Rational redesign of thermophilic PET hydrolase LCCICCG to enhance hydrolysis of high crystallinity polyethylene terephthalates.

Polyethylene terephthalate (PET)-degrading enzymes represent a promising solution to the plastic pollution. However, PET-degrading enzymes, even thermophilic PETase, can effectively degrade low-crystallinity (∼8%) PETs, but exhibit weak depolymerization of more common, high-crystallinity (30-50%) PETs. Here, based on the thermophilic PETase, LCCICCG, we proposed two strategies for rational redesign of LCCICCG using the machine learning tool, Preoptem, combined with evolutionary analysis. Six single-point mutants (S32L, D18T, S98R, T157P, E173Q, N213P) were obtained that exhibit higher catalytic efficiency towards PET powder than wild-type LCCICCG at 75 °C. Additionally, the optimal temperature for degrading 39.07% crystalline PET increased from 65 °C in the wild-type LCCICCG to between 75 and 80 °C in the LCCICCG_I6M mutant that carries all six single-point mutations. Especially, the LCCICCG_I6M mutant has a significantly higher degradation effect on some commonly used bottle-grade plastic powders at 75-80 °C than that of wild type. The enzymatic digestion of ground 31.30% crystalline PET water bottles by LCCICCG_I6M yielded 31.91 ± 0.99 mM soluble products in 24 h, which was 3.64 times that of LCCICCG (8.77 ± 1.52 mM). Overall, this study provides a feasible route for engineering thermostable enzymes that can degrade high-crystallinity PET plastic.

Cadmium-absorptive Bacillus vietnamensis 151-6 reduces the grain cadmium accumulation in rice (Oryza sativa L.): Potential for cadmium bioremediation.

Microbial bioremediation of heavy metal-polluted soil is a promising technique for reducing heavy metal accumulation in crops. In a previous study, we isolated Bacillus vietnamensis strain 151-6 with a high cadmium (Cd) accumulation ability and low Cd resistance. However, the key gene responsible for the Cd absorption and bioremediation potential of this strain remains unclear. In this study, genes related to Cd absorption in B. vietnamensis 151-6 were overexpressed. A thiol-disulfide oxidoreductase gene (orf4108) and a cytochrome C biogenesis protein gene (orf4109) were found to play major roles in Cd absorption. In addition, the plant growth-promoting (PGP) traits of the strain were detected, which enabled phosphorus and potassium solubilization and indole-3-acetic acid (IAA) production. Bacillus vietnamensis 151-6 was used for the bioremediation of Cd-polluted paddy soil, and its effects on growth and Cd accumulation in rice were explored. The strain increased the panicle number (114.82%) and decreased the Cd content in rice rachises (23.87%) and grains (52.05%) under Cd stress, compared with non-inoculated rice in pot experiments. For field trials, compared with the non-inoculated control, the Cd content of grains inoculated with B. vietnamensis 151-6 was effectively decreased in two cultivars (low Cd-accumulating cultivar: 24.77%; high Cd-accumulating cultivar: 48.85%) of late rice. Bacillus vietnamensis 151-6 encoded key genes that confer the ability to bind Cd and reduce Cd stress in rice. Thus, B. vietnamensis 151-6 exhibits great application potential for Cd bioremediation.

MPEPE, a predictive approach to improve protein expression in E. coli based on deep learning.

The expression of proteins in Escherichia coli is often essential for their characterization, modification, and subsequent application. Gene sequence is the major factor contributing expression. In this study, we used the expression data from 6438 heterologous proteins under the same expression condition in E. coli to construct a deep learning classifier for screening high- and low-expression proteins. In conjunction with conserved residue analysis to minimize functional disruption, a mutation predictor for enhanced protein expression (MPEPE) was proposed to identify mutations conducive to protein expression. MPEPE identified mutation sites in laccase 13B22 and the glucose dehydrogenase FAD-AtGDH, that significantly increased both expression levels and activity of these proteins. Additionally, a significant correlation of 0.46 between the predicted high level expression propensity with the constructed models and the protein abundance of endogenous genes in E. coli was also been detected. Therefore, the study provides foundational insights into the relationship between specific amino acid usage, codon usage, and protein expression, and is essential for research and industrial applications.

An operon consisting of a P-type ATPase gene and a transcriptional regulator gene responsible for cadmium resistances in Bacillus vietamensis 151-6 and Bacillus marisflavi 151-25.

BACKGROUND: Cadmium (Cd) is a severely toxic heavy metal to most microorganisms. Many bacteria have developed Cd2+ resistance. RESULTS: In this study, we isolated two different Cd2+ resistance Bacillus sp. strains, Bacillus vietamensis 151-6 and Bacillus marisflavi 151-25, which could be grown in the presence of Cd2+ at concentration up to 0.3 mM and 0.8 mM, respectively. According to the genomic sequencing, transcriptome analysis under cadmium stress, and other related experiments, a gene cluster in plasmid p25 was found to be a major contributor to Cd2+ resistance in B. marisflavi 151-25. The cluster in p25 contained orf4802 and orf4803 which encodes an ATPase transporter and a transcriptional regulator protein, respectively. Although 151-6 has much lower Cd2+ resistance than 151-25, they contained similar gene cluster, but in different locations. A gene cluster on the chromosome containing orf4111, orf4112 and orf4113, which encodes an ATPase transporter, a cadmium efflux system accessory protein and a cadmium resistance protein, respectively, was found to play a major role on the Cd2+ resistance for B. vietamensis 151-6. CONCLUSIONS: This work described cadmium resistance mechanisms in newly isolated Bacillus vietamensis 151-6 and Bacillus marisflavi 151-25. Based on homologies to the cad system (CadA-CadC) in Staphylococcus aureus and analysis of transcriptome under Cd2+ induction, we inferred that the mechanisms of cadmium resistance in B. marisflavi 151-25 was as same as the cad system in S. aureus. Although Bacillus vietamensis 151-6 also had the similar gene cluster to B. marisflavi 151-25 and S. aureus, its transcriptional regulatory mechanism of cadmium resistance was not same. This study explored the cadmium resistance mechanism for B. vietamensis 151-6 and B. marisflavi 151-25 and has expanded our understanding of the biological effects of cadmium.

Identification of the anchoring protein SpoIIIJ for construction of the microbial cell surface display system in Bacillus spp.

Microbial cell surface display technology is a powerful tool for displaying proteins on the surfaces of cells. However, few anchoring proteins can be employed for the display of target proteins on the cell surface of the environmentally benign Gram-positive bacterium Bacillus subtilis. In this study, bioinformatics tools were used to screen all of the encoded proteins of B. subtilis for potential anchoring proteins. A green fluorescent protein (eGFP) reporter system was constructed to evaluate the cell-display efficiency of the selected membrane proteins and their promoters. The anchoring protein SpoIIIJ demonstrated the strongest anchoring activity of all of the selected anchoring proteins from Bacillus spp. cells. A linker was designed to link the anchoring protein SpoIIIJ and eGFP, which had the ability to increase the expression of the fusion protein by 58.32%. Two bio-remediated related proteins (the organophosphorus hydrolase OPHC2 and the metal binding protein CadR) were successfully expressed on the cell surfaces of Bacillus spp. using this system. Therefore, our results suggest that this microbial surface display system may be useful for the expression of target proteins on the cell surfaces and has potential applications in the bioremediation of environmental pollution.

Identification of cadmium-binding proteins from rice (Oryza sativa L.).

Metal-binding proteins play an important role in maintaining intracellular metal homeostasis and eliminating heavy metal toxification. Many metallothioneins (MTs) have been isolated from mammalian sources, which are a family of low molecular weight metal-binding proteins that are rich in cysteine. However, plants contain a different type of cadmium-binding protein that contain fewer cysteine residues. In this study, cadmium affinity chromatography coupled with laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) has been used to separate and identify cadmium-binding proteins from different parts (root, stem, leaf and grain) of rice (Oryza sativa L.) cultivated under cadmium stress conditions. Seven cadmium-binding proteins with low isoelectric points containing relatively few cysteine residues were chosen for expression in Escherichia coli. The cadmium removal efficiency of protein A3AGZ4 (OsJ_10480) from Escherichia coli △zntA-BL21 was the highest (57.35%), which compares favorably with the cadmium removal efficiency of metallothionein MT (48.99%, mt from mouse,) and SMT (55.84%, smt from Sinopotamon honanense). In addition, for the strain A3AGZ4-△zntA-BL21, most of the bound cadmium was found to accumulate in the cytoplasm and not the cell wall. These results indicate that these plant proteins can bind cadmium to reduce heavy metal toxicity, thus contributing towards bioremediation of cadmium in the environment.