Georg Fritz

Precise timing of gene expression is a common feature in natural regulatory networks but is rarely implemented in synthetic pathways, where static control often limits performance. Here, we expand the use of bacterial extracytoplasmic function (ECF) σ factors by integrating their cognate anti-σ factors to create tunable threshold-gated circuits. Anti-σ overexpression has previously limited the utility of such systems due to growth inhibition. We address this by combining targeted truncations of membrane domains with chromosomal integration, yielding a library of ECF/anti-σ pairs that maintain function while minimizing toxicity. In Escherichia coli, these circuits can enable sharper OFF/ON switching, greater dynamic range, and tunable temporal delays compared with ECF-only cascades. In the best case, single-step threshold gates achieve up to 2100-fold induction with delays spanning minutes to hours. Extending the design to two steps enables programmable cascading of gene expression, with delays ranging from 30 to 409 min, although the dynamic range is generally reduced relative to single-step circuits. In the best-performing designs, matching input-output characteristics of promoters preserves dynamic range across cascade levels. Mathematical modeling supports these findings and highlights σ/anti-σ binding affinity as a key parameter for achieving high performance in longer cascades. Together, these results provide design principles for orthogonal, lower-burden timing circuits that enable controlled, sequential gene expression with minimal intervention in synthetic pathways and highlight continuing limitations of these systems.

3,4-Dihydroxybenzoic acid (protocatechuic acid, PCA) is a central intermediate in bacterial aromatic catabolism - a key node for processing aromatic waste streams such as lignin and polyethylene terephthalate (PET) and a compound of growing biotechnological interest for valorising complex aromatic feedstocks. Here, we present a proof-of-concept for upcycling of PCA as a sole carbon source into a non-aromatic, higher-value bioproduct. We show that Vibrio natriegens, an emerging microbial chassis, can use PCA to produce poly-3-hydroxybutyrate (PHB), with no additional carbon source such as yeast extract or peptone. Efficient growth on PCA required careful optimisation of substrate concentration, cultivation conditions, and medium composition, e.g. replacing Fe²⁺ with Fe³⁺. These strategies reduced PCA autoxidation and limited reactive oxygen species (ROS) toxicity. Adaptive laboratory evolution over ~ 1000 generations identified convergent mutations that reveal insights into the architecture of the oxidative stress response in V. natriegens, including catalytic enzymes such as a Dyp-type peroxidase, and multiple regulatory proteins including OxyR, MarR, RpoS, ToxS, and RseB. Further mutations implicate cell wall repair and protein quality control in the PCA stress response. Functionally, the adapted strains produced 2.5-fold more PHB in 80% of the time required by the parental strain. Overall, we demonstrate the potential and current limitations of V. natriegens for the sustainable upcycling of aromatic, waste-derived carbon into value-added bioproducts.

Despite its industrial importance, microbial L-lysine production has largely been confined to classical producer strains, leaving the fast-growing, non-pathogenic marine microorganism V. natriegens largely untapped as an unconventional biosynthetic platform. In this work, we established an L-lysine-overproducing V. natriegens DSM759 strain through a step-wise, systematic rational engineering strategy targeting the native biosynthetic pathway. Guided by our prior systems-level analysis of the strain’s genetic and regulatory architecture, we identified key metabolic bottlenecks and implemented knowledge-driven interventions to relieve pathway constraints. Central to production was alleviation of feedback inhibition in the native key regulatory enzymes, aspartate kinase (AK, lysC ) and dihydrodipicolinate synthase (DHDPS, dapA ). Site-directed amino-acid substitutions, replicating established E. coli feedback-resistance mechanisms, were introduced into conserved regions of the V. natriegens DSM759 enzymes, producing L-lysine-insensitive variants with kinetic parameters comparable to that of corresponding wild type enzymes. Among the tested configurations, the strain co-expressing Vn.lysC2 and Vn.dapA1:E84T reached the highest L-lysine titer (9.0±0.6 mM) and yield (0.11±0.01 mol _Lys mol _Glc ^-1 ), whereas overexpression of additional L-lysine pathway genes provided no further benefit. Leveraging the host’s metabolic versatility, L-lysine synthesis was also demonstrated from the chitin-derived amino-sugar N-acetylglucosamine (0.09±0.00 mol _Lys mol _GlcNAc ^-1 ), highlighting the potential to valorize chitin-rich waste streams from the seafood industry. This work establishes a minimal, rational strategy for L-lysine biosynthesis in V. natriegens DSM759 and positions it as a promising platform for sustainable amino acid production.

We thank Wu and Criddle for their commentary and welcome this scientific dialogue. Our approach was designed to rigorously assess the potential for insect-mediated expanded polystyrene (EPS) degradation by comparing pure and commercial EPS under controlled conditions that eliminated cannibalism artifacts. Our results demonstrate that mealworms mechanically fragment EPS but achieve no genuine biochemical degradation. Pure EPS remained chemically unaffected after gut passage, while commercial EPS showed only modest additive-mediated oxidative changes, and not enzymatic polymer backbone cleavage. Additional studies on superworms (Zophobas morio) with both EPS and polyvinyl chloride (PVC) yielded corresponding results, confirming that the absence of plastic metabolism spans multiple insect species and polymer types. Here we address Wu and Criddle’s concerns regarding mass balance, isotopic interpretation, and analytical methods while demonstrating how experimental artifacts in previous studies generate false evidence for biodegradation. Simple scalability calculations reveal the fundamental impracticality of any insect-based approach: treating one ton of polystyrene would require over sixty million mealworms, producing more than four tons of dead biomass while generating vast quantities of microplastics and achieving zero meaningful degradation. Our controlled methodology establishes that insect-mediated plastic treatment is neither chemically viable nor economically feasible.

Vibrio natriegens, with its exceptionally fast growth rate, has great promise as a revolutionary chassis for synthetic biology, yet the realization of its full potential has been limited by the lack of robust, standardized genetic tools. Here, we present the Vnat Collection, a comprehensive, modular toolkit specifically engineered to overcome these limitations. Leveraging optimized Golden Gate cloning strategies, we introduce improved junction sequences and a highly efficient dropout part system, achieving up to a 300-fold increase in assembly efficiency. Our toolkit significantly expands the synthetic biology toolbox by providing a wide array of characterized inducible promoters, enabling precise, orthogonal gene regulation, and novel operon connectors to streamline the construction of multi-gene pathways critical for metabolic engineering. Furthermore, we enhance genome editing workflows through refined NT-CRISPR methods, incorporating homology-flanked targeting constructs and demonstrating a simplified protocol that eliminates intermediate purification steps. With over 220 rigorously validated modular components, the Vnat Collection establishes an advanced standard for genetic engineering of V. natriegens, empowering researchers to efficiently harness this organism’s unparalleled potential for diverse biotechnology applications.

Acetate can be a sustainable and renewable carbon source which holds significant promise for biotechnological production but is underutilized industrially due to limited microbial efficiency. Vibrio natriegens , recognized for exceptionally fast growth rates, represents a compelling host for developing efficient acetate-based bioprocesses. In this study, adaptive laboratory evolution significantly enhanced V. natriegens ’ ability to grow on acetate as the sole carbon source, achieving an 89% increase in growth rate. Genetic and transcriptomic analyses revealed key adaptations improving acetate uptake and metabolism via increased salt tolerance, boosted Pta/AckA pathway activity, and rewired quorum sensing. Further metabolic engineering and bioprocess optimization enabled the evolved strain to reach high cell densities and efficiently convert acetate into the bioplastic poly-3-hydroxybutyrate (PHB), with productivities up to 0.27 g/L/h and PHB accumulation reaching 45.66% of cell biomass. These advances position V. natriegens as a highly promising microbial platform for sustainable, scalable, and cost-effective biomanufacturing using acetate as green feedstock.