Analysis and engineering of metabolic pathways of Lactobacillus panis PM1

Abstract

Lactobacillus panis PM1 is a novel microorganism isolated from thin stillage (TS), a major by-product resulting from bioethanol fermentation, and was selected as the focus of this thesis due to its ability to produce 1,3-propanediol (1,3-PDO) from glycerol. The purpose of this thesis was to understand the central and auxiliary metabolic pathways of L. panis PM1 and to metabolically-engineer strain PM1 based on the improved metabolic knowledge for industrial applications. The 16S rRNA sequence and carbohydrate fermentation pattern were used to classify L. panis PM1 as belonging to the group III lactobacilli; thus, strain PM1 exclusively fermented glucose to lactate, acetate, and/or ethanol, clearly suggesting that its primary metabolism occurred via the 6-phosphogluconate/phosphoketolase (6-PG/PK) pathway. In contrast to typical group III lactobacilli, for fructose fermentation, L. panis PM1 utilized both the 6-PG/PK and the Embden-Meyerhof pathways, showing distinct strain-specific characteristics (more lactate, less acetate, no mannitol, and sporadic growth). In the PM1 strain, auxiliary metabolic pathways governed end-product formation patterns along with central metabolism. Under aerobic conditions, a coupled NADH oxidase-NADH peroxidase system was a determinant for NAD+ regeneration and was regulated by oxygen availability; however, the accumulation of its major end-product, hydrogen peroxide, eventually resulted in oxidative stress. The citrate-to-succinate route was another important auxiliary pathway in L. panis PM1. This route was directly connected to central energy metabolism, producing extra ATP for survival during the stationary phase, and was regulated by the presence of citrate, acetate, and succinate and a transcriptional repressor (PocR). Lactobacilli panis PM1 produced 1,3-PDO via the glycerol reductive route; however, the absence of the glycerol oxidative route restricted the utilization of glycerol to solely that of electron acceptor. Lower ratio of glucose to glycerol, in combination with PocR, repressed the glycerol reductive route, resulting in less 1,3-PDO production. In an effort to metabolically engineer L. panis PM1, an artificial glycerol oxidative pathway was introduced, and the engineered PM1 strain successfully produced a significant amount of important platform chemicals, including 1,3-PDO, lactate, and ethanol, solely from TS. Overall, this thesis reveals the significant feasibility of utilizing L. panis PM1 for industrial fermentative applications.