The 4-hydroxyvalerate (4HV) is a key intermediate that serves as a precursor for γ-valerolactone (GVL) and terpolymers, contributing to the synthesis of liquid fuels and biodegradable plastics with improved properties. While 4HV is typically derived from levulinic acid (LA) via chemical catalysis, these methods are limited by high costs and operational complexity. In our previous study, we designed an engineered Pseudomonas putida KT2440 with double knockout of pcaIJ and lvaR and introduction of aroG, asbF, and adc, enabling high production of LA from glycerol. Herein, we introduced acyl-CoA synthetase gene (lvaE) and 4-hydroxyvaleryl-CoA dehydratase gene (lvaD) into LA producing strain to construct a 4HV production pathway from glycerol. The strain harboring both lvaD and lvaE consumed the highest amount of LA, and, subsequently, showed the highest 4HV production. In fed batch fermentation, P. putida KT2440 HP403 produced 28.77mM of 4HV from glycerol. To expand its potential applications as a PHA precursor, biologically synthesized 4HV was supplied to the high-performing PHA producer Cupriavidus necator PHB-4/phaCBP-M-CPF4 for polymer synthesis. This produced a terpolymer with the composition P(54.49mol% 3HB-co-6.98mol% 3HV-co-38.53mol% 4HV) with a PHA content of 60.21%. The terpolymer exhibited improved elongation at break and lower glass transition and crystallization temperatures due to increased amorphicity. This study suggests a 4HV bioconversion strategy derived from glycerol by engineered P. putida KT2440 HP403 and demonstrates the production of a high-content P(3HB-co-3HV-co-4HV) terpolymer in C. necator containing high-mole fraction of 4HV, thereby providing a new sustainable approach for PHA production.
The production of recombinant biopharmaceuticals is a billion-dollar industry, with numerous high-value proteins expressed in the bacterium Escherichia coli. Many recombinant protein production (RPP) systems enable the synthesis and purification of large amounts of soluble recombinant protein. However, such systems are often too strong to allow the expression of difficult-to-fold proteins, such as membrane proteins, with high-level RPP resulting in the production of insoluble aggregates (i.e., inclusion bodies), target protein degradation and even cell death. To aid the production of problematic proteins, we have engineered the E. coli lite strains in which the plasmid copy number regulator pcnB has been disrupted to allow expression levels of protein targets to be immediately lowered. Using the E. coli lite strains, we express a variety of proteins including green fluorescent protein (GFP), human growth hormone (hGH) and three toxic membrane proteins (BamAENm, LetBEPEC and YiaM). We show that the E. coli lite strains are able to substantially lower the expression of target proteins and alleviate the cellular toxicity associated with producing membrane-localised proteins. Additionally, we show that the E. coli lite strains are extremely versatile, functioning with a variety of RPP systems, including lac-, T7 RNA polymerase- and paraBAD-based systems, and operate under different expression and growth regimes. Thus, the E. coli lite strains can be used to quickly lower expression levels of complicated target proteins to improve both protein production and cell growth.
Rare earth elements (REE) are increasingly mobilised from mine residues and other industrial sources, creating a need for rapid screening of chemically complex waters where conventional ICP-based analysis is impractical on site. Here, we evaluate a lanthanide-gated enzymatic switch biosensor, for matrix-aware monitoring of mine-influenced waters. We quantified REE-dependent activation and systematic interference from co-occurring metals and anions typical of mine waters, and defined operating windows that preserve lanthanide responsiveness. REE activation was readily detectable at ~100 nM, whereas inhibition emerged in the low-micromolar range and became pronounced near ~10 µM for key interferents, corresponding to a practical tolerance of roughly 1:100 (REE:inhibiting element). Inhibition was strongest for AlIII, FeIII, GaIII, ZnII, and SnII. Combined machine learning, molecular dynamics, and density functional theory analyses indicate distinct inhibition modes, including coordination of FeIII, ZnII, and SnII at the LanM EF4 hand loop and favourable binding of [Al(OH)4]-/[Ga(OH)4]- near the TEM-1 substrate-recognition region. Consistent with these predictions, citrate reduced inhibition from hydrolysing metals, especially AlIII and SnII. Together, these findings define matrix-dependent operating envelopes, mechanistic design rules, and practical mitigation strategies for deploying REE biosensors in chemically complex waters.
