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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., Competing Interests: Declaration of competing interest The authors declare no competing interest., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2021 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Production of proteins and biochemicals in microbial cell factories is often limited by carbon and energy spent on excess biomass formation. To address this issue, we developed several genetic growth switches based on CRISPR interference technology. We demonstrate that growth of Escherichia coli can be controlled by repressing the DNA replication machinery, by targeting dnaA and oriC, or by blocking nucleotide synthesis through pyrF or thyA. This way, total GFP-protein production could be increased by up to 2.2-fold. Single-cell dynamic tracking in microfluidic systems was used to confirm functionality of the growth switches. Decoupling of growth from production of biochemicals was demonstrated for mevalonate, a precursor for isoprenoid compounds. Mass yield of mevalonate was increased by 41%, and production was maintained for more than 45h after activation of the pyrF-based growth switch. The developed methods represent a promising approach for increasing production yield and titer for proteins and biochemicals., (Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Mechanistic biochemical network models describe the dynamics of intracellular metabolite pools in terms of substance concentrations, stoichiometry and reaction kinetics. Data from stimulus response experiments are currently the most informative source for in-vivo parameter estimation in such models. However, only a part of the parameters of classical enzyme kinetic models can usually be estimated from typical stimulus response data. For this reason, several alternative kinetic formats using different "languages" (e.g. linear, power laws, linlog, generic and convenience) have been proposed to reduce the model complexity. The present contribution takes a rigorous "multi-lingual" approach to data evaluation by translating biochemical network models from one kinetic format into another. For this purpose, a new high-performance algorithm has been developed and tested. Starting with a given model, it replaces as many kinetic terms as possible by alternative expressions while still reproducing the experimental data. Application of the algorithm to a published model for Escherichia coli's sugar metabolism demonstrates the power of the new method. It is shown that model translation is a powerful tool to investigate the information content of stimulus response data and the predictive power of models. Moreover, the local and global approximation capabilities of the models are elucidated and some pitfalls of traditional single model approaches to data evaluation are revealed.

The porcine lymphotropic herpesviruses (PLHV) are discussed as possible risk factors in xenotransplantation because of the high prevalence of PLHV-1, PLHV-2 and PLHV-3 in pig populations world-wide and the fact that PLHV-1 has been found to be associated with porcine post-transplant lymphoproliferative disease. To provide structural and functional knowledge on the PLHV immediate-early (IE) transactivator genes, the central regions of the PLHV genomes were characterized by genome walking, sequence and splicing analysis. Three spliced genes were identified (ORF50, ORFA6/BZLF1(h), ORF57) encoding putative IE transactivators, homologous to (i) ORF50 and BRLF1/Rta, (ii) K8/K-bZIP and BZLF1/Zta and (iii) ORF57 and BMLF1 of HHV-8 and EBV, respectively. Expressed as myc-tag or HA-tag fusion proteins, they were located to the cellular nucleus. In reporter gene assays, several PLHV-promoters were mainly activated by PLHV-1 ORF50, to a lower level by PLHV-1 ORFA6/BZLF1(h) and not by PLHV-1 ORF57. However, the ORF57-encoded protein acted synergistically on ORF50-mediated activation.

Phytochromes are photoreceptors that have been found in plants, bacteria, and fungi. Most bacterial and fungal phytochromes are histidine kinases and, for several bacterial phytochromes, light regulation of kinase activity has been demonstrated. Typical histidine kinases are homodimeric proteins in which one subunit phosphorylates the substrate histidine residue of the other subunit; dimerization is an intrinsic property of the histidine kinase itself. Truncated phytochromes which lack the histidine kinase can also form dimers, but the interaction between subunits is modulated by light. This light-dependent dimerization can give a clue to the intramolecular signal transduction of phytochromes which modulates the histidine kinase activity. Size exclusion chromatography, limited proteolysis, and protein crosslinking can be used to study light-induced conformational changes and the interaction of subunits within the homodimer.

Phytochromes are photochromic photoreceptors with a bilin chromophore that have been found in plants and bacteria. Typical bacterial phytochromes are composed of an N-terminal photosensory chromophore module and a C-terminal protein kinase. The former contains the chromophore, which allows phytochromes to adopt the two interconvertible spectral forms, Pr and Pfr. The N-terminal photosensory module of Agrobacterium phytochrome Agp1, Agp1-M15, was used for crystallization studies. The protein was either assembled with the natural chromophore biliverdin or a sterically locked synthetic biliverdin-derivative, termed 15Za. The last-named adduct does not undergo photoisomerization due to an additional carbon chain between the rings C and D of the chromophore. Both adducts could be crystallized, but the resolution was largely improved by the use of 15Za. Crystals of biliverdin-Agp1-M15 diffract to 6A resolution and belong to the tetragonal space group I422 with unit cell dimensions a = b = 171 Angstroms, c = 81 Angstroms, crystals of 15Za-Agp1-M15 belong to the same space group with similar unit cell dimensions a = b = 174 Angstroms, c = 80 Angstroms, but diffract to 3.4 Angstroms resolution. Assuming the asymmetric unit to be occupied by one monomer of 55kDa, the unit cell contains 54-55% solvent with a crystal volume per protein mass, V(m), of 2.7 Angstroms(3) Da(-1).

The porcine lymphotropic herpesvirus 1 (PLHV-1), the first gammaherpesvirus of pigs, has been detected at a high prevalence in healthy pig populations. A porcine gammaherpesvirus has also been detected at high copy numbers in animals suffering from posttransplant lymphoproliferative disease (PTLD). While human PTLD is a EBV-associated complication following clinical transplantation, porcine PTLD is a disease recently described in pigs undergoing experimental allogeneic hematopoietic stem cell transplantation. Here we demonstrate that PLHV-1 and the virus present in porcine PTLD are indistinguishable, and present the characterization of 73 kbp of the genome of PLHV-1. We identified homologs of cellular genes, including a putative G protein-coupled receptor (GCR) as well as a viral homolog of the bcl-2 oncogene (v-bcl-2) and show significant transcription of these genes as well as of several other PLHV-1 genes in lymph nodes of a PTLD-affected pig. These data indicate that PLHV-1 is active during PTLD and may be involved in the etiology of this lymphoproliferative disease.