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The use of synthetic biology to construct engineered strains has provided new perspectives for addressing Pb contamination; however, the large-scale treatment of contaminants is still limited by high operating costs and technological constraints. This study introduces a novel technique for applying engineered yeast in the removal of heavy metals, offering a solution to the cost and process scale challenges associated with utilizing engineered yeast. Hydrogen sulfide-producing engineered yeast strains were constructed based on existing strategies by knocking out the gene encoding the O-acetyl-L-homoserine mercapturic enzyme, which plays a role in sulfate assimilation. To facilitate the transition of engineered yeast from laboratory settings to industrial applications while reducing operating costs and addressing process scale-up issues, we proposes a new operational technology for engineered yeast based on their mechanistic understanding and a response surface optimization approach. The development and application of low-cost engineered media provide important guidance for utilizing engineered yeast to tackle Pb-contaminated wastewater and for the production of PbS crystalline nanomaterials. The industrial culture system was designed using economical materials and, through the response surface methodology, achieved removal rates of 99.02 ± 0.06 % and 80.95 ± 9.68 % of Pb²⁺ from Pb acid electrolyte and industrial Pb wastewater, respectively. This study presents a new technological solution for cost control and process scale-up based on the bioregulatory mechanisms of engineered yeast, laying the groundwork for their industrial application. Furthermore, it offers essential parameters and theoretical support for the industrial applications of engineered yeast in Pb wastewater treatment. [Display omitted] • Revealing the mechanism of H 2 S production in engineered yeast at the protein level. • Theoretical support for efficiently constructing strain to treat Pb-containing wastewater. • Mechanisms of H 2 S production and regulation associated with the MET17 gene in yeast. • Development of low-cost media to alleviate the cost of industrial application of engineered strains. • Further theoretical and applied development of engineered yeasts producing H 2 S. [ABSTRACT FROM AUTHOR]

Summary: A LuxI/R‐like quorum sensing (QS) system (AfeI/R) has been reported in the acidophilic and chemoautotrophic Acidithiobacillus spp. However, the function of AfeI/R remains unclear because of the difficulties in the genetic manipulation of these bacteria. Here, we constructed different afeI mutants of the sulfur‐ and iron‐oxidizer A. ferrooxidans, identified the N‐acyl homoserine lactones (acyl‐HSLs) synthesized by AfeI, and determined the regulatory effects of AfeI/R on genes expression, extracellular polymeric substance synthesis, energy metabolism, cell growth and population density of A. ferrooxidans in different energy substrates. Acyl‐HSLs‐mediated distinct regulation strategies were employed to influence bacterial metabolism and cell growth of A. ferrooxidans cultivated in either sulfur or ferrous iron. Based on these findings, an energy‐substrate‐dependent regulation mode of AfeI/R in A. ferrooxidans was illuminated that AfeI/R could produce different types of acyl‐HSLs and employ specific acyl‐HSLs to regulate specific genes in response to different energy substrates. The discovery of the AfeI/R‐mediated substrate‐dependent regulatory mode expands our knowledge on the function of QS system in the chemoautotrophic sulfur‐ and ferrous iron‐oxidizing bacteria, and provides new insights in understanding energy metabolism modulation, population control, bacteria‐driven bioleaching process, and the coevolution between the acidophiles and their acidic habitats. [ABSTRACT FROM AUTHOR]

Efficient biodiesel production from biomass is essential for the green-oriented energy transition. The current incongruity between fermentation temperature and microbial thermotolerance is one of the key factors restricting biodiesel production, and it is difficult to solve this problem relying only on conventional microbial characterization and breeding methods. We propose a strategy to solve the corn stover cellulose utilization problem by developing thermotolerant biodiesel-production yeast strains. Candida tropicalis was initially improved by adaptive laboratory evolution and then mutated by atmospheric and room-temperature plasma mutagenesis to obtain a thermotolerant strain capable of growing at temperatures up to 45 °C. Two rounds of fluorescence-activated cell sorting were used to select high lipid content cells, which resulted in a 16.8% increase in lipid accumulation capacity compared to the original cells. The simultaneous saccharification fermentation of the generated strain with pretreated corn stover at 44 °C yielded a lipid generation efficiency of 86.2 ± 2 mg/g. This study provides a new method and an alternative strain for the selection and breeding of high-thermotolerance and high biodiesel-production yeasts and offers a feasible strategy for lignocellulosic fermentation for biodiesel production at elevated temperatures using the new industrial strain. [Display omitted] • A new strategy for lignocellulosic fermentation for biodiesel production. • Provides a new method and alternative strain, addressing the temperature asymmetry between microbial and enzyme catalytic (enzymatic) activity during fermentation. • Developing thermotolerant biodiesel-production yeast strains capable of growing at temperatures up to 45 °C. [ABSTRACT FROM AUTHOR]

The sulfur oxidization (Sox) system is the central sulfur oxidization pathway of phototrophic and chemotrophic sulfur-oxidizing bacteria. Regulation and function of the Sox system in the chemotrophic Paracoccus pantotrophus has been elucidated; however, to date, no information is available on the regulation of this system in the chemolithotrophic Acidithiobacillus caldus, which is widely utilized in bioleaching. We described the novel tspSR- sox-like clusters in A. caldus and other chemolithotrophic sulfur-oxidizing bacteria containing Sox systems. The highly homologous σ-dependent two-component signaling system (TspS/R), upstream of the sox operons in these novel clusters, was identified by phylogenetic analyses. A typical σ-dependent promoter, P, was identified upstream of soxX-I in the sox-I cluster of A. caldus MTH-04. The transcriptional start site (G) and the −12/−24 regions (GC/GG) of P were determined by rapid amplification of cDNA ends (5′RACE), and the upstream activator sequences (UASs; TGTCCCAAATGGG ACA) were confirmed by electrophoretic mobility shift assays (EMSAs) in vitro and by UAS-probe-plasmids assays in vivo. Sequence analysis of promoter regions in tspSR- sox-like clusters revealed that there were similar σ-dependent promoters upstream of the soxX genes. Based on our results, we proposed a TspSR-mediated signal transduction and transcriptional regulation pathway for the Sox system in A. caldus. The regulation of σ-dependent two-component systems (TCSs) for Sox pathways were explained for the first time in A. caldus, A. thiooxidans, T. tepidarius, and T. denitrificans, indicating the significance of modulating the sulfur oxidization in these chemolithotrophic sulfur oxidizers. [ABSTRACT FROM AUTHOR]

Acidithiobacillus ferrooxidans is an acidophilic and chemolithotrophic sulfur- and iron-oxidizing bacterium that has been widely used in the bioleaching process for extracting metals. Extracellular polymeric substances (EPS) are essential for bacteria-ore interactions, and the regulation of EPS synthesis could be an important way of influencing the efficiency of the bioleaching process. Therefore, exploring and utilizing the regulatory pathways of EPS synthesis to improve the bacterial bioleaching capability have posed a challenge in the study and application of bioleaching bacteria. Here, several engineering strains were constructed using genetic manipulation methods. And we revealed the regulatory function of the AfeI/R quorum sensing (QS) system in EPS synthesis and biofilm formation of A. ferrooxidans, and the AfeI/R-mediated EPS synthesis could influence bacteria-substrate interactions and the efficiency of bioleaching. Finally, an AfeI/R-mediated bioleaching model was proposed to illustrate the role of QS system in this process. This study provided new insights into and clues for developing highly efficient bioleaching bacteria and modulating the bioleaching process. [ABSTRACT FROM AUTHOR]