Current petroleum mining and conversion to essential fuels and chemicals have drastic environmental consequences, contributing to climate change. In addition, fossil fuels are finite resources, with a fast-approaching shortage. Accordingly, research efforts are increasingly focusing on developing sustainable alternatives for chemical and fuel production. In this context, bioprocesses, relying on microorganisms, have gained particular interest. For example, acetogens use the Wood-Ljungdahl pathway to grow on single carbon C1-gases (CO2 and CO) as their sole carbon source and produce valuable products, such as acetate or ethanol. These autotrophs can, therefore, be exploited for large-scale fermentation processes to produce industrially relevant chemicals from abundant greenhouse gases. In addition, genetic tools have recently been developed to improve these chassis organisms through synthetic biology approaches. This review will focus on the challenges of genetically and metabolically modifying acetogens. It will first discuss the physical and biochemical obstacles complicating successful DNA transfer in these organisms. Current genetic tools developed for several acetogens, crucial for strain engineering to consolidate and expand their catalogue of products, will then be described. Recent tool applications for metabolic engineering purposes to allow redirection of metabolic fluxes or production of non-native compounds will lastly be covered.

The current economic and environmental context requests an accelerating development of sustainable alternatives for the production of various target compounds. Biological processes offer viable solutions and have gained renewed interest in the recent years. For example, photosynthetic chassis organisms are particularly promising for bioprocesses, as they do not require biomass-derived carbon sources and contribute to atmospheric CO2 fixation, therefore supporting climate change mitigation. Marine cyanobacteria are of particular interest for biotechnology applications, thanks to their rich diversity, their robustness to environmental changes, and their metabolic capabilities with potential for therapeutics and chemicals production without requiring freshwater. The additional cyanobacterial properties, such as efficient photosynthesis, are also highly beneficial for biotechnological processes. Due to their capabilities, research efforts have developed several genetic tools for direct metabolic engineering applications. While progress toward a robust genetic toolkit is continuously achieved, further work is still needed to routinely modify these species and unlock their full potential for industrial applications. In contrast to the understudied marine cyanobacteria, genetic engineering and synthetic biology in freshwater cyanobacteria are currently more advanced with a variety of tools already optimized. This mini-review will explore the opportunities provided by marine cyanobacteria for a greener future. A short discussion will cover the advances and challenges regarding genetic engineering and synthetic biology in marine cyanobacteria, followed by a parallel with freshwater cyanobacteria and their current genetic availability to guide the prospect for marine species.

Replicating plasmid shuttle vectors are key tools for efficient genetic and metabolic engineering applications, allowing the development of sustainable bioprocesses using non-model organisms with unique metabolic capabilities. To date, very limited genetic manipulation has been achieved in the thermophilic acetogen, Moorella thermoacetica, partly due to the lack of suitable shuttle vectors. However, M. thermoacetica has considerable potential as an industrial chassis organism, which can only be unlocked if reliable and effective genetic tools are in place. This study reports the construction of a replicating shuttle vector for M. thermoacetica through the identification and implementation of a compatible Gram-positive replicon to allow plasmid maintenance within the host. Although characterisation of plasmid behaviour proved difficult, the designed shuttle vector was subsequently applied for ethanologenesis, i.e., ethanol production in this organism. The non-native ethanologenesis in M. thermoacetica was achieved via plasmid-borne overexpression of the native aldh gene and heterologous expression of Clostridium autoethanogenum adhE1 gene. This result demonstrates the importance of the developed replicating plasmid vector for genetic and metabolic engineering efforts in industrially important M. thermoacetica.