Metabolic Flux Analysis of Simultaneous Production of Vitamin B12 and Propionic Acid in a Coupled Fermentation Process by Propionibacterium freudenreichii.

Yuanyuan Ma,Yuhan Zhang,Xiaolian Li,Yunshan Wang,Ziqiang Wang,Zhiguo Su

doi:10.1007/s12010-021-03584-y

Abstract

The metabolic processes involved in simultaneous production of vitamin B12 and propionic acid by Propionibacterium freudenreichii are very complicated. To further investigate the regulatory mechanism of this metabolism, a simplified metabolic network was established. The effects of glucose feeding, propionic acid removal, and 5,6-dimethylbenzimidazole (DMB) addition on the metabolic flux distribution were investigated. The results showed that synthesis of propionic acid can be increased by increasing the metabolic flux through the oxaloacetate and methylmalonyl-CoA branches in the early and middle stages of the coupled fermentation. After DMB addition, the synthesis of vitamin B12 was significantly enhanced via increased metabolic flux through the δ-aminolevulinate branch, which promoted the synthesis of uroporphyrinogen III, a precursor of vitamin B12. Therefore, the analysis of metabolic flux at key nodes can provide theoretical guidance for the optimization of P. freudenreichii fermentation processes. In an experimental coupled fermentation process, the concentrations of vitamin B12 and propionic acid reached 21.6 and 50.12 g/L respectively, increased by 105.71% and 73.91% compared with batch fermentation, which provides a new strategy for industrial production.

Highlights

Vitamin B12 is a complex cobalt-containing cyclic tetrapyrrole coenzyme that is widely used in the pharmaceutical and food industries
In an experimental coupled fermentation process, the concentrations of vitamin B12 and propionic acid reached 21.6 and 50.12 g/L respectively, increased by 105.71% and 73.91% compared with batch fermentation, which provides a new strategy for industrial production
In the fermentation process of P. freudenreichii, many process variables can affect the production of vitamin B12 and propionic acid, including pH, temperature, fermentation time, inoculum size, the carbon and nitrogen sources and their concentrations, as well as fermentation strategies [25]

Summary

Introduction

Vitamin B12 (cobalamin) is a complex cobalt-containing cyclic tetrapyrrole coenzyme that is widely used in the pharmaceutical and food industries. The industrial production of vitamin B12 by chemical methods is very difficult, complicated and expensive, with > 70 steps. The production of vitamin B12 is exclusively preformed using aerobic and anaerobic fermentation by microorganisms; vitamin B12 synthesis involves > 30 genes [1]. The main strains used for industrial production of vitamin B12 are Pseudomonas denitrificans, Propionibacterium freudenreichii, and Propionibacterium shermanii [2]. Propionic acid and its salts can be used as antibacterial agents, anti-inflammatory drugs, herbicides, food preservatives, and artificial foods [4]. Propionic acid is produced from petroleum-based chemicals via the hydrocarboxylation of ethylene, which causes substantial damage to the environment. Renewable route, the preparation of propionic acid or its salts by microbial fermentation has been investigated extensively and is considered a safe and pollution-free process [5]

Methods

Results

Conclusion