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Lignin is an underutilized raw material. Existing processes in which bacteria degrade lignin and produce added value products are not economically feasible yet due to limited lignin degradation. The aim of our study was to enhance microbial lignin degradation by surface‐display of the bacterial laccase CotA from the thermophilic Bacillus coagulans. Enzyme engineering of surface‐displayed CotA was used to increase its activity at 30 °C for the application at moderate temperatures. Libraries of CotA were created using error‐prone PCR and site saturation mutagenesis. Two different substrates with distinct binding properties in CotA were utilized for activity screening to detect variants with enhanced activity regardless of substrate specificity. Combination of favorable mutations resulted in the variant CotA T260S/L385K/F416R which showed 1.95‐fold and 13.4‐fold activity of CotA with 2,2'‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulfonic acid) (ABTS) and 2,6‐dimethoxyphenol (2,6‐DMP) as substrate, respectively. All mutations were located within the substrate binding site. The KM‐value of CotA T260S/L385K/F416R was increased with ABTS (2.08 mM versus 0.44 mM of ancestor CotA) and decreased with 2,6‐DMP (0.31 mM versus 3.79 mM of ancestor CotA). Surface display of the identified variant CotA T260S/L385K/F416R on Pseudomonas putida enhanced its growth on lignin. [ABSTRACT FROM AUTHOR]

Saccharomyces cerevisiae is a robust cell factory to secrete or surface-display cellulase and amylase for the conversion of agricultural residues into valuable chemicals. Engineering the secretory pathway is a well-known strategy for overproducing these enzymes. Although cell wall biosynthesis can be tightly linked to the secretory pathway by regulation of all involved processes, the effect of its modifications on protein production has not been extensively studied. In this study, we systematically studied the effect of engineering cell wall biosynthesis on the activity of cellulolytic enzyme β-glucosidase (BGL1) by comparing seventy-nine gene knockout S. cerevisiae strains and newly identified that inactivation of DFG5 , YPK1 , FYV5 , CCW12 and KRE1 obviously improved BGL1 secretion and surface-display. Combinatorial modifications of these genes, particularly double deletion of FVY5 and CCW12 , along with the use of rich medium, increased the activity of secreted and surface-displayed BGL1 by 6.13-fold and 7.99-fold, respectively. Additionally, we applied this strategy to improve the activity of the cellulolytic cellobiohydrolase and amylolytic α-amylase. Through proteomic analysis coupled with reverse engineering, we found that in addition to the secretory pathway, regulation of translation processes may also involve in improving enzyme activity by engineering cell wall biosynthesis. Our work provides new insight into the construction of a yeast cell factory for efficient production of polysaccharide degrading enzymes. [Display omitted] • Newly identified gene targets involved in cell wall biosynthesis can improve protein secretion and surface-display. • The double deletion of FVY5 and CCW12 resulted in a 4.42- and 5.13-fold increase in the secretion and surface-display of β-glucosidase (BGL1), respectively. • The use of rich medium further improved BGL1 secretion and surface-display by 31.68% and 46.55%, respectively. • This strategy also led to improvements in the activity of cellobiohydrolase and α-amylase. • Proteomic analysis coupled with reverse engineering provided insights into the mechanism underlying the improved protein secretion or surface-display. [ABSTRACT FROM AUTHOR]

Saccharomyces cerevisiae has been widely used as a microbial cell factory to produce recombinant proteins. Therefore, enhancing the protein production efficiency of yeast cell factories to expand the market demand for protein products is necessary. Recombinant proteins are often retained in the secretory pathway because of the limited protein transport performed by vesicle trafficking. Cell polarization describes the asymmetric organization of the plasma membrane cytoskeleton and organelles and tightly regulates vesicle trafficking for protein transport. Engineering vesicle trafficking has broadly been studied by the overexpression or deletion of key genes involved but not by modifying cell polarization. Here, we used α-amylase as a reporter protein, and its secretion and surface-display were first improved by promoter optimization. To study the effect of engineering cell polarization on protein production, fourteen genes related to cell polarization were overexpressed. BUD1, CDC42, AXL1, and BUD10 overexpression increased the activity of surface-displayed α-amylase, and BUD1, BUD3, BUD4, BUD7, and BUD10 overexpression enhanced secreted α-amylase activity. Furthermore, BUD1 overexpression increased the surface-displayed and secreted α-amylase expression by 56% and 49%, respectively. We also observed that the combinatorial modification and regulation of gene expression improved α-amylase production in a dose-dependent manner. BUD1 and CDC42 co-overexpression increased the α-amylase surface display by 100%, and two genomic copies of BUD1 improved α-amylase secretion by 92%. Furthermore, these modifications were used to improve the surface display and secretion of the recombinant β-glucosidase protein. Our study affords a novel insight for improving the surface display and secretion of recombinant proteins. [ABSTRACT FROM AUTHOR]

CO 2 sequestration is important for reducing greenhouse effects. Carbonic anhydrase (CA) from bacteria has a promising role because it can be modified by genetic techniques and bioengineering. In this study, the CA from B. cereus GLRT202 (Bc-CA) was genetically engineered and anchored on the surface of E. coli by using the N-domain of the ice nucleation protein from P. syringae (INPN). Both surface-displayed and cytosolic Bc-CA yielded high expression levels of CA when induced with 0.5 mM IPTG. It exhibited no adverse influence on the host cell growth. Additionally, surface-displayed Bc-CA enhanced its stability and specificity compared to cytosolic expressed Bc-CA. The CA activity of whole-cell surface-displayed cells was 1.66-fold higher (5.19 U/mL) than that of the cytosolic form. Besides the advantages of higher activity, the whole-cell displaying CA was comparatively stable, with better storage (at 4 ℃) and resting culture stability (at 37 ℃). The whole-cell biocatalyst induced the calcite precipitation, which indicated that the cell facilitated the CO 2 capture. XRD, FTIR, and FESEM characterized calcite precipitates thus obtained. This study demonstrates that Bc-CA can be correctly expressed on the E. coli surface through fusion with the INPN. This leads to an effective whole-cell biocatalyst with enhanced stability and specificity of the enzyme for efficient CO 2 capture applications. [Display omitted] • Carbonic anhydrase from B. cereus GLRT202 was surface displayed on E. coli cells. • The anchorage on the cell was enabled by N-domain of the ice nucleating protein. • The surface-displayed enzyme had no adverse effects on host cell growth. • The enzyme on E. coli cell surface was stable and expressed high activity. • Surface-displayed CA yielded a noticeable CO 2 capture rate via CaCO 3 deposition. [ABSTRACT FROM AUTHOR]

Facing the increasingly prominent tetracycline pollution and the resulting environmental problems, how to find environmental and efficient treatment means is one of the current research hotspots. In this study, the laccase surface-display technology for tetracycline treatment was investigated. Via study, the type of anchoring protein had a minor influence on the laccase ability, while the type of laccase showed a major impact. Bacillus subtilis spore coat protein (CotA) exhibited higher laccase activity, stability, and efficiency in degrading tetracycline than Pleurotus ostreatus laccase 6 (Lacc6). The superiority of bacterial laccase over fungal laccase was elucidated from the perspective of crystal structure. Besides, a variety of technical means were used to verify the success of surface-display. pGSA-CotA surface-displayed bacteria exhibited good tolerance to high temperature, pH, and various heavy metals. Importantly, surface-displayed bacteria showed faster degradation efficiency and better treatment effects than the intracellular expression bacteria in tetracycline degradation. This implies that surface display technology has greater potential for laccase-mediated environmental remediation. Due to the adverse impacts of tetracycline on soil enzyme activity and microorganisms, our study found that pGSA-CotA surface-displayed bacteria can alleviate tetracycline stress in soil and partially activate the soil, thereby increasing soil enzyme activity and certain nitrogen cycling genes. [Display omitted] • The choice of anchoring protein has a minor impact on the laccase's surface-display activity. • Bacterial laccase CotA demonstrates superior tetracycline removal capability. • CotA protein structure determines higher stability and stronger binding ability. • Surface-displayed bacteria exhibited rapid removal of tetracycline from soil. [ABSTRACT FROM AUTHOR]

Semiartificial photosynthetic systems have shown tremendous potential to address global energy issues by combining the exceptional optical properties of nanomaterials with biological whole-cell catalysts. To achieve sustainable energy conversion between inorganic materials and whole cells in semiartificial photosynthetic systems, it is highly desirable to introduce biocompatible nanomaterials with broad light adsorption into the hybrid catalysis system. Herein, we developed a whole-cell biohybrid catalyst with in situ synthesized Ag2S QDs for visible-light-driven photocatalytic organic pollutant degradation. With the synthesized Ag2S QDs on the surface of engineered Escherichia coli cells, the biohybrid system showed superior performance for the photocatalytic degradation of organic pollutants. The photogenerated reactive oxygen species (•O2–) were confirmed to play a pivotal role in removing organic pollutants during the photocatalytic process. The enhanced photocatalytic activity of the E. coli -Ag2S QD biohybrid system could be ascribed to the effective separation and transfer of the interfacial charge carriers. This whole-cell biohybrid system could provide a promising approach for environmental issues in solar energy conversion. [ABSTRACT FROM AUTHOR]

Saccharomyces cerevisiae has been widely used as a microbial cell factory to produce recombinant proteins. Therefore, enhancing the protein production efficiency of yeast cell factories to expand the market demand for protein products is necessary. Recombinant proteins are often retained in the secretory pathway because of the limited protein transport performed by vesicle trafficking. Cell polarization describes the asymmetric organization of the plasma membrane cytoskeleton and organelles and tightly regulates vesicle trafficking for protein transport. Engineering vesicle trafficking has broadly been studied by the overexpression or deletion of key genes involved but not by modifying cell polarization. Here, we used α-amylase as a reporter protein, and its secretion and surface-display were first improved by promoter optimization. To study the effect of engineering cell polarization on protein production, fourteen genes related to cell polarization were overexpressed. BUD1, CDC42, AXL1, and BUD10 overexpression increased the activity of surface-displayed α-amylase, and BUD1, BUD3, BUD4, BUD7, and BUD10 overexpression enhanced secreted α-amylase activity. Furthermore, BUD1 overexpression increased the surface-displayed and secreted α-amylase expression by 56% and 49%, respectively. We also observed that the combinatorial modification and regulation of gene expression improved α-amylase production in a dose-dependent manner. BUD1 and CDC42 co-overexpression increased the α-amylase surface display by 100%, and two genomic copies of BUD1 improved α-amylase secretion by 92%. Furthermore, these modifications were used to improve the surface display and secretion of the recombinant β-glucosidase protein. Our study affords a novel insight for improving the surface display and secretion of recombinant proteins.

Polyethylene terephthalate (PET) is one of the most used plastics which has caused some environmental pollution and social problems. Although many newly discovered or modified PET hydrolases have been reported at present, there is still a lack of comparison between their hydrolytic capacities, as well as the need for new biotechnology to apply them for the PET treatment. Here, we systematically studied the surface-display technology for PET hydrolysis using several PET hydrolases. It is found that anchoring protein types had little influence on the surface-display result under T7 promoter, while the PET hydrolase types were more important. By contrast, the newly reported FAST-PETase showed the strongest hydrolysis effect, achieving 71.3% PET hydrolysis in 24 h by pGSA-FAST-PETase. Via model calculation, FAST-PETase indeed exhibited higher temperature tolerance and catalytic capacity. Besides, smaller particle size and lower crystallinity favored the hydrolysis of PET pellets. Through protein structure comparison, we summarized the common characteristics of efficient PET-hydrolyzing enzymes and proposed three main crystal structures of PET enzymes via crystal structural analysis, with IS PETase being the representative and main structure. Surface co-display of FAST-PETase and MHETase can promote the hydrolysis of PET, and the C-terminal of the fusion protein is crucial for PET hydrolysis. The results of our research can be helpful for PET contamination removal as well as other areas involving the application of enzymes. This research can promote the development of better PET hydrolase and its applications in PET pollution treatment via bacteria surface-display. [Display omitted] • In the PETase surface display, the anchoring protein types had little effect on the results. • Surface-display of FAST-PETase has shown great potential for PET hydrolysis. • There are three main crystal structure patterns of PET hydrolysis enzyme, with IS PEtase being the representative enzyme. • The size and crystallinity of the PET particles play a crucial role in the hydrolysis efficiency of PET. • The surface co-display sequential of FAST-PETase and MHETase has effect on PET hydrolysis. [ABSTRACT FROM AUTHOR]

Highlights: [•] This review focuses on the successful expression of OPH in E. coli for the effective whole-cell biocatalysts to OP chemicals. [•] Through the current studies, confronting limits of the expression of recombinant OPH in E. coli were understood. [•] To overcome the expression limits, the strategies for the surface-displaying of recombinant OPH in E. coli were inquired. [•] The approaches for the secretion of recombinant OPH in E. coli were also addressed. [Copyright &y& Elsevier]

A fed-batch culture strategy for the production of recombinant Escherichia coli cells anchoring surface-displayed transglucosidase for use as a whole-cell biocatalyst for α-arbutin synthesis was developed. Lactose was used as an inducer of the recombinant protein. In fed-batch cultures, dissolved oxygen was used as the feed indicator for glucose, thus accumulation of glucose and acetate that affected the cell growth and recombinant protein production was avoided. Fed-batch fermentation with lactose induction yielded a biomass of 18 g/L, and the cells possessed very high transglucosylation activity. In the synthesis of α-arbutin by hydroquinone glucosylation, the whole-cell biocatalysts showed a specific activity of 501 nkat/g cell and produced 21 g/L of arbutin, which corresponded to 76% molar conversion. A sixfold increased productivity of whole cell biocatalysts was obtained in the fed-batch culture with lactose induction, as compared to batch culture induced by IPTG. [ABSTRACT FROM AUTHOR]

To enhance the use of cellobiose by a recombinant Sachharomyces cerevisiae, the expressed β-glucosidase that hydrolyzes cellobiose was stabilized using a surface-display system. The C-terminal half of α-agglutinin was used as surface-display motif for the expression of β-glucosidase in the cell wall. The surface-displayed β-glucosidase had a half-life time (t1/2) of 100 h in acidic culture broth conditions, while secreted β-glucosidase had a t1/2 of 60 h. With such stabilization of β-glucosidase, the surface-engineered S. cerevisiae utilized 7.5 g cellobiose l-1 over 60 h, while S. cerevisiae secreting β-glucosidase into culture broth used 5.8 g cellobiose l-1 over the same period. [ABSTRACT FROM AUTHOR]