The human microbiota encompasses a diverse collection of microorganisms on and within our body. This includes bacteria, archaea, bacteriophages, eukaryotic viruses, and fungi. Among these, microorganisms inhabiting the gastrointestinal tract form the largest and most diverse cluster (Fan and Pedersen, 2021). It is estimated that the ratio of human to bacterial cells is approximately 1:1 to 1.3 (Sender et al., 2016), and the microbiome carries nearly three orders of magnitude more genes than the human genome (Tierney et al., 2019). The gut microbiota has coevolved with its host, with the host providing a stable environment for microbes, while microbes in turn have profound effects on host biology. They participate in the digestion of complex dietary macronutrients, produce various compounds, influence the host's immune system, regulate gut endocrine function and neurological signalling, modulate drug action and metabolism, and help eliminate toxins. Multiple lines of evidence have demonstrated that disruption of gut microbiome balance, known as dysbiosis, is linked to a wide range of diseases including gastrointestinal disorders and neurological, respiratory, metabolic, hepatic, and cardiovascular illnesses (Koh et al., 2016; Lynch and Pedersen, 2016).

The microbiota influences human health and disease by producing metabolites that can either be harmful and contribute to disease development, or beneficial and provide protection against disease. To date, only a few metabolites generated by the microbiota have been identified. One important process involving the microbiota is the metabolism of dietary fibre (Makki et al., 2018), which leads to the production of short-chain fatty acids (SCFAs), specifically acetate, propionate, and butyrate, and these SCFAs have wide-ranging effects on various aspects of host physiology (Sanna et al., 2019; Tanes et al., 2021). In addition, breakdown of aromatic amino acids from proteins and peptides in the diet by microbial activity can generate diverse metabolites with the potential to regulate immune, metabolic, and neuronal functions (Neis et al., 2015; Roager and Licht, 2018). Furthermore, the microbiota can metabolise dietary choline, phosphatidylcholine, betaine and l-carnitine, producing trimethylamine (TMA). TMA is then converted to trimethylamine N-oxide by flavin monooxygenase 3 in the liver, and this conversion has been associated with multiple cardiometabolic diseases (Brown and Hazen, 2018; Zeisel and Warrier, 2017). Moreover, the microbiota has the capacity to metabolise primary bile acids (BAs), which are host-derived molecules, into secondary BAs through various chemical modifications such as dehydroxylation, oxidation, and epimerisation. These secondary BAs play a vital role in energy balance, host metabolism, and maintaining immune function (Jia et al., 2018).

Phylogenetically, the majority of gut microbes are classified into the Bacteroidetes and Clostridia groups, which together make up more than 80% of the total microbiome (Arumugam et al., 2011). These microorganisms play a crucial role in host physiology by producing a wide variety of metabolites (Donia and Fischbach, 2015; Schroeder and Backhed, 2016). Although culture-independent techniques such as 16S rRNA analysis and multi-omics approaches have been extensively used to investigate the associations between microbial metabolites and host biology, a deeper understanding of the mechanisms underlying the interactions between metabolites and the host/microbiota is needed (Koh and Backhed, 2020). To acquire this knowledge, it is essential to have available genome editing toolbox for manipulating these commensal bacteria. Driven by their significant contributions to human health and the availability of whole genomes, genome editing tools have been developed for Bacteroides spp. and Clostridium spp. to better understand the functions of the microbiome and its interactions with the host. A phylogenetic tree of representative species is shown in Fig. 1. Traditional mutagenesis methods, including homologous recombination-based systems (Bencivenga-Barry et al., 2020; Koropatkin et al., 2008), transposon-based gene mutation systems (Liu et al., 2021; Wu et al., 2015), and phage integrase-assisted genome integration systems (Mimee et al., 2016) have been established for these species. In addition, genome editing tools based on the clustered regularly interspaced short palindromic repeats-associated proteins (CRISPR-Cas) system have also been developed for Bacteroides spp. and Clostridium spp., enabling rapid and efficient modification of their genomes (Guo et al., 2019; Jin et al., 2022; Zheng et al., 2022).

The availability of synthetic biology tools for commensal microbes enables us to manipulate microbe-host and microbe-microbe interactions rationally for biotherapeutic purposes. Gut probiotics including Lactobacillus spp. and Escherichia coli Nissle 1917 have been extensively studied and engineered to address gut inflammation, metabolic diseases, and cancer (Harimoto et al., 2022; Riglar and Silver, 2018). In the case of Bacteroides spp. and Clostridium spp., recent advances in genome editing tools and mechanistic understanding have facilitated engineering of these commensal bacteria for disease treatment by modulating the structure and function of the gut microbiota. Notably, commensal microbes like Bacteroides thetaiotaomicron have been investigated as a potential chassis for engineering live biotherapeutics (Mimee et al., 2016; Taketani et al., 2020). This review summarises recent developments in synthetic biology tools for species within Bacteroides spp. and Clostridium spp., and outlines their applications in understanding the functions of the microbiome, microbiome-host interactions, and engineering bacteria for live biotherapeutics.