Bioscience Evidence 2024, Vol.14, No.2, 81-92 http://bioscipublisher.com/index.php/be 85 CRISPR-Cas9, researchers can knock out genes that divert electrons away from hydrogen production or introduce mutations that enhance the activity of hydrogenases. This precision editing is essential for fine-tuning bacterial metabolic pathways to maximize hydrogen output (Jia et al., 2021). 4.1.2 Gene knockout and overexpression Gene knockout and overexpression are classic genetic engineering techniques that have been extensively used to manipulate the metabolic pathways of anaerobic bacteria. Gene knockout involves the deletion or inactivation of specific genes to eliminate undesirable pathways or reduce the production of competing by-products. Conversely, gene overexpression increases the activity of key enzymes involved in hydrogen production by introducing multiple copies of a gene or using strong promoters to drive higher expression levels. For example, overexpression of hydrogenase genes can significantly boost hydrogen production, while knocking out pathways that lead to the formation of lactate or ethanol can redirect metabolic flux toward hydrogen synthesis (Sekoai and Daramola, 2018). 4.1.3 Synthetic biology approaches Synthetic biology combines principles of engineering with molecular biology to design and construct new biological parts, devices, and systems. In the context of biohydrogen production, synthetic biology approaches involve the construction of artificial metabolic pathways, the design of synthetic promoters for controlled gene expression, and the creation of engineered bacterial strains with optimized metabolic networks. These approaches enable the assembly of novel pathways that do not exist naturally, thereby expanding the capabilities of anaerobic bacteria to produce hydrogen from a wider range of substrates and under various environmental conditions (Kracke et al., 2018). 4.2 Case studies of genetically modified anaerobic bacteria The application of genetic engineering to anaerobic bacteria has led to several successful modifications that have significantly improved biohydrogen production. 4.2.1 Enhanced hydrogen production One notable example is the modification of Clostridium acetobutylicum to overexpress [FeFe]-hydrogenases, resulting in a substantial increase in hydrogen yield. By knocking out competing pathways and enhancing the expression of hydrogenase genes, researchers were able to redirect the metabolic flux toward hydrogen production, achieving higher yields than wild-type strains (Yu et al., 2019). Similarly, Enterobacter cloacae has been engineered to improve its tolerance to oxygen, allowing for more robust hydrogen production under less stringent anaerobic conditions (Lee et al., 2019). 4.2.2 Improved pathway efficiencies Another case study involves the use of CRISPR-Cas9 to edit Thermotoga maritima, a thermophilic bacterium known for its ability to produce hydrogen at high temperatures. By knocking out specific genes involved in competing metabolic pathways, researchers enhanced the efficiency of the butyrate and acetate pathways, leading to improved hydrogen production under industrial conditions (Saidi et al., 2018). Additionally, synthetic biology techniques have been applied to construct artificial operons that combine multiple hydrogen production pathways into a single, highly efficient system, further optimizing the metabolic processes involved (Kracke et al., 2018). 4.3 Challenges and limitations Despite the successes achieved through genetic engineering, several challenges and limitations remain. One significant challenge is the complexity of metabolic networks in anaerobic bacteria. The introduction of genetic modifications can lead to unintended effects, such as the accumulation of toxic intermediates or the disruption of cellular homeostasis, which can negatively impact overall hydrogen production. Another limitation is the difficulty in achieving stable expression of engineered pathways, especially under industrial conditions where factors such as temperature, pH, and substrate availability can vary. Additionally, the
RkJQdWJsaXNoZXIy MjQ4ODYzMg==