Michael Kohlstedt

Metabolic fluxes and beyond—systems biology understanding and engineering of microbial metabolism

The recent years have seen tremendous progress towards the understanding of microbial metabolism on a higher level of the entire functional system. Hereby, huge achievements including the sequencing of complete genomes and efficient post-genomic approaches provide the basis for a new, fascinating era of research—analysis of metabolic and regulatory properties on a global scale. Metabolic flux (fluxome) analysis displays the first systems oriented approach to unravel the physiology of microorganisms since it combines experimental data with metabolic network models and allows determining absolute fluxes through larger networks of central carbon metabolism. Hereby, fluxes are of central importance for systems level understanding because they fundamentally represent the cellular phenotype as integrated output of the cellular components, i.e. genes, transcripts, proteins, and metabolites. A currently emerging and promising area of research in systems biology and systems metabolic engineering is therefore the integration of fluxome data in multi-omics studies to unravel the multiple layers of control that superimpose the flux network and enable its optimal operation under different environmental conditions.

Adaptation of Bacillus subtilis carbon core metabolism to simultaneous nutrient limitation and osmotic challenge: a multi‐omics perspective

The Gram-positive bacterium Bacillus subtilis encounters nutrient limitations and osmotic stress in its natural soil ecosystem. To ensure survival and sustain growth, highly integrated adaptive responses are required. Here, we investigated the system-wide response of B. subtilis to different, simultaneously imposed stresses. To address the anticipated complexity of the cellular response networks, we combined chemostat experiments under conditions of carbon limitation, salt stress and osmoprotection with multi-omics analyses of the transcriptome, proteome, metabolome and fluxome. Surprisingly, the flux through central carbon and energy metabolism is very robust under all conditions studied. The key to achieve this robustness is the adjustment of the biocatalytic machinery to compensate for solvent-induced impairment of enzymatic activities during osmotic stress. Specifically, increased production of several enzymes of central carbon metabolism compensates for their reduced activity in the presence of high salt. A major response of the cell during osmotic stress is the production of the compatible solute proline. This is achieved through the concerted adjustment of multiple reactions around the 2-oxoglutarate node, which drives metabolism towards the proline precursor glutamate. The fine-tuning of the transcriptional and metabolic networks involves functional modules that overarch the individual pathways.

Enabling the valorization of guaiacol-based lignin: Integrated chemical and biochemical production of cis,cis-muconic acid using metabolically engineered Amycolatopsis sp ATCC 39116

Lignin is natures second most abundant polymer and displays a largely unexploited renewable resource for value-added bio-production. None of the lignin-based fermentation processes so far managed to use guaiacol (2-methoxy phenol), the predominant aromatic monomer in depolymerized lignin. In this work, we describe metabolic engineering of Amycolatopsis sp. ATCC 39116 to produce cis,cis-muconic acid (MA), a precursor of recognized industrial value for commercial plastics, from guaiacol. The microbe utilized a very broad spectrum of lignin-based aromatics, such as catechol, guaiacol, phenol, toluene, p-coumarate, and benzoate, tolerated them in elevated amounts and even preferred them over sugars. As a next step, we developed a novel approach for genomic engineering of this challenging, GC-rich actinomycete. The successful introduction of conjugation and blue-white screening, using β-glucuronidase, enabled tailored genomic modifications within ten days. Successive deletion of two putative muconate cycloisomerases from the genome provided the mutant Amycolatopsis sp. ATCC 39116 MA-2, which accumulated 3.1gL-1 MA from guaiacol within 24h, achieving a yield of 96%. The mutant was found also capable to produce MA from a guaiacol-rich true lignin hydrolysate, obtained from pine through hydrothermal conversion. This provides an important proof-of-concept to successfully coupling chemical and biochemical process steps into a value chain from the lignin polymer to an industrial chemical. In addition, Amycolatopsis sp. ATCC 39116 MA-2 was able to produce 2-methyl MA from o-cresol (2-methyl phenol), which opens possibilities towards polymers with novel architecture and properties.

From lignin to nylon: Cascaded chemical and biochemical conversion using metabolically engineered Pseudomonas putida

Cis,cis-muconic acid (MA) is a chemical that is recognized for its industrial value and is synthetically accessible from aromatic compounds. This feature provides the attractive possibility of producing MA from mixtures of aromatics found in depolymerized lignin, the most underutilized lignocellulosic biopolymer. Based on the metabolic pathway, the catechol (1,2-dihydroxybenzene) node is the central element of this type of production process: (i) all upper catabolic pathways of aromatics converge at catechol as the central intermediate, (ii) catechol itself is frequently generated during lignin pre-processing, and (iii) catechol is directly converted to the target product MA by catechol 1,2-dioxygenase. However, catechol is highly toxic, which poses a challenge for the bio-production of MA. In this study, the soil bacterium Pseudomonas putida KT2440 was upgraded to a fully genome-based host for the production of MA from catechol and upstream aromatics. At the core of the cell factories created was a designed synthetic pathway module, comprising both native catechol 1,2-dioxygenases, catA and catA2, under the control of the Pcat promoter. The pathway module increased catechol tolerance, catechol 1,2-dioxygenase levels, and catechol conversion rates. MA, the formed product, acted as an inducer of the module, triggering continuous expression. Cellular energy level and ATP yield were identified as critical parameters during catechol-based production. The engineered MA-6 strain achieved an MA titer of 64.2 g L-1 from catechol in a fed-batch process, which repeatedly regenerated the energy levels via specific feed pauses. The developed process was successfully transferred to the pilot scale to produce kilograms of MA at 97.9% purity. The MA-9 strain, equipped with a phenol hydroxylase, used phenol to produce MA and additionally converted o-cresol, m-cresol, and p-cresol to specific methylated variants of MA. This strain was used to demonstrate the entire value chain. Following hydrothermal depolymerization of softwood lignin to catechol, phenol and cresols, MA-9 accumulated 13 g L-1 MA and small amounts of 3-methyl MA, which were hydrogenated to adipic acid and its methylated derivative to polymerize nylon from lignin for the first time.

A bio-based route to the carbon-5 chemical glutaric acid and to bionylon-6,5 using metabolically engineered Corynebacterium glutamicum

In the present work, we established the bio-based production of glutarate, a carbon-5 dicarboxylic acid with recognized value for commercial plastics and other applications, using metabolically engineered Corynebacterium glutamicum. The mutant C. glutamicum AVA-2 served as a starting point for strain development, because it secreted small amounts of glutarate as a consequence of its engineered 5-aminovalerate pathway. Starting from AVA-2, we overexpressed 5-aminovalerate transaminase (gabT) and glutarate semialdehyde dehydrogenase (gabD) under the control of the constitutive tuf promoter to convert 5-aminovalerate further to glutarate. The created strain GTA-1 formed glutarate as a major product, but still secreted 5-aminovalerate as well. This bottleneck was tackled at the level of 5-aminovalerate re-import. The advanced strain GTA-4 overexpressed the newly discovered 5-aminovalerate importer NCgl0464 and formed glutarate from glucose in a yield of 0.27 mol mol−1. In a fed-batch process, GTA-4 produced more than 90 g L−1 glutarate from glucose and molasses based sugars in a yield of up to 0.70 mol mol−1 and a maximum productivity of 1.8 g L−1 h−1, while 5-aminovalerate was no longer secreted. The bio-based glutaric acid was purified to >99.9% purity. Interfacial polymerization and melt polymerization with hexamethylenediamine yielded bionylon-6,5, a polyamide with a unique structure.

Adaptation of Bacillus subtilis carbon core metabolism to simultaneous nutrient limitation and osmotic challenge

The Gram-positive bacterium Bacillus subtilis encounters nutrient limitations and osmotic stress in its natural soil ecosystem. To ensure survival and sustain growth, highly integrated adaptive responses are required. Here, we investigated the system-wide response of B. subtilis to different, simultaneously imposed stresses. To address the anticipated complexity of the cellular response networks, we combined chemostat experiments under conditions of carbon limitation, salt stress and osmoprotection with multi-omics analyses of the transcriptome, proteome, metabolome and fluxome. Surprisingly, the flux through central carbon and energy metabolism is very robust under all conditions studied. The key to achieve this robustness is the adjustment of the biocatalytic machinery to compensate for solvent-induced impairment of enzymatic activities during osmotic stress. Specifically, increased production of several enzymes of central carbon metabolism compensates for their reduced activity in the presence of high salt. A major response of the cell during osmotic stress is the production of the compatible solute proline. This is achieved through the concerted adjustment of multiple reactions around the 2-oxoglutarate node, which drives metabolism towards the proline precursor glutamate. The fine-tuning of the transcriptional and metabolic networks involves functional modules that overarch the individual pathways.

Adaptation ofBacillus subtiliscarbon core metabolism to simultaneous nutrient limitation and osmotic challenge: a multi-omics perspective: Multi-omics perspective onB. subtilis

The Gram-positive bacterium Bacillus subtilis encounters nutrient limitations and osmotic stress in its natural soil ecosystem. To ensure survival and sustain growth, highly integrated adaptive responses are required. Here, we investigated the system-wide response of B. subtilis to different, simultaneously imposed stresses. To address the anticipated complexity of the cellular response networks, we combined chemostat experiments under conditions of carbon limitation, salt stress and osmoprotection with multi-omics analyses of the transcriptome, proteome, metabolome and fluxome. Surprisingly, the flux through central carbon and energy metabolism is very robust under all conditions studied. The key to achieve this robustness is the adjustment of the biocatalytic machinery to compensate for solvent-induced impairment of enzymatic activities during osmotic stress. Specifically, increased production of several enzymes of central carbon metabolism compensates for their reduced activity in the presence of high salt. A major response of the cell during osmotic stress is the production of the compatible solute proline. This is achieved through the concerted adjustment of multiple reactions around the 2-oxoglutarate node, which drives metabolism towards the proline precursor glutamate. The fine-tuning of the transcriptional and metabolic networks involves functional modules that overarch the individual pathways.