Enhanced biosynthesis of poly(3-hydroxybutyrate) in engineered strains of Pseudomonas putida via increased malonyl-CoA availability.

Malonyl-coenzyme A (CoA) is a key precursor for the biosynthesis of multiple value-added compounds by microbial cell factories, including polyketides, carboxylic acids, biofuels, and polyhydroxyalkanoates. Owing to its role as a metabolic hub, malonyl-CoA availability is limited by competition in several essential metabolic pathways. To address this limitation, we modified a genome-reduced Pseudomonas putida strain to increase acetyl-CoA carboxylation while limiting malonyl-CoA utilization. Genes involved in sugar catabolism and its regulation, the tricarboxylic acid (TCA) cycle, and fatty acid biosynthesis were knocked-out in specific combinations towards increasing the malonyl-CoA pool. An enzyme-coupled biosensor, based on the rppA gene, was employed to monitor malonyl-CoA levels in vivo. RppA is a type III polyketide synthase that converts malonyl-CoA into flaviolin, a red-colored polyketide. We isolated strains displaying enhanced malonyl-CoA availability via a colorimetric screening method based on the RppA-dependent red pigmentation; direct flaviolin quantification identified four engineered strains had a significant increase in malonyl-CoA levels. We further modified these strains by adding a non-canonical pathway that uses malonyl-CoA as precursor for poly(3-hydroxybutyrate) biosynthesis. These manipulations led to increased polymer accumulation in the fully engineered strains, validating our general strategy to boost the output of malonyl-CoA-dependent pathways in P. putida.

Disentangling the Regulatory Response of Agrobacterium tumefaciens CHLDO to Glyphosate for Engineering Whole-Cell Phosphonate Biosensors.

Phosphonates (PHTs), organic compounds with a stable C-P bond, are widely distributed in nature. Glyphosate (GP), a synthetic PHT, is extensively used in agriculture and has been linked to various human health issues and environmental damage. Given the prevalence of GP, developing cost-effective, on-site methods for GP detection is key for assessing pollution and reducing exposure risks. We adopted Agrobacterium tumefaciens CHLDO, a natural GP degrader, as a host and the source of genetic parts for constructing PHT biosensors. In this bacterial species, the phn gene cluster, encoding the C-P lyase pathway, is regulated by the PhnF transcriptional repressor. We selected the phnG promoter, which displays a dose-dependent response to GP, to build a set of whole-cell biosensors. Through stepwise genetic optimization of the transcriptional cascade, we created a whole-cell biosensor capable of detecting GP in the 0.25-50 µM range in various samples, including soil and water.

Systematic mapping of chemoreceptor specificities for Pseudomonas aeruginosa.

IMPORTANCE: Chemotaxis of motile bacteria has multiple physiological functions. It enables bacteria to locate optimal ecological niches, mediates collective behaviors, and can play an important role in infection. These multiple functions largely depend on ligand specificities of chemoreceptors, and the number and identities of chemoreceptors show high diversity between organisms. Similar diversity is observed for the spectra of chemoeffectors, which include not only chemicals of high metabolic value but also bacterial, plant, and animal signaling molecules. However, the systematic identification of chemoeffectors and their mapping to specific chemoreceptors remains a challenge. Here, we combined several in vivo and in vitro approaches to establish a systematic screening strategy for the identification of receptor ligands and we applied it to identify a number of new physiologically relevant chemoeffectors for the important opportunistic human pathogen P. aeruginosa. This strategy can be equally applicable to map specificities of sensory domains from a wide variety of receptor types and bacteria.

Production of (hydroxy)benzoate-derived polyketides by engineered Pseudomonas with in situ extraction.

Polyketides from (hydroxy)benzoates are an interesting group of plant polyphenolic compounds, whose biotechnological production is so far underrepresented due to their challenging heterologous biosynthesis. Efficient heterologous production of 2,4,6-tri- and 2,3',4,6-tetrahydroxybenzophenone, 3,5-dihydroxybiphenyl, and 4-hydroxycoumarin by whole-cell biocatalysis in combination with in situ product extraction with an organic solvent was demonstrated. Production was highly dependent on the used CoA ligase and polyketide synthase type III. Therefore, different combinations of polyketide synthases and benzoate-CoA ligases were evaluated for their biosynthesis performance in the solvent-tolerant Pseudomonas taiwanensis VLB120. A solvent screening yielded 2-undecanone as biocompatible, extraction-efficient solvent with good phase separation. In aqueous-organic two-phase cultivations, this solvent extraction circumvents product instability in the aqueous cultivation medium, and it increases yields by reducing inhibitory effects. Complete de novo synthesis from glucose of all (hydroxy)benzoate-derived polyketides was achieved in two-phase cultivations with metabolically engineered strains. Additionally, mutasynthesis was applied to obtain fluorinated benzophenone derivatives.

A synthetic C2 auxotroph of Pseudomonas putida for evolutionary engineering of alternative sugar catabolic routes.

Acetyl-coenzyme A (AcCoA) is a metabolic hub in virtually all living cells, serving as both a key precursor of essential biomass components and a metabolic sink for catabolic pathways for a large variety of substrates. Owing to this dual role, tight growth-production coupling schemes can be implemented around the AcCoA node. Building on this concept, a synthetic C2 auxotrophy was implemented in the platform bacterium Pseudomonas putida through an in silico-informed engineering approach. A growth-coupling strategy, driven by AcCoA demand, allowed for direct selection of an alternative sugar assimilation route-the phosphoketolase (PKT) shunt from bifidobacteria. Adaptive laboratory evolution forced the synthetic P. putida auxotroph to rewire its metabolic network to restore C2 prototrophy via the PKT shunt. Large-scale structural chromosome rearrangements were identified as possible mechanisms for adjusting the network-wide proteome profile, resulting in improved PKT-dependent growth phenotypes. 13C-based metabolic flux analysis revealed an even split between the native Entner-Doudoroff pathway and the synthetic PKT bypass for glucose processing, leading to enhanced carbon conservation. These results demonstrate that the P. putida metabolism can be radically rewired to incorporate a synthetic C2 metabolism, creating novel network connectivities and highlighting the importance of unconventional engineering strategies to support efficient microbial production.

Synthetic biology landscape and community in Germany.

Despite its start in the early 2000s, synthetic biology is still overall perceived as a young discipline. In some countries, such as the US, synthetic biology is academically and industrially established, while in others, including Germany, it is still an upcoming field of research. Issues with funding schemes, commercial translation of technologies, public perception, and regulations need to be addressed to establish synthetic biology as a key discipline of the 21st century. This perspective article reviews the German and European synthetic biology landscape and how the German Association for Synthetic Biology (GASB) is addressing the above-mentioned challenges with its events and community-building activities.