A high-throughput and selective method for the measurement of surface areas of silver nanoparticles.

A high-throughput and selective method based on biomolecule affinity coordination was employed for measuring nanoparticle surface area in solutions. In this design, silver binding peptides (AgBPs) are immobilized on bacterial cellulose via fusion with cellulose binding domains to capture silver nanoparticles whereas green fluorescent proteins are fused with AgBPs as reporters for surface area quantification.

Recent advances in the biological depolymerization and upcycling of polyethylene terephthalate.

Polyethylene terephthalate (PET) is favored for its exceptional properties and widespread daily use. This review highlights recent advancements that enable the development of biological tools for PET decomposition, transforming PET into valuable platform chemicals and materials in upcycling processes. Enhancing PET hydrolases' catalytic activity and efficiency through protein engineering strategies is a priority, facilitating more effective PET waste management. Efforts to create novel PET hydrolases for large-scale PET depolymerization continue, but cost-effectiveness remains challenging. Hydrolyzed monomers must add additional value to make PET recycling economically attractive. Valorization of hydrolysis products through the upcycling process is expected to produce new compounds with different values and qualities from the initial polymer, making the decomposed monomers more appealing. Advances in synthetic biology and enzyme engineering hold promise for PET upcycling. While biological depolymerization offers environmental benefits, further research is needed to make PET upcycling sustainable and economically feasible.

Microbial biosensors: engineered microorganisms as the sensing machinery.

Whole-cell biosensors are a good alternative to enzyme-based biosensors since they offer the benefits of low cost and improved stability. In recent years, live cells have been employed as biosensors for a wide range of targets. In this review, we will focus on the use of microorganisms that are genetically modified with the desirable outputs in order to improve the biosensor performance. Different methodologies based on genetic/protein engineering and synthetic biology to construct microorganisms with the required signal outputs, sensitivity, and selectivity will be discussed.

Functionalization of OMVs for Biocatalytic Applications.

Outer membrane vesicles (OMVs) are miniature versions of gram-negative bacteria that contain almost the same content as their parent cells, particularly in terms of membrane composition. Using OMVs as biocatalysts is a promising approach due to their potential benefits, including their ability to be handled similarly to bacteria while lacking potentially pathogenic organisms. To employ OMVs as biocatalysts, they must be functionalized with immobilized enzymes to the OMV platform. Various enzyme immobilization techniques are available, including surface display and encapsulation, each with advantages and disadvantages depending on the objectives. This review provides a concise yet comprehensive overview of these immobilization techniques and their applications in utilizing OMVs as biocatalysts. Specifically, we discuss the use of OMVs in catalyzing the conversion of chemical compounds, their role in polymer degradation, and their performance in bioremediation.

Engineering a high-affinity scaffold for non-chromatographic protein purification via intein-mediated cleavage.

While protein purification has long been dominated by standard chromatography, the relatively high cost and complex scale-up have promoted the development of alternative non-chromatographic separation methods. Here we developed a new non-chromatographic affinity method for the purification of proteins expressed in Escherichia coli. The approach is to genetically fuse the target proteins with an affinity tag. Direct purification and recovery can be achieved using a thermo-responsive elastin-like protein (ELP) scaffold containing the capturing domain. Naturally occurring cohesin-dockerin pairs, which are high-affinity protein complex responsible for the formation of cellulosome in anaerobic bacteria, were used as the model. By exploiting the highly specific interaction between the dockerin and cohesin domain from Clostridium thermocellum and the reversible aggregation property of ELP, highly purified and active dockerin-tagged proteins, such as the endoglucanase CelA, chloramphenicol acetyl transferase (CAT), and enhanced green fluorescence protein (EGFP), were recovered directly from crude cell extracts in a single thermal precipitation step with yields achieving over 90%. Incorporation of a self-cleaving intein domain enabled rapid removal of the affinity tag from the target proteins, which was subsequently removed by another cycle of thermal precipitation. This method offers great flexibility as a wide range of affinity tags and ligands can be used.

Co-expression of Arabidopsis thaliana phytochelatin synthase and Treponema denticola cysteine desulfhydrase for enhanced arsenic accumulation.

Arsenic is one of the most hazardous pollutants found in aqueous environments and has been shown to be a carcinogen. Phytochelatins (PCs), which are cysteine-rich and thio-reactive peptides, have high binding affinities for various metals including arsenic. Previously, we demonstrated that genetically engineered Saccharomyces cerevisiae strains expressing phytochelatin synthase (AtPCS) produced PCs and accumulated arsenic. In an effort to further improve the overall accumulation of arsenic, cysteine desulfhydrase, an aminotransferase that converts cysteine into hydrogen sulfide under aerobic condition, was co-expressed in order to promote the formation of larger AsS complexes. Yeast cells producing both AtPCS and cysteine desulfhydrase showed a higher level of arsenic accumulation than a simple cumulative effect of expressing both enzymes, confirming the coordinated action of hydrogen sulfide and PCs in the overall bioaccumulation of arsenic.

Advances in consolidated bioprocessing using synthetic cellulosomes.

The primary obstacle impeding the more widespread use of biomass for energy and chemical production is the absence of a low-cost technology for overcoming their recalcitrant nature. It has been shown that the overall cost can be reduced by using a 'consolidated' bioprocessing (CBP) approach, in which enzyme production, biomass hydrolysis, and sugar fermentation can be combined. Cellulosomes are enzyme complexes found in many anaerobic microorganisms that are highly efficient for biomass depolymerization. While initial efforts to display synthetic cellulosomes have been successful, the overall conversion is still low for practical use. This limitation has been partially alleviated by displaying more complex cellulsome structures either via adaptive assembly or by using synthetic consortia. Since synthetic cellulosome nanostructures have also been created using either protein nanoparticles or DNA as a scaffold, there is the potential to tether these nanostructures onto living cells in order to further enhance the overall efficiency.

Strategies for Multienzyme Assemblies.

Proteins are not designed to be standalone entities and must coordinate their collective action for optimum performance. Nature has developed through evolution the ability to co-localize the functional partners of a cascade enzymatic reaction in order to ensure efficient exchange of intermediates. Inspired by these natural designs, synthetic scaffolds have been created to enhance the overall biological pathway performance. In this chapter, we describe several DNA- and protein-based scaffold approaches to assemble artificial enzyme cascades for a wide range of applications. We highlight the key benefits and drawbacks of these approaches to provide insights on how to choose the appropriate scaffold for different cascade systems.

Simultaneous cell growth and ethanol production from cellulose by an engineered yeast consortium displaying a functional mini-cellulosome.

BACKGROUND: The recalcitrant nature of cellulosic materials and the high cost of enzymes required for efficient hydrolysis are the major impeding steps to their practical usage for ethanol production. Ideally, a recombinant microorganism, possessing the capability to utilize cellulose for simultaneous growth and ethanol production, is of great interest. We have reported recently the use of a yeast consortium for the functional presentation of a mini-cellulosome structure onto the yeast surface by exploiting the specific interaction of different cohesin-dockerin pairs. In this study, we engineered a yeast consortium capable of displaying a functional mini-cellulosome for the simultaneous growth and ethanol production on phosphoric acid swollen cellulose (PASC). RESULTS: A yeast consortium composed of four different populations was engineered to display a functional mini-cellulosome containing an endoglucanase, an exoglucanase and a ß-glucosidase. The resulting consortium was demonstrated to utilize PASC for growth and ethanol production. The final ethanol production of 1.25 g/L corresponded to 87% of the theoretical value and was 3-fold higher than a similar yeast consortium secreting only the three cellulases. Quantitative PCR was used to enumerate the dynamics of each individual yeast population for the two consortia. Results indicated that the slight difference in cell growth cannot explain the 3-fold increase in PASC hydrolysis and ethanol production. Instead, the substantial increase in ethanol production is consistent with the reported synergistic effect on cellulose hydrolysis using the displayed mini-cellulosome. CONCLUSIONS: This report represents a significant step towards the goal of cellulosic ethanol production. This engineered yeast consortium displaying a functional mini-cellulosome demonstrated not only the ability to grow on the released sugars from PASC but also a 3-fold higher ethanol production than a similar yeast consortium secreting only the three cellulases. The use of more complex cellulosomal structures may further improve the overall efficiency for ethanol production.

Fungal Hydrophobin RolA Enhanced PETase Hydrolysis of Polyethylene Terephthalate.

Polyethylene terephthalate (PET) becomes one of the most well-known polyesters and is widely used as packaging material. Recently, polyethylene terephthalate hydrolase (PETase) has emerged as a potential biocatalyst demonstrating the ability to degrade polyethylene terephthalate (PET). We showed that the rate of PETase hydrolysis could be significantly increased in the presence of hydrophobin RolA. Hydrophobins represent a class of small fungal protein that has a high surface-active substance and can spontaneously self-assemble at hydrophilic-hydrophobic interfaces. In this work, a class I hydrophobin named RolA was extracted from the mycelium pellet collected from a fermentation culture of Aspergillus oryzae. The SDS-PAGE analysis of the isolated RolA showed the presence of 11 kDa polypeptide. Recombinant PETase from Ideonella sakaiensis was also successfully expressed in Escherichia coli as a soluble protein with molecular weight approximately 30 kDa. The hydrophobin RolA could enhance the PET hydrolysis in the presence of the recombinant PETase. The hydrolysis of PET bottle by RolA-PETase achieved the highest weight loss of 26% in 4 days. It is speculated that the wetting effect of RolA acts on PET surface converts PET to become hydrophilic that leads PETase easier to contact and attack the surface. Graphical Abstract.

Class I hydrophobins pretreatment stimulates PETase for monomers recycling of waste PETs.

Poly(ethylene terephthalate) hydrolase (PETase) from Ideonella sakaiensis 201-F6 was expressed and purified from Escherichia coli to hydrolyze poly(ethylene terephthalate) (PET) fibers waste for its monomers recycling. Hydrolysis carried out at pH 8 and 30 °C was found to be the optimal condition based on measured monomer mono(2-hydroxyethyl) terephthalate (MHET) and terephthalic acid (TPA) concentrations after 24 h reaction. The intermediate product bis(2-hydroxyethyl) terephthalate (BHET) was a good substrate for PETase because BHET released from PET hydrolysis was efficiently converted into MHET. Only a trace amount of MHET could be further hydrolyzed to TPA. Class I hydrophobins RolA from Aspergillus oryzae and HGFI from Grifola frondosa were expressed and purified from E. coli to pretreat PET surface for accelerating PETase hydrolysis against PET. The weight loss of hydrolyzed PET increased from approximately 18% to 34% after hydrophobins pretreatment. The releases of TPA and MHET from HGFI-pretreated PET were enhanced 48% and 62%, respectively. The selectivity (TPA/MHET ratio) of the hydrolysis reaction was approximately 0.5.

Improving the performance of biotrickling filter microbial fuel cells in treating exhaust gas by adjusting the oxygen content of the anode tank.

A biotrickling filter (BTF) was combined with a microbial fuel cell (MFC) to remove ethyl acetate from exhaust gas while generating electricity in the process. The results indicated that the use of carbide porous ceramic rings (CPCR) as auxiliary anodes produced more biomass and exhibited a high average removal efficiency (98%), making it a superior microorganism growth carrier compared with carbon coke. When CPCR was used as the cathode in the BTF-MFC, the maximum power density (PD) was 5.64-14.8% of that achieved when carbon cloth was used as the cathode, revealing that CPCR is not a suitable cathode. The maximum elimination capacity (EC) and output voltage of the two-stage BTF-MFC (tBTF-MFC) were only 69.4% and 68.4% of those of the single-stage BTF-MFC (sBTF-MFC), presumably because of voltage reversal. Although the output voltage and EC in the tBTF-MFC were less than those in the sBTF-MFC, the follow-up field application involves stacking multiple small MFCs to remove high-concentration pollutants and generate a high power output. Additionally, continuously adding sodium sulfite decreased the average dissolved oxygen; generated an averaged closed-circuit voltage of 477 mV; and produced a maximum PD of 71.7 mW/m3. These findings demonstrated that the aforementioned method can effectively improve the problem of oxygen and MFC anodes competing for electrons, thus delivering a method that enhances MFC performance through controlling the amount of oxygen in practical applications.

Surface display of a functional minicellulosome by intracellular complementation using a synthetic yeast consortium and its application to cellulose hydrolysis and ethanol production.

In this paper, we report the surface assembly of a functional minicellulosome by using a synthetic yeast consortium. The basic design of the consortium consisted of four different engineered yeast strains capable of either displaying a trifunctional scaffoldin, Scaf-ctf (SC), carrying three divergent cohesin domains from Clostridium thermocellum (t), Clostridium cellulolyticum (c), and Ruminococcus flavefaciens (f), or secreting one of the three corresponding dockerin-tagged cellulases (endoglucanase [AT], exoglucanase [EC/CB], or ß-glucosidase [BF]). The secreted cellulases were docked onto the displayed Scaf-ctf in a highly organized manner based on the specific interaction of the three cohesin-dockerin pairs employed, resulting in the assembly of a functional minicellulosome on the yeast surface. By exploiting the modular nature of each population to provide a unique building block for the minicellulosome structure, the overall cellulosome assembly, cellulose hydrolysis, and ethanol production were easily fine-tuned by adjusting the ratio of different populations in the consortium. The optimized consortium consisted of a SC:AT:CB:BF ratio of 7:2:4:2 and produced almost twice the level of ethanol (1.87 g/liter) as a consortium with an equal ratio of the different populations. The final ethanol yield of 0.475 g of ethanol/g of cellulose consumed also corresponded to 93% of the theoretical value. This result confirms the use of a synthetic biology approach for the synergistic saccharification and fermentation of cellulose to ethanol by using a yeast consortium displaying a functional minicellulosome.

Biotransformation of 5-Hydroxymethylfurfural to 2,5-Furandicarboxylic Acid by a Syntrophic Consortium of Engineered Synechococcus elongatus and Pseudomonas putida.

2,5-furandicarboxylic acid (FDCA) is one of the top platform chemicals that can be produced from biomass feedstock. To make the cost of industrial FDCA production compatible with plastics made from fossils, the price of substrates and process complexity should be reduced. The aim of this research is to create a CO2 -driven syntrophic consortium for the catalytic conversion of renewable biomass-derived 5-hydroxymethylfurfural (HMF) to FDCA. Sucrose produced from carbon fixation by the engineered Synechococcus elongatus serves as the sole carbon source for the engineered Pseudomonas putida to catalyze the reaction of HMF to FDCA. The yield of FDCA by the consortium reaches around 70% while the conversion of HMF is close to 100%. With further surface engineering to clump the two strains, the FDCA yield is elevated to almost 100% via the specific association between an Src homology 3 (SH3) domain and its ligand. The syntrophic consortium successfully demonstrates its green and cost-effective characteristics for the conversion of CO2 and biomass into platform chemicals.

Green conversion of 5-hydroxymethylfurfural to furan-2,5-dicarboxylic acid by heterogeneous expression of 5-hydroxymethylfurfural oxidase in Pseudomonas putida S12.

Transforming petrochemical processes into bioprocesses has become an important goal of sustainable development. The chemical synthesis of 2,5-furandicarboxylic acid (FDCA) from 5-hydroxymethylfurfural (HMF) is expensive and environmentally unfavourable. The study aims to investigate a whole-cell biocatalyst for efficient biotransformation of HMF to FDCA. For the first time, a genetically engineered Pseudomonas putida S12 strain expressing 5-hydroxymethylfurfural oxidase (HMFO) was developed for the biocatalytic conversion of HMF to FDCA. This whole-cell biocatalyst produced 35.7 mM FDCA from 50 mM HMF in 24 h without notable inhibition. However, when the initial HMF concentration was elevated to 100 mM, remarkable inhibition on FDCA production was observed, resulting in a reduction of FDCA yield to 42%. We solve this substrate inhibition difficulty by increasing the inoculum density. Subsequently, we used a fed-batch strategy by maintaining low HMF concentration in the culture to maximize the final FDCA titre. Using this approach, 545 mM of FDCA was accumulatively produced after 72 hs, which is the highest production rate per unit mass of cells to the best of our knowledge.

Enhanced Lipid Production in Yarrowia lipolytica Po1g by Over-expressing lro1 Gene under Two Different Promoters.

Yarrowia lipolytica is a well-known oleaginous yeast that naturally accumulates lipids to more than 20% of their dry cell weight. Due to its brief doubling time and Generally Recognized as Safe (GRAS) properties, Y. lipolytica has been exploited for the production of commercially valuable lipids. Among the genes related to the lipid synthesis, the gene YALI0E16797g (LRO1) encoding a major triacylglycerol synthase of Y. lipolytica shows a significant impact during the acylation process. Thus, in the present work, we explore the contributions of hp4d or TEFintron promoters to the response of LRO1 expression on lipid accumulation by molecular cloning technology. Results showed that over-expression of LRO1 led to higher lipid content as well as lipid yield. The one with the hp4d promoter showed the highest lipid content of 12% wt. However, such an enhancement also caused a growth defect of cells. On the other hand, the lipid content of the cells over-expressing LRO1 with TEFintron promoter revealed only a modest increase in lipid content, but it promoted cell growth. Therefore, all things considered the one with the TEFintron promoter showed the highest lipid yield.

Inducing laccase activity in white rot fungi using copper ions and improving the efficiency of azo dye treatment with electricity generation using microbial fuel cells.

This work presents a white rot fungus-microbial fuel cell (WRF-MFC) that uses WRF that is grown at its cathode. Adding Cu2+ to the fungi-containing solid medium stimulated WRF-secreting laccase, which catalyzed the redox reaction in the MFC and thereby promoting the generation of electricity. Adding 12.5 mg L-1 Cu2+ to a G. lucidum-containing medium provided the greatest laccase stimulation and increased the laccase activity by a factor of 1.6. Adding 12.5 mg L-1 Cu2+ to the WRF chamber of WRF-MFC increased its decolorization of Acid Orange 7 (AO-7) and increased its power density to 223 mW m-2, which was 1.77 times that of an MFC without WRF. The enhancement of decolorization and electricity generation improved the performance of the WRF-MFC, indicating that a laccase-catalyzed cathode has great potential effectiveness in microbial fuel cells.

Highly efficient dye removal and lysozyme purification using strong and weak cation-exchange nanofiber membranes.

Electrospinning technology was applied for the preparation of polyacrylonitrile (PAN) nanofiber membrane in this work. After hot pressing, alkaline hydrolysis and neutralization treatment, a weak acid cation exchange membrane (P-COOH) was prepared. By the covalent coupling reaction between the acidic membrane and aminomethane sulfonic acid (AMSA), a strong acidic nanofiber membrane (P-SO3H) was obtained. The surface morphology, chemical structure, and thermal stability of the prepared ion exchange membranes were analyzed via SEM, FTIR and TGA. Analytical results showed that the membranes were prepared successfully and thermally stable. The ion exchange membrane (IEX) was conducted with the newly designed membrane reactor, and different operating conditions affecting the adsorption efficiency of Toluidine Blue dye (TBO) were investigated by dynamic flow process. The results showed that dynamic binding capacity (DBC) of weak and strong IEX membranes for TBO dye was ~170 mg/g in a dynamic flow process. Simultaneously, the ion exchange membranes were also used for purifying lysozyme from chicken egg white (CEW). Results illustrated that the recovery yield and purification factor of lysozyme were 93.43% and 29.23 times (P-COOH); 90.72% and 36.22 times (P-SO3H), respectively. It was revealed that two type ion exchange membranes were very suitable as an adsorber for use in dye waste treatment and lysozyme purification process. P-SO3H strong ion-exchange membrane was more effective either removal of TBO dye or purification of lysozyme. The ion exchange membranes not only effectively purified lysozyme from CEW solution, but also effectively removed dye from wastewater.

Removal of cationic dye waste by nanofiber membrane immobilized with waste proteins.

Water pollution caused by dyes has been a serious problem affecting human health and environment. The surface of polyacrylonitrile (PAN) nanofiber membranes was modified by mild hydrolysis and coupled with bovine serum albumin (BSA) obtained from the laboratory wastes, resulting in the synthesis of P-COOH and P-COOH-BSA nanofibers. The nanofibers with specific functional groups may enhance their potential applications toward the removal of ionic dyes in wastewater. Toluidine blue O (TBO) was applied as an example of cationic dye to evaluate the removal efficiency of P-COOH-BSA nanofiber. Results showed that the equilibrium dissociation constant and maximum removal capacity were 0.48 mg/mL and 434.78 mg/g, respectively, at pH 12, where the TBO removal can be explained based on Langmuir isotherm and pseudo-second-order model. Desorption studies have shown that TBO adsorbed on P-COOH-BSA protein membrane can be completely eluted with either 1 M NaCl or 50% glycerol. The results of repeated studies indicated that after five consecutive adsorption/desorption cycles, the removal efficiency of TBO can be maintained at ~97%. P-COOH-BSA has shown to be promising adsorbent in TBO dye removal from dye wastewater.

Engineering Stable Pseudomonas putida S12 by CRISPR for 2,5-Furandicarboxylic Acid (FDCA) Production.

FDCA (2,5-furandicarboxylic acid) can be enzymatically converted from HMF (5-hydroxymethylfurfural). Pseudomonas putida S12 is promising for FDCA production, but generating stable P. putida S12 is difficult due to its polyploidy and lack of genome engineering tools. Here we showed that coupling CRISPR and λ-Red recombineering enabled one-step gene integration with high efficiency and frequency, and simultaneously replaced endogenous genes in all chromosomes. Using this approach, we generated two stable P. putida S12 strains expressing HMF/furfural oxidoreductase (HMFH) and HMF oxidase (HMFO), both being able to convert 50 mM HMF to ≈42-43 mM FDCA in 24 h. Cosupplementation of MnO2 and CaCO3 to the medium drastically improved the cell tolerance to HMF and enhanced FDCA production. Cointegrating HMFH and HMFT1 (HMF transporter) genes further improved FDCA production, enabling the cells to convert 250 mM HMF to 196 mM (30.6 g/L) FDCA in 24 h. This study implicates the potentials of CRISPR for generating stable P. putida S12 strains for FDCA production.