Genetic tools for the redirection of the central carbon flow towards the production of lactate in the human gut bacterium Phocaeicola (Bacteroides) vulgatus

Abstract

Species of the genera Bacteroides and Phocaeicola play an important role in the human colon. The organisms contribute to the degradation of complex heteropolysaccharides to small chain fatty acids, which are in part utilized by the human body. Furthermore, these organisms are involved in the synthesis of vitamins and other bioactive compounds. Of special interest is Phocaeicola vulgatus, originally classified as a Bacteroides species, due to its abundance in the human intestinal tract and its ability to degrade many plant-derived heteropolysaccharides. We analyzed different tools for the genetic modification of this microorganism, with respect to homologous gene expression of the ldh gene encoding a D-lactate dehydrogenase (LDH). Therefore, the ldh gene was cloned into the integration vector pMM656 and the shuttle vector pG106 for homologous gene expression in P. vulgatus. We determined the ldh copy number, transcript abundance, and the enzyme activity of the wild type and the mutants. The strain containing the shuttle vector showed an approx. 1500-fold increase in the ldh transcript concentration and an enhanced LDH activity that was about 200-fold higher compared to the parental strain. Overall, the proportion of lactate in the general catabolic carbon flow increased from 2.9% (wild type) to 28.5% in the LDH-overproducing mutant. This approach is a proof of concept, verifying the genetic accessibility of P. vulgatus and could form the basis for targeted genetic optimization. Key points • A lactate dehydrogenase was overexpressed in Phocaeicola (Bacteroides) vulgatus. • The ldh transcript abundance and the LDH activity increased sharply in the mutant. • The proportion of lactate in the catabolic carbon flow increased to about 30%.

Clostridia isolated from helminth-colonized humans promote the life cycle of Trichuris species

“…Using this assay, we tested four OA isolates associated with helminths (OA2, OA6, OA8, and OA12) and compared them with two isolates not associated with helminths (OA4 and OA14). We also included two Bacteroidales species, Bacteroides thetaiotaomicron and Phocaeicola vulgatus (previously Bacteroides vulgatus ), 45 which are ubiquitous anaerobes in the human microbiome. In addition, P. vulgatus ’s growth rate was negatively correlated with Trichuris egg burden in the Orang Asli.…”

Functional Fermented Milk with Fruit Pulp Modulates the In Vitro Intestinal Microbiota

“…Although a large number of bacteria, archaea, viruses, and unicellular eukaryotes reside in the human GI tract, only some bacterial genera dominate the human gut, including Bacteroides, Clostridium, Bifidobacterium, and Faecalibacterium [ 55 , 56 ] The Phocaeicola genus comprises Gram-negative, strictly anaerobic bacteria, generally associated with human fecal samples [ 57 , 58 ]. Bacteroides and Phocaeicola represent 30% of the commensal intestinal microbiota, playing an essential role in the gut ecosystem [ 59 , 60 ]. Moreover, Phocaeicola species present glycosyl hydrolases enzymatic activities, which degrade mucin glycans and many plant-derived heteropolysaccharides [ 61 , 62 , 63 ], generating important metabolites such as succinate and propionate [ 64 , 65 ].…”

“…Moreover, Phocaeicola species present glycosyl hydrolases enzymatic activities, which degrade mucin glycans and many plant-derived heteropolysaccharides [ 61 , 62 , 63 ], generating important metabolites such as succinate and propionate [ 64 , 65 ]. There is robust evidence that these gut-associated microbes are involved in the prebiotic metabolisms and bioactive compounds synthesis, which are frequently associated with human and animal health benefits [ 59 , 66 ]. More precisely, from the aforementioned Phocaeicola , a bacterial species of special interest is P. vulgatus , the most abundant microbe within the Bacteroidaceae family normally found in the colon, constituting up to 10 10 cells per gram of stool [ 59 ].…”

“…There is robust evidence that these gut-associated microbes are involved in the prebiotic metabolisms and bioactive compounds synthesis, which are frequently associated with human and animal health benefits [ 59 , 66 ]. More precisely, from the aforementioned Phocaeicola , a bacterial species of special interest is P. vulgatus , the most abundant microbe within the Bacteroidaceae family normally found in the colon, constituting up to 10 10 cells per gram of stool [ 59 ].…”

Bacteroides vulgatus Ameliorates Lipid Metabolic Disorders and Modulates Gut Microbial Composition in Hyperlipidemic Rats

“…As a prominent species of the genus Bacteroides , Bacteroides vulgatus has been described as a prevalent and dominant organism of the colon in our previous work ( 15 ) and other studies ( 16 , 17 ). B. vulgatus is equipped with enzyme systems to degrade complex polysaccharides ( 18 ) and is known to produce acetate, propionate, butyrate, and lactate ( 19 , 20 ). To date, B. vulgatus has been intensively explored as a treatment for intestinal inflammatory diseases, while its effects are strain dependent ( 21 , 22 ).…”