Biological degradation of PET plastic holds great potential for plastic recycling. However, the high costs associated with preparing free enzymes for degrading PET make it unfeasible for industrial applications. Hence, we developed various cell catalysts by surface-displaying PETase mutants and MHETase using autotransporters in E. coli and P. putida. The efficiency of surface display was enhanced through modifying the host, co-expressing molecular chaperones, and evoluting the autotransporter. In strain EC9F, PET degradation rate was boosted to 3.85 mM/d, 51-fold and 23-fold increase compared to free enzyme and initial strain ED1, respectively. The reusability of cell catalyst EC9F was demonstrated with over 38 % and 30 % of its initial activity retained after 22 cycles of BHET degradation and 3 cycles of PET degradation. The highest reported PET degradation rate of 4.95 mM/d was achieved by the dual-enzyme cascade catalytic system EC9F+EM2+R, a mixture of cell catalyst EC9F and EM2 with surfactant rhamnolipid.

Cadmium (Cd) is one of the common heavy metal pollutants in soil, which can induce various diseases and pose a serious threat to human health. Metallothioneins (MTs) are well-known for their excellent metal binding ability due to a high content of cysteine, which has great potential for heavy metal chelation. In this study, we used the Escherichia coli (E. coli) surface display system LPP-OmpA to construct a recombinant plasmid pBSD-LCF encoding LPP-OmpA-CUP1-Flag fusion protein. Then we displayed the metallothionein CUP1 from Saccharomyces cerevisiae on E. coli DH5α surface for Cd removing. The feasibility of surface display of metallothionein CUP1 in recombinant E. coli DH5α (pBSD-LCF) by Lpp-OmpA system was proved by flow cytometry and western blot analysis, and the specificity of the fusion protein in the recombinant strain was also verified. The results showed that the Cd2+ resistance capacity of DH5α (pBSD-LCF) was highly enhanced by about 200%. Fourier-transform infrared spectroscopy showed that sulfhydryl and sulfonyl groups were involved in Cd2+ binding to cell surface of DH5α (pBSD-LCF). Meanwhile, Cd removal rate by DH5α (pBSD-LCF) was promoted to 95.2%. Thus, the recombinant strain E. coli DH5α (pBSD-LCF) can effectively chelate environmental metals, and the cell surface expression of metallothionein on E. coli can provide new ideas and directions for heavy metals remediation.

Although many efforts have been devoted to the modification of polyethylene terephthalate (PET) hydrolases for improving the efficiency of PET degradation, the catalytic performance of these enzymes at near-ambient temperatures remains a challenge. Herein, a multi-enzyme cascade system (PT-EC) was developed and validated by assembling three well-developed PETases, PETaseEHA, Fast-PETase, and Z1-PETase, respectively, together with carboxylesterase TfCa, and hydrophobic binding module CBM3a using scaffold proteins. The resulting PT-ECEHA, PT-ECFPE, PT-ECZPE all demonstrated outstanding PET degradation efficacy. Notably, PT-ECEHA exhibited a 16.5-fold increase in product release compared to PETaseEHA, and PT-ECZPE yielded the highest amount of product. Subsequently, PT-ECs were displayed on the surface of Escherichia coli, respectively, and their degradation efficiency toward three PET types was investigated. The displayed PT-ECEHA exhibited a 20-fold increase in degradation efficiency with PET film compared to the surface-displayed PETaseEHA. Remarkably, an almost linear increase in product release was observed for the displayed PT-ECZPE over a one-week degradation period, reaching 11.56 ± 0.64 mM after 7 days. TfCaI69W/L281Y evolved using a docking-based virtual screening strategy showed a further 2.5-fold increase in the product release of PET degradation. Collectively, these advantages of PT-EC demonstrated the potential of a multi-enzyme cascade system for PET bio-cycling.

The biology-material hybrid method for chemical-electricity conversion via microbial fuel cells (MFCs) has garnered significant attention in addressing global energy and environmental challenges. However, the efficiency of these systems remains unsatisfactory due to the complex manufacturing process and limited biocompatibility. To overcome these challenges, here, we developed a simple bio-inorganic hybrid system for bioelectricity generation in Shewanella oneidensis (S. oneidensis) MR-1. A biocompatible surface display approach was designed, and silver-binding peptide AgBP2 was expressed on the cell surface. Notably, the engineered Shewanella showed a higher electrochemical sensitivity to Ag+, and a 60 % increase in power density was achieved even at a low concentration of 10 μM Ag+. Further analysis revealed significant upregulations of cell surface negative charge intensity, ATP metabolism, and reducing equivalent (NADH/NAD+) ratio in the engineered S. oneidensis-Ag nanoparticles biohybrid. This work not only provides a novel insight for electrochemical biosensors to detect metal ions, but also offers an alternative biocompatible surface display approach by combining compatible biomaterials with electricity-converting bacteria for advancements in biohybrid MFCs.

Heavy metal resistance mechanisms and heavy metal response genes are crucial for microbial utilization in heavy metal remediation. Here, Corynebacterium crenatum was proven to possess good tolerance in resistance to copper. Then, the transcriptomic responses to copper stress were investigated, and the vital pathways and genes involved in copper resistance of C. crenatum were determined. Based on transcriptome analysis results, a total of nine significantly upregulated DEGs related to metal ion transport were selected for further study. Among them, GY20_RS0100790 and GY20_RS0110535 belong to transcription factors, and GY20_RS0110270, GY20_RS0100790, and GY20_RS0110545 belong to copper-binding peptides. The two transcription factors were studied for the function of regulatory gene expression. The three copper-binding peptides were displayed on the C. crenatum surface for a copper adsorption test. Furthermore, the nine related metal ion transport genes were deleted to investigate the effect on growth in copper stress. This investigation provided the basis for utilizing C. crenatum in copper bioremediation.

BACKGROUND: The accumulation of fast-growing polyethylene terephthalate (PET) wastes has posed numerous threats to the environments and human health. Enzymatic degradation of PET is a promising approach for PET waste treatment. Currently, the efficiency of various PET biodegradation systems requires further improvements.
RESULTS: In this work, we engineered whole cell systems with co-display of strong adhesive proteins and the most active PETase for PET biodegradation in E. coli cells. Adhesive proteins of cp52k and mfp-3 and Fast-PETase were simultaneously displayed on the surfaces of E. coli cells, and the resulting cells displaying mfp-3 showed 50% increase of adhesion ability compared to those without adhesive proteins. Consequently, the degradation rate of E. coli cells co-displaying mfp-3 and Fast-PETase for amorphous PET exceeded 15% within 24 h, exhibiting fast and thorough PET degradation.
CONCLUSIONS: Through the engineering of co-display systems in E. coli cells, PET degradation efficiency was significantly increased compared to E. coli cells with sole display of Fast-PETase and free enzyme. This feasible E. coli co-display system could be served as a convenient tool for extending the treatment options for PET biodegradation.

Bacterial cis-epoxysuccinic acid hydrolases (CESHs) are intracellular enzymes used in the industrial production of enantiomeric tartaric acids. The enzymes are mainly used as whole-cell catalysts because of the low stability of purified CESHs. However, the low cell permeability is the major drawback of the whole-cell catalyst. To overcome this problem, we developed whole-cell catalysts using various surface display systems for CESH[L] which produces L(+)-tartaric acid. Considering that the display efficiency depends on both the carrier and the passenger, we screened five different anchoring motifs in Escherichia coli. Display efficiencies are significantly different among these five systems and the InaPbN-CESH[L] system has the highest whole-cell enzymatic activity. Conditions for InaPbN-CESH[L] production were optimized and a maturation step was discovered which can increase the whole-cell activity several times. After optimization, the total activity of the InaPbN-CESH[L] surface display system is higher than the total lysate activity of an intracellular CESH[L] overexpression system, indicating a very high CESH[L] display level. Furthermore, the whole-cell InaPbN-CESH[L] biocatalyst exhibited good storage stability at 4 °C and considerable reusability. Thereby, an efficient whole-cell CESH[L] biocatalyst was developed in this study, which solves the cell permeability problem and provides a valuable system for industrial L(+)-tartaric acid production.

Biofuel production from lignocellulose feedstocks is sustainable and environmentally friendly. However, the lignocellulosic pretreatment could produce fermentation inhibitors causing multiple stresses and low yield. Therefore, the engineering construction of highly resistant microorganisms is greatly significant. In this study, a composite functional chimeric cellulosome equipped with laccase, versatile peroxidase, and lytic polysaccharide monooxygenase was riveted on the surface of Saccharomyces cerevisiae to construct a novel yeast strain YI/LVP for synergistic lignin degradation and cellulosic ethanol production. The assembly of cellulosome was assayed by immunofluorescence microscopy and flow cytometry. During the whole process of fermentation, the maximum ethanol concentration and cellulose conversion of engineering strain YI/LVP reached 8.68 g/L and 83.41%, respectively. The results proved the availability of artificial chimeric cellulosome containing lignin-degradation enzymes for cellulosic ethanol production. The purpose of the study was to improve the inhibitor tolerance and fermentation performance of S. cerevisiae through the construction and optimization of a synergistic lignin-degrading enzyme system based on cellulosome.

Baculovirus has been widely used for foreign protein expression in biomedical studies, and budded virus (BV) surface display has developed into an important research tool for heterogenous membrane protein studies. The basic strategy of surface display is to construct a recombinant virus where the target gene is fused with a complete or partial gp64 gene. In this study, we further investigate and develop this BV surface displaying strategy. We constructed stable insect cell lines to express the target protein flanking with different regions of signal peptide (SP) and GP64 transmembrane domain (TMD). Subsequently, recombinant BmNPV was used to infect the cell, and the integration of heterogeneous protein into BV was detected. The results indicated that deletion of the n-region of SP (SPΔn) decreased the incorporation rate more than that of the full-length SP. However, the incorporation rate of the protein fused with h and c-region deletion of SP (SPΔh-c) was significantly enhanced by 35-40 times compare to full-length SP. Moreover, the foreign protein without SP and TMD failed to display on the BV, while the integration of foreign proteins with GP64 TMD fusion at the c-terminal was significantly enhanced by 12-26 times compared to the control. Thus, these new strategies developed the BV surface display system further.

Lignin peroxidase (LiP) has a good application prospect in lignin degradation, environmental treatment, straw feed, and other industries. However, its application is constrained by the high price and low stability of enzyme preparation. In this study, the Escherichia coli-Bacillus subtilis (E. coli-B. subtilis) shuttle expression vector pHS-cotG-lip was constructed and displayed on the surface of Bacillus subtilis spores. The analysis of enzymatic properties showed that the optimal catalytic temperature and pH of the immobilized LiP were 55 °C and 4.5, respectively. Compared with free LiP (42 °C and pH4.0), the optimal reaction temperature increased by 13 °C. After incubation at 70 °C for 1 h, its activity remained above 30%, while the free LiP completely lost its activity under the same conditions. Adding Mn2+, DL-lactic acid, and PEG-4000 increased the CotG-LiP enzyme activity to 313%, 146%, and 265%, respectively. The recyclability of spore display made the fusion protein CotG-LiP retain more than 50% enzyme activity after four cycles. The excellent recycling rate indicated that LiP displayed on the spore surface had a good application prospect in sewage treatment and other fields, and also provided a reference for the rapid and low-cost immobilized production of enzyme preparations.