Search

Agricultural products, have a high phytate content that has a hidden potential as a renewable source of phosphate. We present the first biotechnological route for the production of food-grade, organic polyphosphate (polyP) from deoiled seeds or bran, included in a vision for a circular phosphorus economy. The three-step production process includes phosphorus mobilization (e.g., 37 mg PO43−/g bran) using phytase enzymes. Non-genetically modified phosphate starved Saccharomyces cerevisiae is then fed with soluble P-extracts. The yeast intracellularly polymerizes the phosphate to polyP (≥ 30% mol polyP in yeast per mol PO43− in medium) and polyP-rich yeast extract or pure polyP is purified from the biomass. We demonstrate that the obtained polyP-rich yeast extract is an excellent green surrogate for polyP from fossil P-sources in meat manufacturing. The valorization of phytate–P while producing P-depleted biomass as a demanded feed in livestock production is shown. Our sustainable process enables the production of food-grade polyP from renewable resources and is contributing to the sustainable management of the dwindling nutrient phosphorus.

Summary: Bile acid (BA) metabolism is a complex system that includes a wide variety of primary and secondary, as well as conjugated and unconjugated BAs that undergo continuous enterohepatic circulation (EHC). Alterations in both composition and dynamics of BAs have been associated with various diseases. However, a mechanistic understanding of the relationship between altered BA metabolism and related diseases is lacking. Computational modeling may support functional analyses of the physiological processes involved in the EHC of BAs along the gut-liver axis. In this study, we developed a physiologically based model of murine BA metabolism describing synthesis, hepatic and microbial transformations, systemic distribution, excretion, and EHC of BAs at the whole-body level. For model development, BA metabolism of specific pathogen-free (SPF) mice was characterized in vivo by measuring BA levels and composition in various organs, expression of transporters along the gut, and cecal microbiota composition. We found significantly different BA levels between male and female mice that could only be explained by adjusted expression of the hepatic enzymes and transporters in the model. Of note, this finding was in agreement with experimental observations. The model for SPF mice could also describe equivalent experimental data in germ-free mice by specifically switching off microbial activity in the intestine. The here presented model can therefore facilitate and guide functional analyses of BA metabolism in mice, e.g., the effect of pathophysiological alterations on BA metabolism and translation of results from mouse studies to a clinically relevant context through cross-species extrapolation.

Plastic mulch films and biofertilizers (processed sewage sludge, compost or manure) have helped to increase crop yields. However, there is increasing evidence that these practices significantly contribute to microplastic contamination in agricultural soils, affecting biodiversity and soil health. Here, we draw attention to the use of hydrolase enzymes that depolymerize polyester-based plastics as a bioremediation technique for agricultural soils (in situ), biofertilizers and irrigation water (ex situ), and discuss the need for fully biodegradable plastic mulches. We also highlight the need for ecotoxicological assessment of the proposed approach and its effects on different soil organisms. Enzymes should be optimized to work effectively and efficiently under the conditions found in natural soils (typically, moist solids at an ambient temperature with low salinity). Such optimization is also necessary to ensure that already distressed ecosystems are not disrupted any further.

Lignin is a ubiquitously available and sustainable feedstock that is underused as its depolymerization yields a range of aromatic monomers that are challenging substrates for microbes. In this study, we investigated the growth of Pseudomonas taiwanensis VLB120 on biomass-derived aromatics, namely, 4-coumarate, ferulate, 4-hydroxybenzoate, and vanillate. The wild type strain was not able to grow on 4-coumarate and ferulate. After integration of catabolic genes for breakdown of 4-coumarate and ferulate, the metabolically engineered strain was able to grow on these aromatics. Further, the specific growth rate of the strain was enhanced up to 3-fold using adaptive laboratory evolution, resulting in increased tolerance towards 4-coumarate and ferulate. Whole-genome sequencing highlighted several different mutations mainly in two genes. The first gene was actP, coding for a cation/acetate symporter, and the other gene was paaA coding for a phenyl acetyl-CoA oxygenase. The evolved strain was further engineered for rhamnolipid production. Among the biomass-derived aromatics investigated, 4-coumarate and ferulate were promising substrates for product synthesis. With 4-coumarate as the sole carbon source, a yield of 0.27 (Cmolrhl/Cmol4-coumarate) was achieved, corresponding to 28% of the theoretical yield. Ferulate enabled a yield of about 0.22 (Cmolrhl/Cmolferulate), representing 42% of the theoretical yield. Overall, this study demonstrates the use of biomass-derived aromatics as novel carbon sources for rhamnolipid biosynthesis.

Microbial co-cultures have been used in several biotechnological applications. Within these co-cultures, the microorganisms tend to interact with each other and perform complex actions. Investigating metabolic interactions in microbial co-cultures is crucial in designing microbial consortia. Here, we present a pipeline integrating modelling and experimental approaches to understand metabolic interactions between organisms in a community. We define a new index named “Metabolic Support Index (MSI)”, which quantifies the benefits derived by each organism in the presence of the other when grown as a co-culture. We computed MSI for several experimentally demonstrated co-cultures and showed that MSI, as a metric, accurately identifies the organism that derives the maximum benefit. We also computed MSI for a commonly used yeast co-culture consisting of Saccharomyces cerevisiae and Pichia stipitis and observed that the latter derives higher benefit from the interaction. Further, we designed two-stage experiments to study mutual interactions and showed that P. stipitis indeed derives the maximum benefit from the interaction, as shown from our computational predictions. Also, using our previously developed computational tool MetQuest, we identified all the metabolic exchanges happening between these organisms by analysing the pathways spanning the two organisms. By analysing the HPLC profiles and studying the isotope labelling, we show that P. stipitis consumes the ethanol produced by S. cerevisiae when grown on glucose-rich medium under aerobic conditions, as also indicated by our in silico pathway analyses. Our approach represents an important step in understanding metabolic interactions in microbial communities through an integrated computational and experimental workflow.

Recent advances in the availability and applicability of genetic tools for non-conventional yeasts have raised high hopes regarding the industrial applications of such yeasts; however, quantitative physiological data on these yeasts, including intracellular flux distributions, are scarce and have rarely aided in the development of novel yeast applications. The compartmentation of eukaryotic cells adds to model complexity. Model constraints are ideally based on biochemical evidence, which is rarely available for non-conventional yeast and eukaryotic cells. A small-scale model for 13C-based metabolic flux analysis of central yeast carbon metabolism was developed that is universally valid and does not depend on localization information regarding amino acid anabolism. The variable compartmental origin of traced metabolites is a feature that allows application of the model to yeasts with uncertain genomic and transcriptional backgrounds. The presented test case includes the baker's yeast Saccharomyces cerevisiae and the methylotrophic yeast Hansenula polymorpha. Highly similar flux solutions were computed using either a model with undefined pathway localization or a model with constraints based on curated (S. cerevisiae) or computationally predicted (H. polymorpha) localization information, while false solutions were found with incorrect localization constraints. These results indicate a potentially adverse effect of universally assuming Saccharomyces-like constraints on amino acid biosynthesis for non-conventional yeasts and verify the validity of neglecting compartmentation constraints using a small-scale metabolic model. The model was specifically designed to investigate the intracellular metabolism of wild-type yeasts under various growth conditions but is also expected to be useful for computing fluxes of other eukaryotic cells.

Ustilago trichophora RK089 has been found recently as a good natural malic acid producer from glycerol. This strain has previously undergone adaptive laboratory evolution for enhanced substrate uptake rate resulting in the strain U. trichophora TZ1. Medium optimization and investigation of process parameters enabled titers and rates that are able to compete with those of organisms overexpressing major parts of the underlying metabolic pathways. Metabolic engineering can likely further increase the efficiency of malate production by this organism, provided that basic genetic tools and methods can be established for this rarely used and relatively obscure species. Here we investigate and adapt existing molecular tools from U. maydis for use in U. trichophora. Selection markers from U. maydis that confer carboxin, hygromycin, nourseothricin, and phleomycin resistance are applicable in U. trichophora. A plasmid was constructed containing the ip-locus of U. trichophora RK089, resulting in site-specific integration into the genome. Using this plasmid, overexpression of pyruvate carboxylase, two malate dehydrogenases (mdh1, mdh2), and two malate transporters (ssu1, ssu2) was possible in U. trichophora TZ1 under control of the strong Petef promoter. Overexpression of mdh1, mdh2, ssu1, and ssu2 increased the product (malate) to substrate (glycerol) yield by up to 54% in shake flasks reaching a titer of up to 120 g L−1. In bioreactor cultivations of U. trichophora TZ1 Petefssu2 and U. trichophora TZ1 Petefmdh2 a drastically lowered biomass formation and glycerol uptake rate resulted in 29% (Ssu1) and 38% (Mdh2) higher specific production rates and 38% (Ssu1) and 46% (Mdh2) increased yields compared to the reference strain U. trichophora TZ1. Investigation of the product spectrum resulted in an 87% closed carbon balance with 134 g L−1 malate and biomass (73 g L−1), succinate (20 g L−1), CO2 (17 g L−1), and α-ketoglutarate (8 g L−1) as main by-products. These results open up a wide range of possibilities for further optimization, especially combinatorial metabolic engineering to increase the flux from pyruvate to malic acid and to reduce by-product formation.

Soil microorganisms mineralize lignin-derived aromatic carbon sources using oxidative catabolic pathways, such as the β-ketoadipate pathway. Although this aromatic pathway is one of the best-studied pathways in biochemistry, the complete pathway, including its regulation by aromatic carbon sources, has not been integrated into the metabolic network. In particular, information about the in vivo operation (e.g., kinetics and flux capacity) of the pathway is lacking. In this contribution, we use kinetic modeling and thermodynamic analysis to evaluate the in vivo operation of this key aromatic multi-step pathway. The resulting ab initio deterministic model of benzoate degradation via the β-ketoadipate (ortho-cleavage) pathway in Pseudomonas putida KT2440 is presented. The kinetic model includes mechanistic rate expressions for the enzymes and transport processes. The design and experimental validation of the model are driven by data generated from short-term perturbation experiments in a benzoate-limited continuous culture. The results of rigorous modeling of the in vivo dynamics provide strong support for flux regulation by the benzoate transporter and the enzymes forming and cleaving catechol. Revisiting the β-ketoadipate pathway might be valuable for applications in different fields, such as biochemistry and metabolic engineering, that use lignin monomers as a carbon source. Keywords: Metabolic modeling, Pseudomonas putida, Aromatic metabolism, Transient response, Intracellular metabolites

Microbial carbon dioxide assimilation and conversion to chemical platform molecules has the potential to be developed as economic, sustainable processes. The carbon dioxide assimilation can proceed by a variety of natural pathways and recently even synthetic CO2 fixation routes have been designed. Early assessment of the performance of the different carbon fixation alternatives within biotechnological processes is desirable to evaluate their potential. Here we applied stoichiometric metabolic modeling based on physiological and process data to evaluate different process variants for the conversion of C1 carbon compounds to the industrial relevant platform chemical succinic acid. We computationally analyzed the performance of cyanobacteria, acetogens, methylotrophs, and synthetic CO2 fixation pathways in Saccharomyces cerevisiae in terms of production rates, product yields, and the optimization potential. This analysis provided insight into the economic feasibility and allowed to estimate the future industrial applicability by estimating overall production costs. With reported, or estimated data of engineered or wild type strains, none of the simulated microbial succinate production processes showed a performance allowing competitive production. The main limiting factors were identified as gas and photon transfer and metabolic activities whereas metabolic network structure was not restricting. In simulations with optimized parameters most process alternatives reached economically interesting values, hence, represent promising alternatives to sugar-based fermentations. Keywords: Carbon fixation, Metabolic engineering, Flux Balance Analysis, Succinic acid, Profitability, C1 carbon source