Design introduction

Overview

This project harnesses the unique capabilities of Acidithiobacillus ferrooxidans as a chassis organism to develop an innovative biotechnological approach for the selective extraction of gold from pyrite. By constructing three specialized modules, we aim to enhance bioleaching efficiency while ensuring environmental safety.

Module 1: c-di-GMP Module

The first module focuses on boosting the levels of cyclic di-GMP (c-di-GMP) within Acidithiobacillus ferrooxidans. Elevated c-di-GMP concentrations promote robust biofilm formation, which is critical for enhancing the microorganism's ability to leach metals from mineral substrates. While this module significantly improves bioleaching efficiency, it is essential to recognize that the increase in c-di-GMP may have non-specific effects, potentially influencing various biological processes within the microbial community. For example, high levels of c-di-GMP reduce bacterial growth.

Module 2: Gold-Responsive Module

The second module is designed to enable the specific detection and extraction of gold. This module is strategically positioned upstream of the c-di-GMP module to ensure coordinated operation. When the presence of gold is sensed, this module activates biofilm formation, thereby maximizing the efficiency of gold extraction. This targeted approach is crucial for minimizing the impact on non-target metals and optimizing the recovery of gold from pyrite.

Module 3: Suicide Safety Module

Safety is a paramount concern in any biotechnological application. The suicide safety module serves to regulate the growth and metabolic activity of A. ferrooxidans. It is programmed to trigger a self-destruction mechanism under specific conditions, such as the absence of gold or exposure to adverse environmental factors. This precautionary measure is designed to mitigate potential ecological risks associated with the release of genetically modified organisms into the environment.

By integrating these three modules, our project aims to provide a sustainable and efficient method for gold extraction from pyrite using A. ferrooxidans. This approach not only enhances the recovery of valuable resources but also prioritizes environmental safety, making it a promising solution for bioremediation and bioleaching applications. Our research paves the way for further advancements in microbial mining technologies, contributing to more sustainable practices in the resource extraction industry.

Background Why we chose Acidithiobacillus ferrooxidans

Biomining involves two processes - biological leaching and biological oxidation. In bioleaching, microorganisms dissolve metal sulfides (MS) through metabolic activity, releasing the target metal into the solution. Biological oxidation occurs in the process of bioleaching, which exposes the target metal by metabolic activities of microorganisms to facilitate further extraction of the metal. In biomining, the oxidation pathway of iron and sulfur during metabolic activity can produce Fe³⁺ and H⁺ to decompose minerals, releasing the target metal into the solution, that’s why the process is crucial. They can also provide the extreme environment required for cell survival, and also provide the energy required for microbial growth^[1].

Bioleaching is the key link in biomining, which is divided into two groups of contact leaching and non-contact leaching. In the contact leaching, Extracellular Polymeric Substances (EPS) produced by microorganisms and the enriched positive charges Fe³⁺ and H⁺ promote cells to attach to the surface of MS to form biofilms. The enriched Fe³⁺ and H⁺ by biofilms further promote the attack on MS, catalyzing its dissolution and releasing Fe²⁺, reduced inorganic sulfur compounds (RISCs) and target metals^[2]. Therefore, improving the EPS produced by mining microorganisms through genetic engineering can significantly improve the efficiency of biological mining^[3][4].

In our project, Acidithiobacillus ferrooxidans ATCC 23270, the contact leaching type strain, is selected. A. ferrooxidans can directly adsorb on mineral surfaces to form biofilm, which can improve leaching efficiency^[6]. A. ferrooxidans forms a monolayer biofilm composed by bacterial cells embedded in an EPS matrix whose role is fundamental in creating a special microenvironment that favors mineral dissolution by oxidation^[7][8]. A. ferrooxidans can be used to leach gold from pyrite, which naturally occurs in large quantities in the form of monomers^[9]. According to statistics, more than 5% of the world's gold is produced in this way every year^[5]. Therefore, we plan to optimize the biofilm formation of A. ferrooxidans to enhance the bioleaching.

Strategy 1 Improving biomining efficiency by modulating the c-di-GMP pathway

Bacteria switch their lifestyles between planktonic and sessile modes in response to various environmental cues. This switch is often regulated by sophisticated intracellular signaling networks that modulate the levels of small molecules. Among them, c-di-GMP networks is the most-studied chemical signaling systems.

The c-di-GMP monomer exhibits two-fold symmetry, with two GMP moieties that are fused by a 5'-3' macrocyclic ring. The synthesis of c-di-GMP is catalysed by diguanylate cyclases (DGCs) through the cooperative action of their two catalytic GGDEF domains. Specific phosphodiesterases (PDEs) that contain EAL or HD-GYP domains hydrolyse c-di-GMP into GMP. Through binding to effector molecules, c-di-GMP regulates diverse cellular processes, including motility, adherence, biofilm formation, virulence, development and cell cycle progression^[10].

Earlier studies have shown that c-di-GMP plays an important role in mediating several cellular functions and various aspects of bacterial physiology ^[11][12]. High levels of c-di-GMP promote biofilm formation by increasing cell aggregation and exopolysaccharide production, whereas low levels of c-di-GMP reduce biofilm formation by decreasing bacterial motility and extracellular DNA production^[13].

Current research has demonstrated the presence of a c-di-GMP (cyclodiguanosine monophosphate) metabolic pathway in the genome of Acidithiobacillus ferrooxidans, which includes five open reading frames (ORFs) that encode metabolic enzymes, diguanylate cyclases (DGCs), and phosphodiesterases (PDEs) involved in the c-di-GMP pathway. Details are presented in Table 1^[14] .

Gene	Identified domains	Identitied function
AFE_0053	GGDEF/EAL	DGC
AFE_1360	GGDEF/EAL	DGC
AFE_1373	GGDEF/EAL	DGC
AFE_1379	GGDEF/EAL	DGC
AFE_1852	EAL	PDE

Furthermore, existing research has found that overexpression of the Escherichia coli DGC (YedQ) in Comamonas testosteroni can significantly increase the concentration of c-di-GMP and enhance the biofilm formation process, with a concurrent increase in the concentration of extracellular polymeric substances (EPS)^[15]. We have optimized the codon usage of the yedQ gene, a highly efficient c-di-GMP synthesis module, and then introduced it along with the DGC from the Acidithiobacillus ferrooxidans genome into the target bacteria. The aim is to enhance the biofilm formation efficiency of the bacterium through the overexpression of DGC, thereby improving the efficiency of bioleaching processes.

Strategy 2 Specific biomining to Aurum

Compared to traditional industrial leaching for metal extraction, bio-mining technology is characterized by low energy consumption, mild conditions, and environmental friendliness^[5][6].However, creating acidic conditions for acidophilic mining microorganisms to complete leaching still requires a significant amount of cost and energy, which greatly reduces the economic value created by microbial mining. Therefore, it is urgent to develop a technical method for recycling high-value precious metals.

From ancient times to the present, gold has always been a highly regarded precious metal, not only because of its high value but also because of its wide application and research in jewelry, nano-domains, and biomedicine^[16]. In most natural environments, the occurrence of gold is always accompanied by small amounts of copper or silver. Selectively and effectively distinguishing gold from other metals is often a challenging issue^[17]. Although there are various physicochemical methods available for the detection of heavy metal ions, including gold, these methods generally rely on expensive equipment and complex procedures, making them impractical for use outside the laboratory environment. Therefore, metal biosensors with high selectivity, high sensitivity, and in-situ monitoring capabilities have come into view with their unique advantages^[18][19].

We have noticed from existing research the specific gold-binding MerR family transcription factor GolS, which has been identified in Salmonella sp. bacteria. This specific gold-binding protein can achieve specific binding of Au[III] and activate the transcription of downstream genes^[17]. Based on this, we designed genetic circuits (such as enhancing the iron-sulfur metabolic pathway to provide more energy for microorganisms, enhancing the expression of biofilms to improve leaching efficiency, designing more efficient extracellular electron transfer channels to accelerate the exposure of target metals, and adding biomineralization modules to make target metals easier to extract, etc.)^[5], and carried out synthetic biology modification of mining microorganisms, which can further expand the functions of mining microorganisms. This design is applied to engineered, standardized synthetic biology technology, which will create more possibilities for the future of synthetic biology in the field of bio-mining. In this project, we designed genetic circuits and tested the specificity and sensitivity of GolS binding to Au[III].

Strategy Killing Switch

For different applications of our engineered bacteria, we designed different kill switches to ensure bio-safety.

We introduce the inverter into our kill switch systems which consist of a cI repressor and a P_R from E. coli λ phage, where cI repressor can inhibit P_R promoter. In this case, activation of the upstream promoter of cI repressor will lead to high expression of it, thereby reducing the expression efficiency of downstream P_R promoter. By using an inverter, inducible promoters can obtain two-way uses, while reducing the complexity of circuit construction and eliminating the need of precise control of steady state. It is a basis of our kill switches design.

Since our engineered bacteria in detection system will work in a certain device, we need to make the engineered bacteria survive only in a special environment where some particular substances exist.

We introduce the inverter to make engineered bacteria survive when certain inducers are present and die when they do not exist. The circuit in the Fig 6. could make engineered bacteria survive when the concentration of IPTG and Au[Ⅲ] is high enough, but die when is low. Once engineered bacteria escape to the external environment, or the concentration of Au[Ⅲ] in the environment is very low , the expression of mazF will cause the engineered bacteria die due to MazF protein's toxicity.

Reference
[1] Zhan, Y., Yang, M., Zhang, S., Zhao, D., Duan, J., Wang, W., Yan, L., 2019. Iron and sulfur oxidation pathways of Acidithiobacillus ferrooxidans. World J. Microbiol. Biotechnol. 35, 1–12.
[2] Srichandan, H., Mohapatra, R.K., Parhi, P.K., Mishra, S., 2019. Bioleaching approach for extraction of metal values from secondary solid wastes: a critical review. Hydrometallurgy 189, 105122.
[3] Kaksonen, A.H., Lakaniemi, A.M., Tuovinen, O.H., 2020. Acid and ferric sulfate bioleaching of uranium ores: a review. J. Clean. Prod., 121586
[4] Vardanyan, N., Badalyan, H., Markosyan, L., Vardanyan, A., Zhang, R., Sand, W., 2020. Newly isolated Acidithiobacillus sp. Ksh from Kashen copper ore: peculiarities of EPS and colloidal exopolysaccharide. Front. Microbiol. 11, 1802.
[5] Chen J, Liu Y, Diep P, et al. Genetic engineering of extremely acidophilic Acidithiobacillus species for biomining: Progress and perspectives[J]. Journal of Hazardous Materials, 2022, 438: 129456.
[6] Rohwerder, T., Gehrke, T., Kinzler, K. and Sand, W. (2003) Bioleaching review part A: progress in bioleaching: fundamentals and mechanisms of bacterial metal sulfide oxidation. Appl Microbiol Biotechnol 63, 239–248.
[7] Harneit, K., Goksel, A., Kock, D., Klock, J.H., Gehrke, T. and Sand, W. (2006) Adhesion to metal sulfide surfaces by cells of Acidithiobacillus ferrooxidans,Acidithiobacillus thiooxidans and Leptospirillum ferrooxidans. Hydrometallurgy 83, 245–254.
[8] Sand, W. and Gehrke, T. (2006) Extracellular polymeric substances mediate bioleaching ⁄ biocorrosion via interfacial processes involving iron(III) ions and acidophilic bacteria. Res Microbiol 157, 49–56.
[9] Johnson D B. Biomining—biotechnologies for extracting and recovering metals from ores and waste materials[J]. Current opinion in biotechnology, 2014, 30: 24-31.
[10] Jenal U, Reinders A, Lori C. Cyclic di-GMP: second messenger extraordinaire[J]. Nat Rev Microbiol, 2017,15(5):271-284.
[11] Cotter P A, Stibitz S. c-di-GMP-mediated regulation of virulence and biofilm formation[J]. Curr Opin Microbiol, 2007,10(1):17-23.
[12] Hengge R. Principles of c-di-GMP signalling in bacteria[J]. Nat Rev Microbiol, 2009,7(4):263-273.
[13] Hu Y, Wu Y, Mukherjee M, et al. A near-infrared light responsive c-di-GMP module-based AND logic gate in Shewanella oneidensis[J]. Chem Commun (Camb), 2017,53(10):1646-1648.
[14] Ruiz L M, Castro M, Barriga A, et al. The extremophile Acidithiobacillus ferrooxidans possesses ac‐di‐GMP signalling pathway that could play a significant role during bioleaching of minerals[J]. Letters in applied microbiology, 2012, 54(2): 133-139.
[15] Yang S, Wu Y, Qu C, et al. Quantitative analysis of the surficial and adhesion properties of the Gram-negative bacterial species Comamonas testosteroni modulated by c-di-GMP[J]. Colloids Surf B Biointerfaces, 2021,198:111497.
[16] Dai B, Wang Q, Yu F, et al. Effect of Au nano-particle aggregation on the deactivation of the AuCl3/AC catalyst for acetylene hydrochlorination[J]. Scientific reports, 2015, 5(1): 10553.
[17] Wei W, Zhu T, Wang Y, et al. Engineering a gold-specific regulon for cell-based visual detection and recovery of gold[J]. Chemical Science, 2012, 3(6): 1780-1784.
[18] Verma N, Singh M. Biosensors for heavy metals[J]. Biometals, 2005, 18: 121-129.
[19] Kim H J, Jeong H, Lee S J. Synthetic biology for microbial heavy metal biosensors[J]. Analytical and bioanalytical chemistry, 2018, 410: 1191-1203.