Journal of biotechnology最新文献

Two biosensors with enzymes immobilized on fibrous cellulose powder and paper have been developed for the colorimetric detection of biogenic amines, which are of great interest for food and health security. These systems exploited diamine oxidase (DAO) and soybean peroxidase (SBP) catalytic activities. DAO oxidizes the amines, producing ammonia and hydrogen peroxide; the latter activates SBP, catalyzing the oxidation of the co-substrates with the formation of a purple product, easily detectable by UV-visible spectrometry. For most of the examined amines, the quantification resulted in being fast and effective even at low concentrations, reaching calculated Limits Of Detection (LOD) between 10^-6-10^-8 M. However, proper methodological adjustments were needed for catecholamines, implying the exclusive use of SBP-based materials. Biosensors' characterizations showed the rise of peculiar phenomena during their preparation: paper underwent hydrophobization and shrinkage after the oxidation step, while DAO caused unexpected fiber thinning in paper and fibers' coalescence of powdery cellulose. Moreover, infrared spectroscopy and calorimetry demonstrated modifications in hydrogen networks after cellulose and paper treatments. Overall, these changes did not affect the potential of biosensors, which are sensitive, portable, and biodegradable, although there is room for improvement in lowering catecholamine LOD and broadening their promising applicability by testing real matrices.

Endo-inulinases are key biocatalysts for converting inulin into prebiotic inulooligosaccharides (IOS). However, industrial implementation is hindered by a mismatch between inulin solubility and enzyme operating temperatures. Natural inulin dissolves well at high temperatures (≥70 °C) but poorly at the moderate temperatures (30-50 °C) typical for most endo-inulinases. Therefore, identifying thermophilic endo-inulinases is crucial. Herein, a novel thermophilic endo-inulinase InuG with exceptional thermostability was identified. Our strategy used a known endo-inulinase as a molecular probe to source homologs, prioritizing candidates from their source microorganisms with high optimal growth temperatures. Recombinant InuG was gene-synthesized, expressed in Escherichia coli and purified. InuG-His₆ hydrolyzed inulin, yielding inulotriose (F₃) as the dominant product. The enzyme exhibited superior properties to all reported endo-inulinases, with an optimal temperature of 70 °C and an exceptional half-life of 82 h at 70 °C. Molecular-dynamics simulations revealed that tight hydrophobic packing, reduced flexible segments, and rigid loop regions collectively underpin this extreme thermostability. Furthermore, we elucidated the functional role of the conserved bacterial domain LamG and the structural mechanism conferring InuG's product preference for F₃. These properties position InuG as a robust biocatalyst for IOS production at high-temperature, offering immediate advantages for industrial food and feed applications.

The spatial organization of multi-enzyme cascades offers a powerful strategy to overcome diffusion limitations and cofactor regeneration bottlenecks in industrial biocatalysis. In this study, a cellulosome-inspired complex was engineered to facilitate the efficient, self-sufficient oxidation of isopropanol (IPA). By exploiting high-affinity cohesin-dockerin interactions, a dockerin-fused secondary alcohol dehydrogenase (S-ADH_doc) was co-immobilized with a dockerin-fused alkyl hydroperoxide reductase (AhpF_doc), serving as a robust NADH regeneration partner. Kinetic characterization of the individual recombinant modules revealed that S-ADH_doc exhibited a Vmax of 5.59 ± 0.80 μmol·minute^-1 and a turnover number (kcat) of 46.6 s^-1. Crucially, the regeneration module, AhpF_doc, displayed superior kinetics with a Vmax of 27.0 ± 0.60 μmol·minute^-1 and a kcat of 225 s^-1, ensuring that cofactor recycling was not rate-limiting. Upon assembly, the complex demonstrated synergistic catalytic efficiency driven by proximity-induced substrate channeling and rapid electron transfer and thereby improving the overall enzyme kinetics (Vmax of 1834.8 μmol·minute^-1). The engineered system exhibited high selectivity for IPA against structurally related alcohols and demonstrated remarkable operational stability, retaining over 85% of its initial activity after 15 days with high reproducibility (RSD = 3.87%). These findings underscore the potential of artificial scaffoldin-based architectures as versatile, scalable platforms for constructing stable enzymatic modules for use in industrial bioprocess monitoring and sustainable chemical synthesis.

Strictosidinic acid is a key intermediate in the biosynthetic pathway of camptothecin, a plant alkaloid that serves as a precursor for semisynthetic anticancer drugs. At the moment, camptothecin is mainly sourced from trees, causing limited supply and high costs. Improving access to strictosidinic acid would help to elucidate yet unknown biosynthetic steps downstream of this intermediate and in the long term enable sustainable production of camptothecin in heterologous hosts. While structurally similar to the common monoterpene indole alkaloid precursor strictosidine, strictosidinic acid has not been the target of metabolic engineering efforts before. Here, we present a strategy to produce strictosidinic acid from glucose and tryptophan in engineered yeast. First, we create a basic strain that generates 75 mg/L strictosidine. We further optimise this strain by introducing a membrane steroid binding protein and a second copy of the farnesyl pyrophosphate synthase mutant gene ERG20^WW, boosting strictosidine levels by 5.5-fold to 398 mg/L. At these higher titres, a previously overlooked shunt product, (2E,6E)-2,6-dimethylocta-2,6-dienedioic acid (DOA), was identified that diverts flux from the pathway. Lastly, we reprogrammed our strictosidine strain to strictosidinic acid production by four genomic modifications. Final fed-batch cultivation in shake flasks resulted in 843 mg/L strictosidine or 548 mg/L strictosidinic acid, respectively, after 168 h. Taken together, our work now grants access to strictosidinic acid by metabolic engineering, while revealing strategies to further enhance the production of strictosidine and related monoterpene indole alkaloids. These findings will help to produce plant alkaloids in microbial cell factories in the future at scale.

Terephthalic acid (TPA) is an aromatic dicarboxylic acid that is widely used as a monomer in the production of polyesters such as polyethylene terephthalate (PET) and poly (butylene adipate-co-terephthalate) (PBAT). However, its chemical stability causes TPA to persistently accumulate in the natural environment after plastic degradation and cause secondary pollution. Therefore, we engineered a Pseudomonas putida KT2440 strain capable of utilizing TPA as a substrate to produce levulinic acid (LA), a key intermediate in the synthesis of polymers, pharmaceuticals, solvents, plasticizers, and biofuels. Although P. putida KT2440 possesses robust central metabolic pathways, it lacks the enzymatic steps required for LA biosynthesis. To establish an LA producing pathway from TPA, phospho-2-dehydro-3-deoxyheptonate aldolase (AroG), 3-dehydroshikimate dehydratase (AsbF), and acetoacetate decarboxylase (Adc) were integrated into the pcaIJ locus and introduced tpaK, tphA, and tphB into a pcaIJ and lvaR knocked out P. putida KT2440 (GP102). The engineered P. putida KT2440 GP102 strain used TPA as the sole carbon source and produced 4.9 mM of LA from 6 mM of TPA after optimization. Under continuous TPA feeding, a maximum of 8.75 mM LA was produced, however, substrate accumulation inhibited cell growth and reduced LA productivity, resulting in 7.44 mM LA at 96 h. To address this limitation, glycerol was supplemented with TPA as a co-substrate, which enhanced bacterial growth and TPA utilization and yielded 25.40 mM of LA at 96 h. This study is the first to report biological conversion of TPA to LA in P. putida KT2440 and demonstrates the potential of TPA as a valuable platform chemical for sustainable bioprocessing.

Sendai virus (SeV)-based vectors are recognized as potential tools in gene therapy and regenerative medicine as they can express transgenes without chromosomal insertion. We previously reported that a replication-defective and persistent SeV (SeVdp) vector capable of long-term and multiple transgene expression can promote reprogramming of somatic cells into induced pluripotent stem (iPS) cells. Importantly, siRNA- and miRNA-mediated suppression of the SeV RNA-dependent RNA polymerase facilitates removal of SeVdp vectors from reprogrammed cells, resulting in establishing transgene-free iPS cells. However, these approaches are considerably dependent on transfection efficiency and intracellular miRNA activity, respectively. In this study, we assessed a simple approach to eliminate SeVdp vectors from infected cells using antiviral agents. GHP-88309, an antiviral compound against a broad range of paramyxoviruses, effectively inhibited SeV replication and enabled the removal of SeVdp vectors. Notably, this compound allowed complete elimination of a BRN4-expressing SeVdp vector from neural stem cells after enforced differentiation of embryonic stem cells. Our findings suggest that GHP-88309 would be an effective agent to enhance the utility and flexibility of SeVdp vectors in various biological and medical applications including transcription factor-mediated cell differentiation.