Enhancing the endo-activity of the thermophilic chitinase to yield chitooligosaccharides with high degrees of polymerization.

Thermophilic endo-chitinases are essential for production of highly polymerized chitooligosaccharides, which are advantageous for plant immunity, animal nutrition and health. However, thermophilic endo-chitinases are scarce and the transformation from exo- to endo-activity of chitinases is still a challenging problem. In this study, to enhance the endo-activity of the thermophilic chitinase Chi304, we proposed two approaches for rational design based on comprehensive structural and evolutionary analyses. Four effective single-point mutants were identified among 28 designed mutations. The ratio of (GlcNAc)3 to (GlcNAc)2 quantity (DP3/2) in the hydrolysates of the four single-point mutants undertaking colloidal chitin degradation were 1.89, 1.65, 1.24, and 1.38 times that of Chi304, respectively. When combining to double-point mutants, the DP3/2 proportions produced by F79A/W140R, F79A/M264L, F79A/W272R, and M264L/W272R were 2.06, 1.67, 1.82, and 1.86 times that of Chi304 and all four double-point mutants exhibited enhanced endo-activity. When applied to produce chitooligosaccharides (DP ≥ 3), F79A/W140R accumulated the most (GlcNAc)4, while M264L/W272R was the best to produce (GlcNAc)3, which was 2.28 times that of Chi304. The two mutants had exposed shallower substrate-binding pockets and stronger binding abilities to shape the substrate. Overall, this research offers a practical approach to altering the cutting pattern of a chitinase to generate functional chitooligosaccharides.

Harnessing microbial interactions with rice: Strategies for abiotic stress alleviation in the face of environmental challenges and climate change.

Rice, which feeds more than half of the world's population, confronts significant challenges due to environmental and climatic changes. Abiotic stressors such as extreme temperatures, drought, heavy metals, organic pollutants, and salinity disrupt its cellular balance, impair photosynthetic efficiency, and degrade grain quality. Beneficial microorganisms from rice and soil microbiomes have emerged as crucial in enhancing rice's tolerance to these stresses. This review delves into the multifaceted impacts of these abiotic stressors on rice growth, exploring the origins of the interacting microorganisms and the intricate dynamics between rice-associated and soil microbiomes. We highlight their synergistic roles in mitigating rice's abiotic stresses and outline rice's strategies for recruiting these microorganisms under various environmental conditions, including the development of techniques to maximize their benefits. Through an in-depth analysis, we shed light on the multifarious mechanisms through which microorganisms fortify rice resilience, such as modulation of antioxidant enzymes, enhanced nutrient uptake, plant hormone adjustments, exopolysaccharide secretion, and strategic gene expression regulation, emphasizing the objective of leveraging microorganisms to boost rice's stress tolerance. The review also recognizes the growing prominence of microbial inoculants in modern rice cultivation for their eco-friendliness and sustainability. We discuss ongoing efforts to optimize these inoculants, providing insights into the rigorous processes involved in their formulation and strategic deployment. In conclusion, this review emphasizes the importance of microbial interventions in bolstering rice agriculture and ensuring its resilience in the face of rising environmental challenges.

Protein-protein interaction and site prediction using transfer learning.

The advanced language models have enabled us to recognize protein-protein interactions (PPIs) and interaction sites using protein sequences or structures. Here, we trained the MindSpore ProteinBERT (MP-BERT) model, a Bidirectional Encoder Representation from Transformers, using protein pairs as inputs, making it suitable for identifying PPIs and their respective interaction sites. The pretrained model (MP-BERT) was fine-tuned as MPB-PPI (MP-BERT on PPI) and demonstrated its superiority over the state-of-the-art models on diverse benchmark datasets for predicting PPIs. Moreover, the model's capability to recognize PPIs among various organisms was evaluated on multiple organisms. An amalgamated organism model was designed, exhibiting a high level of generalization across the majority of organisms and attaining an accuracy of 92.65%. The model was also customized to predict interaction site propensity by fine-tuning it with PPI site data as MPB-PPISP. Our method facilitates the prediction of both PPIs and their interaction sites, thereby illustrating the potency of transfer learning in dealing with the protein pair task.

MECE: a method for enhancing the catalytic efficiency of glycoside hydrolase based on deep neural networks and molecular evolution.

The demand for high efficiency glycoside hydrolases (GHs) is on the rise due to their various industrial applications. However, improving the catalytic efficiency of an enzyme remains a challenge. This investigation showcases the capability of a deep neural network and method for enhancing the catalytic efficiency (MECE) platform to predict mutations that improve catalytic activity in GHs. The MECE platform includes DeepGH, a deep learning model that is able to identify GH families and functional residues. This model was developed utilizing 119 GH family protein sequences obtained from the Carbohydrate-Active enZYmes (CAZy) database. After undergoing ten-fold cross-validation, the DeepGH models exhibited a predictive accuracy of 96.73%. The utilization of gradient-weighted class activation mapping (Grad-CAM) was used to aid us in comprehending the classification features, which in turn facilitated the creation of enzyme mutants. As a result, the MECE platform was validated with the development of CHIS1754-MUT7, a mutant that boasts seven amino acid substitutions. The kcat/Km of CHIS1754-MUT7 was found to be 23.53 times greater than that of the wild type CHIS1754. Due to its high computational efficiency and low experimental cost, this method offers significant advantages and presents a novel approach for the intelligent design of enzyme catalytic efficiency. As a result, it holds great promise for a wide range of applications.

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.

Combined assembly of long and short sequencing reads improve the efficiency of exploring the soil metagenome.

BACKGROUND: Advances in DNA sequencing technologies have transformed our capacity to perform life science research, decipher the dynamics of complex soil microbial communities and exploit them for plant disease management. However, soil is a complex conglomerate, which makes functional metagenomics studies very challenging. RESULTS: Metagenomes were assembled by long-read (PacBio, PB), short-read (Illumina, IL), and mixture of PB and IL (PI) sequencing of soil DNA samples were compared. Ortholog analyses and functional annotation revealed that the PI approach significantly increased the contig length of the metagenomic sequences compared to IL and enlarged the gene pool compared to PB. The PI approach also offered comparable or higher species abundance than either PB or IL alone, and showed significant advantages for studying natural product biosynthetic genes in the soil microbiomes. CONCLUSION: Our results provide an effective strategy for combining long and short-read DNA sequencing data to explore and distill the maximum information out of soil metagenomics.

A novel thermophilic chitinase directly mined from the marine metagenome using the deep learning tool Preoptem.

Chitin is abundant in nature and its degradation products are highly valuable for numerous applications. Thermophilic chitinases are increasingly appreciated for their capacity to biodegrade chitin at high temperatures and prolonged enzyme stability. Here, using deep learning approaches, we developed a prediction tool, Preoptem, to screen thermophilic proteins. A novel thermophilic chitinase, Chi304, was mined directly from the marine metagenome. Chi304 showed maximum activity at 85 â„ƒ, its Tm reached 89.65 ± 0.22℃, and exhibited excellent thermal stability at 80 and 90 °C. Chi304 had both endo- and exo-chitinase activities, and the (GlcNAc)2 was the main hydrolysis product of chitin-related substrates. The product yields of colloidal chitin degradation reached 97% within 80 min, and 20% over 4 days of reaction with crude chitin powder. This study thus provides a method to mine the novel thermophilic chitinase for efficient chitin biodegradation.

The endophytic bacterium Bacillus koreensis 181-22 promotes rice growth and alleviates cadmium stress under cadmium exposure.

Recently, cadmium (Cd) contamination in paddy soils has become a highly concerning pollution problem. Endophytic microbes in rice not only affect the plant growth but also contribute to ion absorption by the roots. Therefore, they are a promising, ecologically sound means of reducing the Cd transport from soils to shoots and grains of the plant. In this study, a Cd-resistant endophytic bacterium, named 181-22, with high Cd absorption capacity (90.8%) was isolated from the roots of rice planting in heavily Cd-contaminated paddy soils and was identified as Bacillus koreensis CGMCC 19,468. The strain significantly increased fresh weight of roots and shoots (44.4% and 42.7%) and dry weight of roots and shoots (71.3% and 39.9%) and decreased Cd content in the rice roots (12.8%), shoots (34.3%), and grains (39.1%) under Cd stress compared to uninoculated plant by colonizing rice roots via seed inoculation. Moreover, colonization of 181-22 reprogrammed rice physiology to alleviate Cd stress by increasing pigment and total protein content, regulating Cd-induced oxidative stress enzymes such as superoxide dismutase and catalase and reducing malondialdehyde. Thus, B. koreensis 181-22 has the potential to protect rice against Cd stress and can be used as a biofertilizer to bioremediate paddy soils contaminated with Cd. KEY POINTS: • Bacillus koreensis 181-22 colonized the inside of rice roots at high numbers via seed inoculation. • B. koreensis 181-22 promoted rice growth and decreased Cd accumulation in grains. • B. koreensis 181-22 regulated the physiological response to alleviated Cd stress in rice.

Cadmium Pollution Impact on the Bacterial Community Structure of Arable Soil and the Isolation of the Cadmium Resistant Bacteria.

Microorganisms play an important role in the remediation of cadmium pollution in the soil and their diversity can be affected by cadmium. In this study, the bacterial community in arable soil samples collected from two near geographical sites, with different degrees of cadmium pollution at three different seasons, were characterized using Illumina MiSeq sequencing. The result showed that cadmium is an important factor to affect the bacterial diversity and the microbial communities in the high cadmium polluted area (the site H) had significant differences compared with low cadmium polluted area (the site L). Especially, higher concentrations of Cd significantly increased the abundance of Proteobacteria and Gemmatimonas whereas decreased the abundance of Nitrospirae. Moreover, 42 Cd-resistant bacteria were isolated from six soil samples and evaluated for potential application in Cd bioremediation. Based on their Cd-MIC [minimum inhibitory concentration (MIC) of Cd2+], Cd2+ removal rate and 16S rDNA gene sequence analyses, three Burkholderia sp. strains (ha-1, hj-2, and ho-3) showed very high tolerance to Cd (5, 5, and 6 mM) and exhibited high Cd2+ removal rate (81.78, 79.37, and 63.05%), six Bacillus sp. strains (151-5,151-6,151-13, 151-20, and 151-21) showed moderate tolerance to Cd (0.8, 0.4, 0.8, 0.4, 0.6, and 0.4 mM) but high Cd2+ removal rate (84.78, 90.14, 82.82, 82.39, 81.79, and 84.17%). Those results indicated that Burkholderia sp. belonging to the phylum Proteobacteria and Bacillus sp. belonging to the phylum Firmicutes have developed a resistance for cadmium and may play an important role in Cd-contaminated soils. Our study provided baseline data for bacterial communities in cadmium polluted soils and concluded that Cd-resistant bacteria have potential for bioremediation of Cd-contaminated soils.

Protein engineering of stable IsPETase for PET plastic degradation by Premuse.

Poly(ethylene terephthalate) (PET) is used widely by human beings, but is very difficult to degrade. Up to now, the PET degradation effect of PETase from Ideonella sakaiensis 201-F6 (IsPETase) variants with low stability and activity was not ideal. In this study, a mutation design tool, Premuse, was developed to integrate the sequence alignment and quantitative selection of the preferred mutations based on natural sequence evolution. Ten single point mutants were selected from 1486 homologous sequences using Premuse, and then two mutations (W159H and F229Y) with improved stability were screened from them. The derived double point mutant, W159H/F229Y, exhibited a strikingly enhanced enzymatic performance. Its Tm and catalytic efficiency values (kcat/Km) respectively increased by 10.4 °C and 2.0-fold using p-NPP as the substrate compared with wild type. The degradation activity for amorphous PET was increased by almost 40-fold in comparison with wild type at 40 °C in 24 h. Additionally, the variant could catalyze biodegradation of PET bottle preform at a mean rate of 23.4 mgPET/h/mgenzyme. This study allowed us to design the mutation more efficiently, and provides a tool for achieving biodegradation of PET pollution under mild natural environments.

Construction of a mApple-D6A3-mediated biosensor for detection of heavy metal ions.

Pollution of heavy metals in agricultural environments is a growing problem to the health of the world's human population. Green, low-cost, and efficient detection methods can help control such pollution. In this study, a protein biosensor, mApple-D6A3, was built from rice-derived Cd2+-binding protein D6A3 fused with the red fluorescent protein mApple at the N-terminus to detect the contents of heavy metals. Fluorescence intensity of mApple fused with D6A3 indicated the biosensor's sensitivity to metal ions and its intensity was more stable under alkaline conditions. mApple-D6A3 was most sensitive to Cu2+, then Ni2+, then Cd2+. Isothermal titration calorimetry experiments demonstrated that mApple-D6A3 successfully bound to each of these three metal ions, and its ability to bind the ions was, from strongest to weakest, Cu2+ > Cd2+ > Ni2+. There were strong linear relationships between the fluorescence intensity of mApple-D6A3 and concentrations of Cd2+ (0-100 µM), Cu2+ (0-60 µM) and Ni2+ (0-120 µM), and their respective R2 values were 0.994, 0.973 and 0.973. When mApple-D6A3 was applied to detect concentrations of heavy metal ions in water (0-0.1 mM) or culture medium (0-1 mM), its accuracy for detection attained more than 80%. This study demonstrates the potential of this biosensor as a tool for detection of heavy metal ions.

Identification of a chitosanase from the marine metagenome and its molecular improvement based on evolution data.

Chitooligosaccharides have important application value in the fields of food and agriculture. Chitosanase can degrade chitosan to obtain chitooligosaccharides. The marine metagenome contains many genes related to the degradation of chitosan. However, it is difficult to mine valuable genes from large gene resources. This study proposes a method to screen chitosanases directly from the marine metagenome. Chitosanase gene chis1754 was identified from the metagenome and heterologously expressed in Escherichia coli. The optimal temperature and pH of CHIS1754 were 55 °C and 5.5, respectively. A mutant, CHIS1754T, with 15 single point mutations designed based on molecular evolution data was also expressed in E. coli. The results indicated that the thermal stability of CHIS1754T was significantly improved, as the Tm showed an increase of ~ 7.63 °C. Additionally, the kcat/Km of CHIS1754T was 4.8-fold higher than that of the wild type. This research provides new theories and foundations for the excavation, modification, and industrial application of chitosanases. KEY POINTS: A chitosanase gene, chis1754, was firstly identified from marine metagenome. A multi-site mutant was designed to improve enzyme stability and activity. The kcat/Kmof the designed mutant was 4.8-fold higher than that of the wild type.

Highly efficient production of chitooligosaccharides by enzymes mined directly from the marine metagenome.

The products of chitin degradation, chitosan and chitooligosaccharides, are valuable to the food and agriculture industries. The bio-enzymatic degradation of chitin can overcome the shortcomings of chemical degradation methods. This study identified two novel enzymes involved in chitin degradation from the marine metagenome: chitin deacetylase CDA20 and chitosanase CHIS5. Published chitin deacetylases (CDAs) are generally active against acetylated oligosaccharides with degrees of polymerization ≥ 2 or N-acetyl-d-glucosamine (GlcNAc). However, the deacetylase CDA20 effectively removed the acetyl groups from GlcNAc and chitobiose simultaneously. The chitosanase CHIS5 is an endo-type chitosanase and degraded chitosan into chitooligosaccharides with degrees of polymerization of 2-5. When used in combination, CHIS5 preferentially hydrolyzed chitosan to acetylated chitooligosaccharides, and then CDA20 removed the acetyl group to produce chitooligosaccharides. Our research has identified valuable enzymes related to chitin degradation encoded in the marine metagenome and broadens the theoretical basis for chitin biodegradation by bio-enzymes.

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 and characterization of sequence signatures in the Bacillus subtilis promoter Pylb for tuning promoter strength.

OBJECTIVE: To thoroughly characterize the Pylb promoter and identify the elements that affect the promoter activity. RESULT: The sequences flanking the - 35 and - 10 box of the Pylb promoter were divided into six segments, and six random-scanning mutant promoter libraries fused to an enhanced green fluorescent protein EGFP were made and analyzed by flow cytometry. Our results showed that the four nucleotides flanking the - 35 box could mostly influence the promoter activity, and this influence was related to the GC content. The promoters mutated in these regions were successfully used for expressing the gene ophc2 encoding organophosphorus hydrolase (OPHC2) and the gene katA encoding catalase (KatA). CONCLUSION: Our work identified and characterized the sequence signatures of the Pylb promoter that could tune the promoter strength, providing further information for the potential application of this promoter. Meanwhile, the sequence signatures have the potential to be used for tuning gene expression in enzyme production, metabolic engineering, and synthetic biology.

Enhancing thermostability of a psychrophilic alpha-amylase by the structural energy optimization in the trajectories of molecular dynamics simulations.

The cold-adapted alpha-amylase (PHA) from Pseudoalteromonas haloplanktis is a psychrophilic enzyme which demonstrates high activity at low temperatures, but poor thermostability. Most of the method only employed the crystal structure to design the target protein. However, the trajectory of protein molecular dynamics (MD) simulation contained clues about the protein stability. In this study, we combined MD simulation and energy optimization methods to design mutations located at non-conserved residues. Two single point mutants (S255K, S340P) and one integrated mutant (S255K/S340P) enhanced thermostability without affecting the optimal catalytic activity. After incubation at 40 °C for 80 min, the residual activities of mutants S255K, S340P and S255K/S340P were 1.6-, 2.4-, and 2.6-fold greater than that of the wild type (WT). Additionally, the catalytic efficiency values (kcat/Km) of S255K, S340P, and S255K/S340P also increased 1.9-, 2.0-, and 2.7-fold when compared to WT.

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.

Improving Cadmium Resistance in Escherichia coli Through Continuous Genome Evolution.

Cadmium (Cd) is a heavy metal that is extremely toxic to many organisms; however, microbes are highly adaptable to extreme conditions, including heavy metal contamination. Bacteria can evolve in the natural environment, generating resistant strains that can be studied to understand heavy-metal resistance mechanisms, but obtaining such adaptive strains usually takes a long time. In this study, the genome replication engineering assisted continuous evolution (GREACE) method was used to accelerate the evolutionary rate of the Escherichia coli genome to screen for E. coli mutants with high resistance to cadmium. As a result, a mutant (8mM-CRAA) with a minimum inhibitory concentration (MIC) of 8 mM cadmium was generated; this MIC value was approximately eightfold higher than that of the E. coli BL21(DE3) wild-type strain. Sequencing revealed 329 single nucleotide polymorphisms (SNPs) in the genome of the E. coli mutant 8mM-CRAA. These SNPs as well as RNA-Seq data on gene expression induced by cadmium were used to analyze the genes related to cadmium resistance. Overexpression, knockout and mutation of the htpX (which encodes an integral membrane heat shock protein) and gor (which encodes glutathione reductase) genes revealed that these two genes contribute positively to cadmium resistance in E. coli. Therefore, in addition to the previously identified cadmium resistance genes zntA and capB, many other genes are also involved in bacterial cadmium resistance. This study assists us in understanding the mechanism of microbial cadmium resistance and facilitating the application of heavy-metal remediation.