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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%.
Highlights
The human colon contains a versatile and complex microbial flora, and the majority of these organisms proliferate only under anaerobic conditions (Sender et al 2016; Lu and Imlay 2021)
Plasmid pG106 comprises a 5 kb Mob-Rep region, which contains an oriV for the initiation of plasmid replication in P. vulgatus
Stable transformation of pG106 into P. vulgatus was achieved by electroporation
Summary
The human colon contains a versatile and complex microbial flora, and the majority of these organisms proliferate only under anaerobic conditions (Sender et al 2016; Lu and Imlay 2021). Species of the genera Bacteroides and Phocaeicola are ubiquitous commensals, comprising about thirty percent of the human gut microbiota (Salyers 1984). These microorganisms play an essential role in the colonic. The RBS from species of the family Bacteroidaceae has a lower GC content, which results in a lower tendency to form secondary structures (Accetto and Avguštin 2011). These unique promoters and RBS structures make it difficult to transfer genetic systems evolved in bacteria of other phyla (Smith et al 1992). The heterogeneity highlights the need for genetic studies on different species belonging to the family Bacteroidaceae
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