Nicolai Kallscheuer

Construction of a Corynebacterium glutamicum platform strain for the production of stilbenes and (2S)-flavanones

Corynebacterium glutamicum is an important organism in industrial biotechnology for the microbial production of bulk chemicals, in particular amino acids. However, until now activity of a complex catabolic network for the degradation of aromatic compounds averted application of C. glutamicum as production host for aromatic compounds of pharmaceutical or biotechnological interest. In the course of the construction of a suitable C. glutamicum platform strain for plant polyphenol production, four gene clusters comprising 21 genes involved in the catabolism of aromatic compounds were deleted. Expression of plant-derived and codon-optimized genes coding for a chalcone synthase (CHS) and a chalcone isomerase (CHI) in this strain background enabled formation of 35mg/L naringenin and 37mg/L eriodictyol from the supplemented phenylpropanoids p-coumaric acid and caffeic acid, respectively. Furthermore, expression of genes coding for a 4-coumarate: CoA-ligase (4CL) and a stilbene synthase (STS) led to the production of the stilbenes pinosylvin, resveratrol and piceatannol starting from supplemented phenylpropanoids cinnamic acid, p-coumaric acid and caffeic acid, respectively. Stilbene concentrations of up to 158mg/L could be achieved. Additional engineering of the amino acid metabolism for an optimal connection to the synthetic plant polyphenol pathways enabled resveratrol production directly from glucose. The construction of these C. glutamicum platform strains for the synthesis of plant polyphenols opens the door towards the microbial production of high-value aromatic compounds from cheap carbon sources with this microorganism.

A Novel Synthetic Pathway Enables Microbial Production of Polyphenols Independent from the Endogenous Aromatic Amino Acid Metabolism

Numerous plant polyphenols have potential applications as pharmaceuticals or nutraceuticals. Stilbenes and flavonoids as most abundant polyphenols are synthesized from phenylpropanoids, which are exclusively derived from aromatic amino acids in nature. Several microorganisms were engineered for the synthesis of biotechnologically interesting plant polyphenols; however, low activity of heterologous ammonia lyases, linking endogenous microbial aromatic amino acid biosynthesis to phenylpropanoid synthesis, turned out to be the limiting step during microbial synthesis. We here developed an alternative strategy for polyphenol production from cheap benzoic acids by reversal of a β-oxidative phenylpropanoid degradation pathway avoiding any ammonia lyase activity. The synthetic pathway running in the non-natural direction is feasible with respect to thermodynamics and involved reaction mechanisms. Instantly, product titers of 5 mg/L resveratrol could be achieved in recombinant Corynebacterium glutamicum strains indicating that phenylpropanoid synthesis from 4-hydroxybenzoic acid can in principle be implemented independently from aromatic amino acids and ammonia lyase activity.

Functional expression of plant-derived O-methyltransferase, flavanone 3-hydroxylase, and flavonol synthase in Corynebacterium glutamicum for production of pterostilbene, kaempferol, and quercetin

Plant polyphenols receive significant attention due to their anti-oxidative and health-promoting properties, and several microorganisms are currently engineered towards producing these valuable compounds. Previously, Corynebacterium glutamicum has been engineered for synthesizing polyphenol core structures such as the stilbene resveratrol and the (2S)-flavanone naringenin. Decoration of these compounds by O-methylation or hydroxylation would provide access to polyphenols of even higher commercial interest. In this study, introduction of a heterologous O-methyltransferase into a resveratrol-producing C. glutamicum strain allowed synthesis of 42mg/L (0.16mM) of the di-O-methylated pterostilbene from p-coumaric acid. A prerequisite for reaching this product titer was a fusion of O-methyltransferase with the maltose-binding protein of Escherichia coli lacking its signal peptide, thereby increasing the solubility of the O-methyltransferase. Furthermore, expression of heterologous dioxygenase genes in (2S)-flavanone-producing C. glutamicum strains enabled the production of flavanonols and flavonols starting from the phenylpropanoids p-coumaric acid and caffeic acid. For the flavonols kaempferol and quercetin, maximum product titers of 23mg/L (0.08mM) and 10mg/L (0.03mM) could be achieved, respectively. The obtained results demonstrate that C. glutamicum is a suitable host organism for the production of more complex plant polyphenols.

BacHBerry: BACterial Hosts for production of Bioactive phenolics from bERRY fruits

BACterial Hosts for production of Bioactive phenolics from bERRY fruits (BacHBerry) was a 3-year project funded by the Seventh Framework Programme (FP7) of the European Union that ran between November 2013 and October 2016. The overall aim of the project was to establish a sustainable and economically-feasible strategy for the production of novel high-value phenolic compounds isolated from berry fruits using bacterial platforms. The project aimed at covering all stages of the discovery and pre-commercialization process, including berry collection, screening and characterization of their bioactive components, identification and functional characterization of the corresponding biosynthetic pathways, and construction of Gram-positive bacterial cell factories producing phenolic compounds. Further activities included optimization of polyphenol extraction methods from bacterial cultures, scale-up of production by fermentation up to pilot scale, as well as societal and economic analyses of the processes. This review article summarizes some of the key findings obtained throughout the duration of the project.

Production of plant-derived polyphenols in microorganisms: current state and perspectives

Plants synthesize several thousand different polyphenols of which many have the potential to aid in preventing or treating cancer, cardiovascular, and neurodegenerative diseases. However, plants usually contain complex polyphenol mixtures impeding access to individual compounds in larger quantities. In contrast, functional integration of biosynthetic plant polyphenol pathways into microorganisms allows for the production of individual polyphenols as chemically distinct compounds, which can be synthesized in large amounts and can be more easily isolated. Over the last decade, microbial synthesis of many plant polyphenols could be achieved, and along the way, many decisive bottlenecks in the endogenous microbial host metabolism as well as in the heterologous plant pathways could be identified. In this review, we present recent advancements in metabolic engineering of microorganisms for the production of plant polyphenols and discuss how current challenges could be addressed in the future.

Reversal of β-oxidative pathways for the microbial production of chemicals and polymer building blocks

β-Oxidation is the ubiquitous metabolic strategy to break down fatty acids. In the course of this four-step process, two carbon atoms are liberated per cycle from the fatty acid chain in the form of acetyl-CoA. However, typical β-oxidative strategies are not restricted to monocarboxylic (fatty) acid degradation only, but can also be involved in the utilization of aromatic compounds, amino acids and dicarboxylic acids. Each enzymatic step of a typical β-oxidation cycle is reversible, offering the possibility to also take advantage of reversed metabolic pathways for applied purposes. In such cases, 3-oxoacyl-CoA thiolases, which catalyze the final chain-shortening step in the catabolic direction, mediate the condensation of an acyl-CoA starter molecule with acetyl-CoA in the anabolic direction. Subsequently, the carbonyl-group at C3 is stepwise reduced and dehydrated yielding a chain-elongated product. In the last years, several β-oxidation pathways have been studied in detail and reversal of these pathways already proved to be a promising strategy for the production of chemicals and polymer building blocks in several industrially relevant microorganisms. This review covers recent advancements in this field and discusses constraints and bottlenecks of this metabolic strategy in comparison to alternative production pathways.

Impact of the cultivation strategy on resveratrol production from glucose in engineered Corynebacterium glutamicum

The health benefits of polyphenols such as stilbenes and flavonoids for humans are increasingly attracting attention. Resveratrol is a well-characterized naturally-occurring stilbene and potent anti-oxidant, which is used as food supplement and cosmetic ingredient. Several microorganisms including Corynebacterium glutamicum were engineered for resveratrol production from glucose. Based on the cultivation of a resveratrol-producing C. glutamicum strain in shake flasks, different strategies for improving production under controlled conditions at bioreactor scale were tested. To this end, different cultivation parameters including substrate concentration and operation modes (batch and fed-batch) were evaluated. Whereas the highest biomass concentration was observed during fed-batch fermentation, the maximum resveratrol production was achieved in batch mode. The maximal titer obtained was 12mgL-1 of resveratrol without the addition of the fatty acid synthase inhibitor cerulenin, which was previously shown to be crucial for production with C. glutamicum. The specific growth rate during production seems to have a significant effect in resveratrol production and apparently low specific growth rates may redirect the metabolic bottleneck from p-coumaric acid formation to malonyl-CoA or ATP availability. We also show that high oxygen concentrations in the bioreactor negatively affected the obtained resveratrol titers with C. glutamicum, which is most likely due to the strong tendency of resveratrol to oxidize or oligomerize. Thus, up-scaling of the resveratrol production process is technically challenging and individual process parameters have to be optimized cautiously.

OptPipe - a pipeline for optimizing metabolic engineering targets

BackgroundWe propose OptPipe - a Pipeline for Optimizing Metabolic Engineering Targets, based on a consensus approach. The method generates consensus hypotheses for metabolic engineering applications by combining several optimization solutions obtained from distinct algorithms. The solutions are ranked according to several objectives, such as biomass and target production, by using the rank product tests corrected for multiple comparisons.ResultsOptPipe was applied in a genome-scale model of Corynebacterium glutamicum for maximizing malonyl-CoA, which is a valuable precursor for many phenolic compounds. In vivo experimental validation confirmed increased malonyl-CoA level in case of ΔsdhCAB deletion, as predicted in silico.ConclusionsA method was developed to combine the optimization solutions provided by common knockout prediction procedures and rank the suggested mutants according to the expected growth rate, production and a new adaptability measure. The implementation of the pipeline along with the complete documentation is freely available at https://github.com/AndrasHartmann/OptPipe.

Production of plant metabolites with applications in the food industry using engineered microorganisms

Secondary plant metabolites are extensively used in todays food industries, for example, as coloring-agents, flavouring-agents or texturizing agents. In particular, metabolites with antioxidative properties find applications as preservatives or anti-browning agents. Today, extraction from plant material represents the major source of these metabolites, but progress in the field of metabolic engineering also enabled the microbial production of these valuable compounds as a more economic and ecological alternative. This review article presents the current state of metabolic engineering of microorganisms for production of plant metabolites with applications in the food industries. We focus on compounds, which are already used in food applications, discuss current limitations of microbial plant metabolite production, and outline strategies on how these challenges can be addressed in the future.

Produktion pflanzlicher Polyphenole mit Corynebacterium glutamicum

Many plant polyphenols demonstrate important pharmacological activities, but extraction from natural plant sources is usually limited by low abundance. After identification and elimination of the responsible degradation pathways, production of a broad range of different stilbenes and flavonoids could be achieved in Corynebacterium glutamicum. Furthermore, a synthetic pathway could be established in C. glutamicum, which allows for plant polyphenol synthesis from cheap 4-hydroxybenzoic acids.