# Strategies for Enhancing Extracellular Electron Transfer in Environmental Biotechnology: A Review
## 3 Strategies for Enhancing EET in Environmental Biotechnology  
### Genetic Engineering Approaches  
(a) Overexpression of key EET genes  
Overexpression of specific EET genes can have a major impact on the efficiency and effectiveness of EET processes. Microorganisms can improve their ability to transmit electrons across cell membranes and interact with external electron acceptors or donors by boosting the expression levels of essential genes [34]. This strategy has been extensively studied and applied in various environmental biotechnology applications. MtrC, which encodes a _c_-type cytochrome protein found in _Shewanella_ species, is one example of a critical gene involved in EET. The upregulation of cytochrome c, specifically CymA, in the bacterium _S. oneidensis_ MR-1, has resulted in an enhancement of both bioelectricity production and cell proliferation within a microbial fuel cell [38]. To enhance the expression of cytochrome c and optimize electron transition, [PERSON] et al. [39] employed error-prone PCR to introduce random mutations into ccmH. Subsequently, they investigated the resulting increment in cytochrome synthesis. The findings of the study demonstrated that the introduction of the mutant ccmH gene significantly augmented the efficacy of the genetic circuit responsible for regulating EET, resulting in a notable 77% rise in current. The electron transfer process acquired by the metabolism of cells from the interior membrane protein, which is the CymA, to terminal periplasmic reductases is of significant importance in the chain of electron transport in _S. oneidensis_. When the CymA undergoes mutation, there was an observed reduction in the current generation by around 80%. On the other hand, MFCs that were inoculated with genetically modified strains that overexpress CymA demonstrated an increased maximum power output of 0.13 mW and a higher specific growth rate of 0.087 h\({}^{-1}\). In comparison, the wild-type MFCs had an optimal energy output of 0.11 mW and an increase of 0.043 h\({}^{-1}\)[38]. Enhancing EET can also be achieved by strategically improving the network of periplasmic cytochromes. A diverse array of c-type cytochromes is spatially scattered inside the periplasm, collectively establishing an intricate network for electron transport. To simplify the pathway of electrons in the periplasm and mitigate the possibility of the competitive response of MR-1, a study conducted gene knockouts of the periplasmic cytochrome genes, which are _nrfA_, _ccpA_, _napB_, and _napA_, and subsequently introduced the expression of cytochromes _cctA_ at the respective knockout locations. According to [PERSON] et al. [40], the findings of the study indicated a positive correlation between the copy number of _cctA_ and the EET rate. Moreover, it was observed that the current exhibited a 23% rise in response to this relationship. Hence, the simplification of the periplasmic cytochrome network has the potential to modify the extracellular redox potential and expedite the process of electron transfer. The overexpression of these key EET genes can be achieved through various genetic engineering techniques, such as plasmid-based gene expression systems or genome editing tools like CRISPR-Cas9 [41]. By manipulating the expression levels of these genes, it can be possible to fine-tune the EET capabilities of microorganisms and optimize their performance in environmental biotechnology applications. Exogenous EET refers to the introduction of external electron transfer mediators or genetically engineered microorganisms into an environment to enhance electron transfer capabilities. This process can be introduced through various means, such as the addition of redox-active compounds or the genetic modification of microorganisms to express specific EET-related proteins. The introduction of exogenous EET can have several benefits in environmental biotechnology. Firstly, it can enhance the overall efficiency of electron transfer processes.