Synthetic microalgal-bacterial symbiotic system integrating oxidase-protease fusion enzyme for simultaneous removal of recalcitrant nitrogen-containing organic compounds and PET microplastics.
Aug 2026· Bioresource Technology· Vol 463, pp.
135685
· 0 citations· 30 references
Medicine
Abstract
Industrial wastewaters-particularly those from the printing and dyeing sector-contain complex mixtures of recalcitrant nitrogen-containing organic compounds and microplastics that resist conventional treatment. Here we report a previously undescribed oxidase-protease fusion enzyme (A20674), discovered through transcriptomic analysis of wastewater-acclimated Chlorella vulgaris. Domain dissection reveals that the oxidase-like region drives broad-spectrum removal of N-heterocyclic and aromatic compounds, while the protease-like domain removes organic nitrogen. Engineered overexpression of this enzyme boosted organic nitrogen removal up to fifty-five-fold (final concentration 13-100 mg/L) across different industrial wastewaters. Capitalizing on this metabolic specialization, we constructed a synthetic microalgal-bacterial consortium in which Pseudomonas putida uses microalgal extracellular polysaccharides (EPS) as a carbon source to sustain growth, while supplying indole-3-acetic acid that stimulates microalgal biomass and EPS production. Reciprocal engineering of EPS overproduction in C. vulgaris and enhanced polysaccharide-catabolism in P. putida amplified this mutualistic loop. An evolved, PETase/MHETase-expressing P. putida strain simultaneously acquired elevated IAA output, further strengthening the symbiosis. The optimized consortium reduced organic nitrogen concentrations by sixteen-fold to discharge-compliant levels (≤5 mg/L), enhanced removal of recalcitrant organic nitrogen compounds by five-fold (final concentration 34 mg/L), and improved PET microplastic removal by nine-fold (initial concentration 1 g/L and final concentration 684 mg/L) in printing and dyeing wastewater. These findings uncover a bifunctional enzyme architecture for degrading structurally diverse industrial pollutants, and establish a synthetic-ecology framework for integrated removal of dissolved nitrogen-containing organic compounds and particulate microplastics-a combination unattainable by any single organism or conventional treatment process.
Conventional biological treatment of ester-type volatile organic compounds (VOCs) is challenged by low mineralization rates or uncontrolled CO2 emissions when high mineralization occurs. In this study, an algal-bacterial symbiosis (ABS) system with a high carbon fixation rate and bidirectional interaction was successfully established in a phototrophic-biotrickling filter (PBTF), enabling low-carbon treatment of VOCs. Metabolic reconstruction based on metagenomic and metatranscriptomic analyses suggested that endogenous indole-3-acetic acid (IAA) may contribute to synergistic interactions between microalgae and bacteria. The concentration of endogenous IAA reached 446.16 ± 40.03 mg/m3 (packing material), with the expression of IAA-related genes exhibiting a 3.23-fold increase; this resulted in highly active cross-kingdom signal transduction between microalgae and bacteria. Consequently, the biosynthesis of extracellular polymeric substances (EPSs) increased to 973.52 ± 277.36 mg/m3 (packing material), leading to the formation of a dense EPS-derived physical barrier that protected ABS from n-butyl acetate-induced toxicity. Additionally, the presence of microalgae increased the abundance of phototrophic bacteria (e.g., Leptolyngbya) and the expression of associated carbon-related genes (rbcL/S) by 2.54-fold. As a result, the synergistic ABS mode ensured efficient n-butyl acetate removal (99.43 ± 0.50%) and CO2 fixation (705.33 ± 17.70 g/(m3 packing material·d), thus maintaining the stability of the PBTF. Overall, the dual-enhancement system of ABS in a PBTF presents an effective low-carbon VOC removal strategy, paving the way for broader, stable applications of this system in the treatment of ester-type VOCs.
W. Wang, Yuhan Xiao, Ziyi Guo et al.· ACS Sustainable Chemistry &a...· 0 citations
Muconic acid is an industrially valuable molecule that can be biologically produced from diverse biogenic and waste-derived feedstocks, including sugars and lignin- and plastic-derived aromatic compounds. However, accumulation of protocatechuate (PCA) has been observed in multiple microbes engineered for muconate production when the PCA decarboxylase, AroY, is used. This raises the question of whether PCA decarboxylation represents a rate-limiting step and how this bottleneck might be alleviated, especially given the toxicity and reactivity of PCA and catechol intermediates. To address this, we performed adaptive laboratory evolution (ALE) on a strain of Pseudomonas putida originally engineered for muconate production from aromatic compounds, but with catBC restored, to select for improved conversion of PCA and, in separate lineages, 4-hydroxybenzoate. Contrary to our expectations, the predominant beneficial mutations localized to the catA1 cassette encoding catechol 1,2-dioxygenase, rather than aroY or its associated cofactor biosynthesis genes. Transcriptomic analysis revealed elevated catA1 expression in evolved isolates from ALE, and introduction of these mutations improved productivity in strains designed for muconate production from both aromatic and sugar substrates. Quantitative proteomics and biochemical assays demonstrated that the mutations also led to increased CatA1 protein abundance and modest enhancements in catalytic efficiency, respectively, with strain phenotypes largely driven by high CatA1 levels and potentially synergistic kinetic improvements. Additional reverse-engineering studies identified variants with modest effects on muconate accumulation, including those with potential to enhance biosynthesis of the prenylated FMN cofactor of AroY. Collectively, these results indicate that catechol, not PCA, is the principal bottleneck in muconate production via the PCA decarboxylation route originally demonstrated by Draths et al., refining our understanding of pathway limitations and offering new strategies for improving rate, yield, and strain resilience in muconate bioproduction. Highlights Accumulation of metabolic intermediates was alleviated by adaptive laboratory evolution Sequencing, proteomics, and enzyme kinetics revealed mechanisms for adaptation Increased CatA1 expression reduced bottlenecks and improved muconate production
Alissa C. Bleem, Tracy L. Hodges, Torrey M Lind et al.· bioRxiv· 2 citations
Cyanophycin granule polypeptide (CGP), also known as multi-L-arginyl-poly(aspartic acid), is a biodegradable biopolymer composed primarily of aspartic acid and arginine. Due to its versatile functional properties, CGP has attracted increasing interest for potential applications in food, medicine, cosmetics, agriculture, and corrosion inhibition. The objective of this study was to biosynthesize a biologically derived corrosion-inhibiting biomaterial using recombinant Escherichia coli BL21(DE3) expressing cyanophycin synthetase (CphA), followed by cost-effective induction strategy. Specifically, this work aimed to establish a high-cell-density cultivation process capable of achieving improved CGP production while reducing dependence on costly inducers such as IPTG. Initial shake-flask experiments demonstrated that lactose induction resulted in higher CGP production and biomass formation compared to IPTG induction. In addition, supplementation with phosphate, ribose, trace elements, yeast extract, and tryptone further improved CGP accumulation. Based on these findings, a high-cell-density fed-batch fermentation strategy using lactose as both inducer and carbon source was developed, maximum gravimetrically recovered crude soluble and insoluble CGP-containing fractions of 35.4 and 17.8 g/L. Product characterization was further supported by FTIR, XRD, MALDI-MS. Furthermore, the recovered CGP-related material was evaluated in preliminary corrosion inhibition experiments under acidic conditions and showed significant reduction in corrosion rates compared with untreated control samples, indicating its potential for future corrosion-protection applications.
S. Kafle, Anirudh Mukunth, Arum Han et al.· Bioprocess and biosystems en...· 0 citations
Muconic acid is a versatile platform chemical that can be biologically produced from lignocellulosic substrates, including from lignin-related aromatic compounds. Pseudomonas putida has been previously engineered to convert lignin-related aromatic compounds to muconate at quantitative molar yields. This high atom efficiency requires a supplemental carbon and energy source to support bacterial growth, and central carbon metabolic efficiency and its interaction with aromatic catabolism are underexplored. Here, we applied proteomics, metabolomics, and 13C-fluxomics to quantitatively compare central carbon and energy metabolism in wild-type P. putida KT2440 and a muconate-producing strain, P. putida CJ781. During cultivation on glucose and 4-hydroxybenzoate, CJ781 showed increased glucose uptake, reconfigured central fluxes, and increased extracellular leakage of aliphatic acids relative to wild type. These altered fluxes supported a 3-fold higher ATP pool, in excess of demand. Pyruvate and acetate secretion in CJ781 was mitigated by debottlenecking TCA-cycle entry via citrate synthase overexpression. Furthermore, tuned expression of the catechol dioxygenase and protocatechuate decarboxylase enabled the production of 36.3 g L-1 muconate at 1.1 g L-1 h-1. Overall, this work reveals how P. putida redirects carbon and energy fluxes to support aromatic bioconversion for improved bioproduction from renewable feedstocks.
R. Wilkes, P. Suthers, A. Borchert et al.· bioRxiv· 3 citations
Laccases are versatile oxidoreductases found in bacteria, fungi, and plants., which have a wide range of industrial applications, including the removal of pharmaceutical micropollutants from wastewater. Bacterial laccases are particularly attractive because of their high catalytic activity and stability over broad temperature and pH ranges. One example is the copper efflux oxidase (CueO) from
Escherichia coli
, which has been fused to the adhesion-promoting peptide Macaque Histatin (MacHis) because immobilized enzymes have proven especially effective for solid-phase wastewater treatment. However, the practical application of this enzyme is constrained by the relatively low production yields of 25–50 mg/L when expressed in recombinant
E. coli
or
Pichia pastoris
.
Here, we used transient expression in Nicotiana benthamiana to demonstrate that the engineered CueO-MacHis can be produced at substantially higher levels, reaching 250–320 (286 ± 35) mg/kg biomass in the prokaryotic-like environment of chloroplasts in 6-week-old plants, which are typically used for production purposes. Subsequent systematic optimization of the transient expression process based on statistical experimental designs increased the accumulation of recombinant CueO-MacHis approximately threefold to 846 ± 57 mg/kg biomass. Furthermore, we established a simplified and cost-effective purification strategy that leverages the enzyme’s temperature and pH stability to clarify extracts before anion exchange chromatography, achieving 76% recovery and 92% purity. Moreover, the plant-derived CueO-MacHis had a specific activity of 7.22 ± 1.55 U/mg, which is in the same range as that reported for other CueO enzymes.
Our approach paves the way for the large-scale production of CueO-MacHis as a technical enzyme for industrial applications.
Henrik Nausch, C. Bernau, Dirk Scheffler et al.· Frontiers in Plant Science· 0 citations
The economic viability of methanol-based biomanufacturing, particularly with green methanol as feedstock, is often limited by the low value of single-product processes. Here, we developed an integrated co-production strategy in the methylotrophic yeast Pichia pastoris (Komagataella phaffii) for the simultaneous conversion of methanol into three products: the sweetener erythritol, the industrial biocatalyst β-mannanase, and single-cell protein (SCP) biomass. This new strategy explores the inherent spatial and functional separation between the ER-Golgi secretory pathway for enzyme production and the cytosolic pathway for chemical synthesis, thereby reducing interference between the two pathways. The engineered co-production strain achieved β-mannanase and erythritol titers comparable to those of the corresponding β-mannanase- and erythritol-producing reference strains in both shake-flask and fed-batch fermentor cultures. Furthermore, transcriptomic analysis revealed distinct regulatory responses related to enzyme production and erythritol synthesis, supporting the limited cross-pathway interference between the two pathways. In addition, we showed that ultrafiltration enabled efficient downstream separation of the small-molecule erythritol from the secreted β-mannanase, with recovery efficiencies exceeding 89%. These results demonstrate a feasible strategy for methanol valorization into multiple value-added products and expand the potential of methylotrophic yeasts for integrated biomanufacturing.
Jiayu Fang, Shuxian Wang, Guoxia Liu et al.· Bioresources and Bioprocessi...· 0 citations
