Journal Article

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

Lück, Rebecca, Deppenmeier, Uwe

2022

Overview

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%.

• Publisher: Springer Berlin Heidelberg,Springer,Springer Nature B.V
• Subject: Abundance / Anaerobic bacteria / Analysis / Applied Genetics and Molecular Biotechnology / Bacteroides / Bioactive compounds / Biodegradation / Biomedical and Life Sciences / Biotechnology / Carbon / Colon / Copy number / D-Lactate dehydrogenase / Dehydrogenase / Dehydrogenases / Enzymatic activity / Enzyme activity / Fatty acids / Gene expression / Genera / genes / Genetic aspects / Genetic engineering / Genetic modification / Genetic vectors / Homology / Humans / Identification and classification / intestinal microorganisms / L-Lactate dehydrogenase / Lactate dehydrogenase / Lactic Acid / LDH gene / Life Sciences / Methods / Microbial Genetics and Genomics / Microbiology / Microbiota (Symbiotic organisms) / Mutants / Optimization / Plants / Species classification / Transcription / Vitamins