUSIONS: The here shown findings confirm multiple reporter strains to be a noninvasive tool for monitoring cellular characteristics and identifying potential subpopulations in bioprocesses. In combination with experiments in scale-down setups, these can be utilized for a better physiological understanding of bioprocesses and support future scale-up procedures.

Biotechnological mass production of DNA origami.

DNA nanotechnology, in particular DNA origami, enables the bottom-up self-assembly of micrometre-scale, three-dimensional structures with nanometre-precise features. These structures are customizable in that they can be site-specifically functionalized or constructed to exhibit machine-like or logic-gating behaviour. Their use has been limited to applications that require only small amounts of material (of the order of micrograms), owing to the limitations of current production methods. But many proposed applications, for example as therapeutic agents or in complex materials, could be realized if more material could be used. In DNA origami, a nanostructure is assembled from a very long single-stranded scaffold molecule held in place by many short single-stranded staple oligonucleotides. Only the bacteriophage-derived scaffold molecules are amenable to scalable and efficient mass production; the shorter staple strands are obtained through costly solid-phase synthesis or enzymatic processes. Here we show that single strands of DNA of virtually arbitrary length and with virtually arbitrary sequences can be produced in a scalable and cost-efficient manner by using bacteriophages to generate single-stranded precursor DNA that contains target strand sequences interleaved with self-excising 'cassettes', with each cassette comprising two Zn2+-dependent DNA-cleaving DNA enzymes. We produce all of the necessary single strands of DNA for several DNA origami using shaker-flask cultures, and demonstrate end-to-end production of macroscopic amounts of a DNA origami nanorod in a litre-scale stirred-tank bioreactor. Our method is compatible with existing DNA origami design frameworks and retains the modularity and addressability of DNA origami objects that are necessary for implementing custom modifications using functional groups. With all of the production and purification steps amenable to scaling, we expect that our method will expand the scope of DNA nanotechnology in many areas of science and technology.

Bioproduction and applications of aldobionic acids with a focus on maltobionic and cellobionic acid.

Aldobionic acids are sugar acids which consist of a disaccharide with an anomeric acid group. The most famous is lactobionic acid (LBA). LBA is used in many applications such as food and beverages, pharmaceuticals and medicine, cosmetics or chemical processes. During the last decade, all these industries are observing a shift of consumer preferences towards plant-based options. Thus, the biotechnological industry is trying to replace the animal-derived LBA. Maltobionic acid (MBA) and cellobionic acid (CBA) are two stereoisomers of LBA which have emerged as vegan alternatives. However, MBA and CBA face different obstacles related to their industrial production. While traditionally used electrochemical or chemical catalysis often rely on cost intensive and/or hazardous catalysts, novel production methods with microorganisms are still poorly studied. In the first part, this paper discusses both alternatives in terms of their characteristics and applications. In the second part, it reviews the long-studied chemical production and the novel bioproduction methods, which are based on enzymatic and microbial systems. This review concludes with a discussion of future work needed to bring their production to the industrial scale.

Phage-free production of artificial ssDNA with Escherichia coli.

Artificial single-stranded DNA (ssDNA) with user-defined sequences and lengths up to the kilobase range is increasingly needed in mass quantities to realize the potential of emerging technologies such as genome editing and DNA origami. However, currently available biotechnological approaches for mass-producing ssDNA require dedicated, and thus costly, fermentation infrastructure, because of the risk of cross-contaminating manufacturer plants with self-replicating phages. Here we overcome this problem with an efficient, scalable, and cross-contamination-free method for the phage-free biotechnological production of artificial ssDNA with Escherichia coli. Our system utilizes a designed phagemid and an optimized helper plasmid. The phagemid encodes one gene of the M13 phage genome and a freely chosen custom target sequence, while the helper plasmid encodes the other genes of the M13 phage. The phagemid particles produced with this method are not capable of self-replication in the absence of the helper plasmid. This enables cross-contamination-free biotechnological production of ssDNA at any contract manufacturer. Furthermore, we optimized the process parameters to reduce by-products and increased the maximal product concentration up to 83 mg L-1 of ssDNA in a stirred-tank bioreactor, thus realizing up to a 40-fold increase in maximal product concentration over previous scalable phage-free ssDNA production methods.

Metabolic control analysis enables rational improvement of E. coli L-tryptophan producers but methylglyoxal formation limits glycerol-based production.

BACKGROUND: Although efficient L-tryptophan production using engineered Escherichia coli is established from glucose, the use of alternative carbon sources is still very limited. Through the application of glycerol as an alternate, a more sustainable substrate (by-product of biodiesel preparation), the well-studied intracellular glycolytic pathways are rerouted, resulting in the activity of different intracellular control sites and regulations, which are not fully understood in detail. Metabolic analysis was applied to well-known engineered E. coli cells with 10 genetic modifications. Cells were withdrawn from a fed-batch production process with glycerol as a carbon source, followed by metabolic control analysis (MCA). This resulted in the identification of several additional enzymes controlling the carbon flux to L-tryptophan. RESULTS: These controlling enzyme activities were addressed stepwise by the targeted overexpression of 4 additional enzymes (trpC, trpB, serB, aroB). Their efficacy regarding L-tryptophan productivity was evaluated under consistent fed-batch cultivation conditions. Although process comparability was impeded by process variances related to a temporal, unpredictable break-off in L-tryptophan production, process improvements of up to 28% with respect to the L-tryptophan produced were observed using the new producer strains. The intracellular effects of these targeted genetic modifications were revealed by metabolic analysis in combination with MCA and expression analysis. Furthermore, it was discovered that the E. coli cells produced the highly toxic metabolite methylglyoxal (MGO) during the fed-batch process. A closer look at the MGO production and detoxification on the metabolome, fluxome, and transcriptome level of the engineered E. coli indicated that the highly toxic metabolite plays a critical role in the production of aromatic amino acids with glycerol as a carbon source. CONCLUSIONS: A detailed process analysis of a new L-tryptophan producer strain revealed that several of the 4 targeted genetic modifications of the E. coli L-tryptophan producer strain proved to be effective, and, for others, new engineering approaches could be derived from the results. As a starting point for further strain and process optimization, the up-regulation of MGO detoxifying enzymes and a lowering of the feeding rate during the last third of the cultivation seems reasonable.

Machine learning-based protein crystal detection for monitoring of crystallization processes enabled with large-scale synthetic data sets of photorealistic images.

Since preparative chromatography is a sustainability challenge due to large amounts of consumables used in downstream processing of biomolecules, protein crystallization offers a promising alternative as a purification method. While the limited crystallizability of proteins often restricts a broad application of crystallization as a purification method, advances in molecular biology, as well as computational methods are pushing the applicability towards integration in biotechnological downstream processes. However, in industrial and academic settings, monitoring protein crystallization processes non-invasively by microscopic photography and automated image evaluation remains a challenging problem. Recently, the identification of single crystal objects using deep learning has been the subject of increased attention for various model systems. However, the advancement of crystal detection using deep learning for biotechnological applications is limited: robust models obtained through supervised machine learning tasks require large-scale and high-quality data sets usually obtained in large projects through extensive manual labeling, an approach that is highly error-prone for dense systems of transparent crystals. For the first time, recent trends involving the use of synthetic data sets for supervised learning are transferred, thus generating photorealistic images of virtual protein crystals in suspension (PCS) through the use of ray tracing algorithms, accompanied by specialized data augmentations modelling experimental noise. Further, it is demonstrated that state-of-the-art models trained with the large-scale synthetic PCS data set outperform similar fine-tuned models based on the average precision metric on a validation data set, followed by experimental validation using high-resolution photomicrographs from stirred tank protein crystallization processes.

Control of parallelized bioreactors I: dynamic scheduling software for efficient bioprocess management in high-throughput systems.

The shift towards high-throughput technologies and automation in research and development in industrial biotechnology is highlighting the need for increased automation competence and specialized software solutions. Within bioprocess development, the trends towards miniaturization and parallelization of bioreactor systems rely on full automation and digital process control. Thus, mL-scale, parallel bioreactor systems require integration into liquid handling stations to perform a range of tasks stretching from substrate addition to automated sampling and sample analysis. To orchestrate these tasks, the authors propose a scheduling software to fully leverage the advantages of a state-of-the-art liquid handling station (LHS) and to enable improved process control and resource allocation. Fixed sequential order execution, the norm in LHS software, results in imperfect timing of essential operations like feeding or Ph control and execution intervals thereof, that are unknown a priori. However, the duration and control of, e.g., the feeding task and their frequency are of great importance for bioprocess control and the design of experiments. Hence, a software solution is presented that allows the orchestration of the respective operations through dynamic scheduling by external LHS control. With the proposed scheduling software, it is possible to define a dynamic process control strategy based on data-driven real-time prioritization and transparent, user-defined constraints. Drivers for a commercial 48 parallel bioreactor system and the related sensor equipment were developed using the SiLA 2 standard greatly simplifying the integration effort. Furthermore, this paper describes the experimental hardware and software setup required for the application use case presented in the second part.

Lab-scale photobioreactor systems: principles, applications, and scalability.

Phototrophic microorganisms that convert carbon dioxide are being explored for their capacity to solve different environmental issues and produce bioactive compounds for human therapeutics and as food additives. Full-scale phototrophic cultivation of microalgae and cyanobacteria can be done in open ponds or closed photobioreactor systems, which have a broad range of volumes. This review focuses on laboratory-scale photobioreactors and their different designs. Illuminated microtiter plates and microfluidic devices offer an option for automated high-throughput studies with microalgae. Illuminated shake flasks are used for simple uncontrolled batch studies. The application of illuminated bubble column reactors strongly emphasizes homogenous gas distribution, while illuminated flat plate bioreactors offer high and uniform light input. Illuminated stirred-tank bioreactors facilitate the application of very well-defined reaction conditions. Closed tubular photobioreactors as well as open photobioreactors like small-scale raceway ponds and thin-layer cascades are applied as scale-down models of the respective large-scale bioreactors. A few other less common designs such as illuminated plastic bags or aquarium tanks are also used mainly because of their relatively low cost, but up-scaling of these designs is challenging with additional light-driven issues. Finally, this review covers recommendations on the criteria for photobioreactor selection and operation while up-scaling of phototrophic bioprocesses with microalgae or cyanobacteria.

Control of parallelized bioreactors II: probabilistic quantification of carboxylic acid reductase activity for bioprocess optimization.

Autonomously operated parallelized mL-scale bioreactors are considered the key to reduce bioprocess development cost and time. However, their application is often limited to products with very simple analytics. In this study, we investigated enhanced protein expression conditions of a carboxyl reductase from Nocardia otitidiscaviarum in E. coli. Cells were produced with exponential feeding in a L-scale bioreactor. After the desired cell density for protein expression was reached, the cells were automatically transferred to 48 mL-scale bioreactors operated by a liquid handling station where protein expression studies were conducted. During protein expression, the feed rate and the inducer concentration was varied. At the end of the protein expression phase, the enzymatic activity was estimated by performing automated whole-cell biotransformations in a deep-well-plate. The results were analyzed with hierarchical Bayesian modelling methods to account for the biomass growth during the biotransformation, biomass interference on the subsequent product assay, and to predict absolute and specific enzyme activities at optimal expression conditions. Lower feed rates seemed to be beneficial for high specific and absolute activities. At the optimal investigated expression conditions an activity of [Formula: see text] was estimated with a [Formula: see text] credible interval of [Formula: see text]. This is about 40-fold higher than the highest published data for the enzyme under investigation. With the proposed setup, 192 protein expression conditions were studied during four experimental runs with minimal manual workload, showing the reliability and potential of automated and digitalized bioreactor systems.

Byproduct-free geraniol glycosylation by whole-cell biotransformation with recombinant Escherichia coli.

OBJECTIVE: Geraniol, a fragrance of great importance in the consumer goods industry, can be glucosylated by the UDP-glucose-dependent glucosyltransferase VvGT14a from Vitis vinifera, yielding more stable geranyl glucoside. Escherichia coli expressing VvGT14a is a convenient whole-cell biocatalyst for this biotransformation due to its intrinsic capability for UDP-glucose regeneration. The low water solubility and high cytotoxicity of geraniol can be overcome in a biphasic system where the non-aqueous phase functions as an in situ substrate reservoir. However, the effect of different process variables on the biphasic whole-cell biotransformation is unknown. Thus, the goal of this study was to identify potential bottlenecks during biotransformation with in situ geraniol supply via isopropyl myristate as second non-aqueous phase. RESULTS: First, insufficient UDP-glucose supply could be ruled out by measurement of intracellular UDP-glucose concentrations. Instead, oxygen supply was determined as a bottleneck. Moreover, the formation of the byproduct geranyl acetate by chloramphenicol acetyltransferase (CAT) was identified as a constraint for high product yields. The use of a CAT-deficient whole-cell biocatalyst prevented the formation of geranyl acetate, and geranyl glucoside could be obtained with 100% selectivity during a biotransformation on L-scale. CONCLUSION: This study is the first to closely analyze the whole-cell biotransformation of geraniol with Escherichia coli expressing an UDP-glucose-dependent glucosyltransferase and can be used as an optimal starting point for the design of other glycosylation processes.

Metabolic control analysis of L-tryptophan producing Escherichia coli applying targeted perturbation with shikimate.

L-tryptophan production from glycerol with Escherichia coli was analysed by perturbation studies and metabolic control analysis. The insertion of a non-natural shikimate transporter into the genome of an Escherichia coli L-tryptophan production strain enabled targeted perturbation within the product pathway with shikimate during parallelised short-term perturbation experiments with cells withdrawn from a 15 L fed-batch production process. Expression of the shikimate/H+-symporter gene (shiA) from Corynebacterium glutamicum did not alter process performance within the estimation error. Metabolic analyses and subsequent extensive data evaluation were performed based on the data of the parallel analysis reactors and the production process. Extracellular rates and intracellular metabolite concentrations displayed evident deflections in cell metabolism and particularly in chorismate biosynthesis due to the perturbations with shikimate. Intracellular flux distributions were estimated using a thermodynamics-based flux analysis method, which integrates thermodynamic constraints and intracellular metabolite concentrations to restrain the solution space. Feasible flux distributions, Gibbs reaction energies and concentration ranges were computed simultaneously for the genome-wide metabolic model, with minimum bias in relation to the direction of metabolic reactions. Metabolic control analysis was applied to estimate elasticities and flux control coefficients, predicting controlling sites for L-tryptophan biosynthesis. The addition of shikimate led to enhanced deviations in chorismate biosynthesis, revealing a so far not observed control of 3-dehydroquinate synthase on L-tryptophan formation. The relative expression of the identified target genes was analysed with RT-qPCR. Transcriptome analysis revealed disparities in gene expression and the localisation of target genes to further improve the microbial L-tryptophan producer by metabolic engineering.

Development and characterization of Escherichia coli triple reporter strains for investigation of population heterogeneity in bioprocesses.

BACKGROUND: Today there is an increasing demand for high yielding robust and cost efficient biotechnological production processes. Although cells in these processes originate from isogenic cultures, heterogeneity induced by intrinsic and extrinsic influences is omnipresent. To increase understanding of this mechanistically poorly understood phenomenon, advanced tools that provide insights into single cell physiology are needed. RESULTS: Two Escherichia coli triple reporter strains have been designed based on the industrially relevant production host E. coli BL21(DE3) and a modified version thereof, E. coli T7E2. The strains carry three different fluorescence proteins chromosomally integrated. Single cell growth is followed with EmeraldGFP (EmGFP)-expression together with the ribosomal promoter rrnB. General stress response of single cells is monitored by expression of sigma factor rpoS with mStrawberry, whereas expression of the nar-operon together with TagRFP657 gives information about oxygen limitation of single cells. First, the strains were characterized in batch operated stirred-tank bioreactors in comparison to wildtype E. coli BL21(DE3). Afterwards, applicability of the triple reporter strains for investigation of population heterogeneity in bioprocesses was demonstrated in continuous processes in stirred-tank bioreactors at different growth rates and in response to glucose and oxygen perturbation simulating gradients on industrial scale. Population and single cell level physiology was monitored evaluating general physiology and flow cytometry analysis of fluorescence distributions of the triple reporter strains. Although both triple reporter strains reflected physiological changes that were expected based on the expression characteristics of the marker proteins, the triple reporter strain based on E. coli T7E2 showed higher sensitivity in response to environmental changes. For both strains, noise in gene expression was observed during transition from phases of non-growth to growth. Apparently, under some process conditions, e.g. the stationary phase in batch cultures, the fluorescence response of EmGFP and mStrawberry is preserved, whereas TagRFP657 showed a distinct response. CONCLUSIONS: Single cell growth, general stress response and oxygen limitation of single cells could be followed using the two triple reporter strains developed in this study. They represent valuable tools to study population heterogeneity in bioprocesses significantly increasing the level of information compared to the use of single reporter strains.

Comparative evaluation of Aspergillus niger strains for endogenous pectin-depolymerization capacity and suitability for D-galacturonic acid production.

Pectinaceous agricultural residues rich in D-galacturonic acid (D-GalA), such as sugar beet pulp, are considered as promising feedstocks for waste-to-value conversions. Aspergillus niger is known for its strong pectinolytic activity. However, while specialized strains for production of citric acid or proteins are well characterized, this is not the case for the production of pectinases. We, therefore, systematically compared the pectinolytic capabilities of six A. niger strains (ATCC 1015, ATCC 11414, NRRL 3122, CBS 513.88, NRRL 3, and N402) using controlled batch cultivations in stirred-tank bioreactors. A. niger ATCC 11414 showed the highest polygalacturonase activity, specific protein secretion, and a suitable morphology. Furthermore, D-GalA release from sugar beet pulp was 75% higher compared to the standard lab strain A. niger N402. Our study, therefore, presents a robust initial strain selection to guide future process improvement of D-GalA production from agricultural residues and identifies a high-performance base strain for further genetic optimizations.

L-Erythrulose production with a multideletion strain of Gluconobacter oxydans.

Many ketoses or organic acids can be produced by membrane-associated oxidation with Gluconobacter oxydans. In this study, the oxidation of meso-erythritol to L-erythrulose was investigated with the strain G. oxydans 621HΔupp BP.8, a multideletion strain lacking the genes for eight membrane-bound dehydrogenases. First batch biotransformations with growing cells showed re-consumption of L-erythrulose by G. oxydans 621HΔupp BP.8 in contrast to resting cells. The batch biotransformation with 2.8 g L-1 resting cells of G. oxydans 621HΔupp BP.8 in a DO-controlled stirred-tank bioreactor resulted in 242 g L-1 L-erythrulose with a product yield of 99% (w/w) and a space-time yield of 10 g L-1 h-1. Reaction engineering studies showed substrate excess inhibition as well as product inhibition of G. oxydans 621HΔupp BP.8 in batch biotransformations. In order to overcome substrate inhibition, a continuous membrane bioreactor with full cell retention was applied for meso-erythritol oxidation with resting cells of G. oxydans 621HΔupp BP.8. At a mean hydraulic residence time of 2 h, a space-time yield of 27 g L-1 h-1 L-erythrulose was achieved without changing the product yield of 99% (w/w) resulting in a cell-specific product yield of up to 4.4 gP gX-1 in the steady state. The product concentration (54 g L-1 L-erythrulose) was reduced in the continuous biotransformation process compared with the batch process to avoid product inhibition.

Asymmetric Whole-Cell Bio-Reductions of (R)-Carvone Using Optimized Ene Reductases.

(2R,5R)-dihydrocarvone is an industrially applied building block that can be synthesized by site-selective and stereo-selective C=C bond bio-reduction of (R)-carvone. Escherichia coli (E. coli) cells overexpressing an ene reductase from Nostoc sp. PCC7120 (NostocER1) in combination with a cosubstrate regeneration system proved to be very effective biocatalysts for this reaction. However, the industrial applicability of biocatalysts is strongly linked to the catalysts' activity. Since the cell-internal NADH concentrations are around 20-fold higher than the NADPH concentrations, we produced E. coli cells where the NADPH-preferring NostocER1 was exchanged with three different NADH-accepting NostocER1 mutants. These E. coli whole-cell biocatalysts were used in batch operated stirred-tank reactors on a 0.7 l-scale for the reduction of 300 mM (R)-carvone. 287 mM (2R,5R)-dihydrocarvone were formed within 5 h with a diasteromeric excess of 95.4% and a yield of 95.6%. Thus, the whole-cell biocatalysts were strongly improved by using NADH-accepting enzymes, resulting in an up to 2.1-fold increased initial product formation rate leading to a 1.8-fold increased space-time yield when compared to literature.

Fed-batch production of l-tryptophan from glycerol using recombinant Escherichia coli.

l-tryptophan is an essential amino acid of high industrial interest that is routinely produced by microbial processes from glucose as carbon source. Glycerol is an alternative substrate providing a variety of economic and metabolic advantages. Process performance of the recombinant l-tryptophan producer Escherichia coli NT367 was studied in controlled fed-batch processes. The chromosome of the recombinant l-tryptophan producer was equipped with additional genes coding for enzymes of the aromatic amino acids biosynthetic pathway and l-serine biosynthesis, including genes for feedback-resistant enzyme variants ( trpE fbr , aroFBL, and serA fbr ), deletions of enzymatic steps for the degradation of precursors or the product l-tryptophan ( sdaB and tnaA), and alterations in the regulation of l-tryptophan metabolism (deletion of trpL and trpR). The impact of glycerol supply rates as well as the application of a multicopy plasmid (pF112- aroFBL -kan) were investigated in fully controlled stirred-tank bioreactors on a 15 L scale. The combination of E. coli NT367 carrying pF112- aroFBL -kan and an appropriate biomass-specific glycerol supply-rate resulted in the highest final product concentration of 12.5 g L -1 l-tryptophan with the lowest concentrations of other aromatic amino acids. Fed-batch production of l-tryptophan from glycerol was shown for the first time with recombinant E. coli.