CRISPR/Cas12a-Mediated Genome Editing in Thioalkalivibrio versutus.

Haloalkaliphilic Thioalkalivibrio versutus, a dominant species for sulfide removal, has attracted increasing attention. However, research on T. versutus is limited by the lack of genetic manipulation tools. In this work, we developed a CRISPR/AsCas12a-mediated system in T. versutus for an efficient and implementable genome editing workflow. Compared to the CRISPR/Cas9-mediated system, the CRISPR/AsCas12a system exhibited enhanced editing efficiency. Additionally, as Cas12a is capable of processing the crRNA maturation independently, the CRISPR/AsCas12a system allowed multiplex gene editing and large-fragment DNA knockout by expressing more than one crRNA under the control of one promoter. Using the CRISPR/AsCas12a system, five key genes of the elemental sulfur oxidation pathway were knocked out. Simultaneous deletion of the rhd and tusA genes disrupted the ability of T. versutus to metabolize elemental sulfur, resulting in a 24.7% increase in elemental sulfur generation and a 15.2% reduction in sulfate production. This genome engineering strategy significantly improved our understanding of sulfur metabolism in Thioalkalivibrio spp.

Hydrophobic cell surface display system of PETase as a sustainable biocatalyst for PET degradation.

Remarkably, a hydrolase from Ideonella sakaiensis 201-F6, termed PETase, exhibits great potential in polyethylene terephthalate (PET) waste management due to it can efficiently degrade PET under moderate conditions. However, its low yield and poor accessibility to bulky substrates hamper its further industrial application. Herein a multigene fusion strategy is introduced for constructing a hydrophobic cell surface display (HCSD) system in Escherichia coli as a robust, recyclable, and sustainable whole-cell catalyst. The truncated outer membrane hybrid protein FadL exposed the PETase and hydrophobic protein HFBII on the surface of E. coli with efficient PET accessibility and degradation performance. E. coli containing the HCSD system changed the surface tension of the bacterial solution, resulting in a smaller contact angle (83.9 ± 2° vs. 58.5 ± 1°) of the system on the PET surface, thus giving a better opportunity for PETase to interact with PET. Furthermore, pretreatment of PET with HCSD showed rougher surfaces with greater hydrophilicity (water contact angle of 68.4 ± 1° vs. 106.1 ± 2°) than the non-pretreated ones. Moreover, the HCSD system showed excellent sustainable degradation performance for PET bottles with a higher degradation rate than free PETase. The HCSD degradation system also had excellent stability, maintaining 73% of its initial activity after 7 days of incubation at 40°C and retaining 70% activity after seven cycles. This study indicates that the HCSD system could be used as a novel catalyst for efficiently accelerating PET biodegradation.

Potential Use of Microbial Enzymes for the Conversion of Plastic Waste Into Value-Added Products: A Viable Solution.

The widespread use of commercial polymers composed of a mixture of polylactic acid and polyethene terephthalate (PLA-PET) in bottles and other packaging materials has caused a massive environmental crisis. The valorization of these contaminants via cost-effective technologies is urgently needed to achieve a circular economy. The enzymatic hydrolysis of PLA-PET contaminants plays a vital role in environmentally friendly strategies for plastic waste recycling and degradation. In this review, the potential roles of microbial enzymes for solving this critical problem are highlighted. Various enzymes involved in PLA-PET recycling and bioconversion, such as PETase and MHETase produced by Ideonella sakaiensis; esterases produced by Bacillus and Nocardia; lipases produced by Thermomyces lanuginosus, Candida antarctica, Triticum aestivum, and Burkholderia spp.; and leaf-branch compost cutinases are critically discussed. Strategies for the utilization of PLA-PET's carbon content as C1 building blocks were investigated for the production of new plastic monomers and different value-added products, such as cyclic acetals, 1,3-propanediol, and vanillin. The bioconversion of PET-PLA degradation monomers to polyhydroxyalkanoate biopolymers by Pseudomonas and Halomonas strains was addressed in detail. Different solutions to the production of biodegradable plastics from food waste, agricultural residues, and polyhydroxybutyrate (PHB)-accumulating bacteria were discussed. Fuel oil production via PLA-PET thermal pyrolysis and possible hybrid integration techniques for the incorporation of thermostable plastic degradation enzymes for the conversion into fuel oil is explained in detail.

Recent advances in microbial capture of hydrogen sulfide from sour gas via sulfur-oxidizing bacteria.

Biological desulfurization offers several remarkably environmental advantages of operation at ambient temperature and atmospheric pressure, no demand of toxic chemicals as well as the formation of biologically re-usable sulfur (S0), which has attracted increasing attention compared to conventionally physicochemical approaches in removing hydrogen sulfide from sour gas. However, the low biomass of SOB, the acidification of process solution, the recovery of SOB, and the selectivity of bio-S0 limit its industrial application. Therefore, more efforts should be made in the improvement of the BDS process for its industrial application via different research perspectives. This review summarized the recent research advances in the microbial capture of hydrogen sulfide from sour gas based on strain modification, absorption enhancement, and bioreactor modification. Several efficient solutions to limitations for the BDS process were proposed, which paved the way for the future development of BDS industrialization.

Revealing sulfate role in empowering the sulfur-oxidizing capacity of Thioalkalivibrio versutus D301 for an enhanced desulfurization process.

Haloalkaliphilic Thioalkalivibrio, a dominant genus for sulfide removal, has attracted growing interest. However, the bacterial biological response to this process's final product, sulfate, has not been well-studied. Here, thiosulfate oxidation and sulfur formation by T. versutus D301 were being enhanced with increasing sulfate supply. With the addition of 0.73 M sulfate, the thiosulfate utilization rate and sulfur production were improved by 68.1% and 120.1% compared with carbonate-grown control at the same salinity (1.8 M). For sulfate-grown cells, based on metabolic analysis, the downregulation of central carbon metabolism indicated that sulfate triggered a decrease in energy conservation efficiency. Additionally, the gene expression analysis further revealed that sulfate induced the inhibition of sulfur to sulfate oxidation, causing the upregulation of thiosulfate to sulfur oxidation for providing cells with additional energy. This study enhances researchers' understanding regarding the sulfate effect on the bio-desulfurization process and presents a new perspective of optimizing the biotechniques.

Recent advances in biocatalysts engineering for polyethylene terephthalate plastic waste green recycling.

The massive waste of poly(ethylene terephthalate) (PET) that ends up in the landfills and oceans and needs hundreds of years for degradation has attracted global concern. The poor stability and productivity of the available PET biocatalysts hinder their industrial applications. Active PET biocatalysts can provide a promising avenue for PET bioconversion and recycling. Therefore, there is an urgent need to develop new strategies that could enhance the stability, catalytic activity, solubility, productivity, and re-usability of these PET biocatalysts under harsh conditions such as high temperatures, pH, and salinity. This has raised great attention in using bioengineering strategies to improve PET biocatalysts' robustness and catalytic behavior. Herein, historical and forecasting data of plastic production and disposal were critically reviewed. Challenges facing the PET degradation process and available strategies that could be used to solve them were critically highlighted and summarized. In this review, we also discussed the recent progress in enzyme bioengineering approaches used for discovering new PET biocatalysts, elucidating the degradation mechanism, and improving the catalytic performance, solubility, and productivity, critically assess their strength and weakness and highlighting the gaps of the available data. Discovery of more potential PET hydrolases and studying their molecular mechanism extensively via solving their crystal structure will widen this research area to move forward the industrial application. A deeper knowledge of PET molecular and degradation mechanisms will give great insight into the future identification of related enzymes. The reported bioengineering strategies during this review could be used to reduce PET crystallinity and to increase the operational temperature of PET hydrolyzing enzymes.

Improving confirmed nanometric sulfur bioproduction using engineered Thioalkalivibrio versutus.

Complicated production procedures and superior characteristics of nano-sized sulfur elevate its price to 25-40 fold higher than micrograde kind. Also, natural gas hydrogen sulfide levels are restricted because of its toxic environmental consequences. Thioalkalivibrio versutus is a polyextremophilic industrial autotroph with high natural gas desulfurization capability. Here, nanometric (>50 nm) sulfur bioproduction using T. versutus while desulfurizing natural gas was validated. Also, this production was enhanced by 166.7% via lowering sulfate production by 55.1%. A specially-developed CRISPR system, with 42% editing efficiency, simplified the genome editing workflow scheme for this challenging bacterium. In parallel, sulfur metabolism was uncovered using proteins mining and transcriptome studies for defining sulfate-producing key genes (heterodisulfide reductase-like complex, sulfur dioxygenase, sulfite dehydrogenase and sulfite oxidase). This study provided cost-effective nanometric sulfur production and improved this production using a novel CRISPR strategy, which could be suitable for industrial polyextremophiles, after uncovering sulfur pathways in T. versutus.