Development of a Propionibacterium-Escherichia coli Shuttle Vector for Metabolic Engineering of Propionibacterium jensenii, an Efficient Producer of Propionic Acid

Abstract

dPropionic acid (PA) is an important chemical building block and is widely applied for organic synthesis, food, feedstuff, and pharmaceuticals. To date, the strains that can efficiently produce PA have included Propionibacterium thoenii, P. freudenreichii, and P. acidipropionici. In this report, we show that P. jensenii ATCC 4868 is also able to produce PA in much higher yields than the previously reported strains. To further improve the production capacity, a P. jensenii-Escherichia coli shuttle vector was developed for the metabolic engineering of P. jensenii. Specifically, a 6.9-kb endogenous plasmid, pZGX01, was isolated from P. acidipropionici ATCC 4875 and sequenced. Since the sequencing analysis indicated that pZGX01 could encode 11 proteins, the transcriptional levels of the corresponding genes were also investigated. Then, a P. jensenii-Escherichia coli shuttle vector was constructed using the pZGX01 plasmid, the E. coli pUC18 plasmid, and a chloramphenicol resistance gene. Interestingly, not only could the developed shuttle vector be transformed into P. jensenii ATCC 4868 and 4870, but it also could be transformed into freudenreichii ATCC 6207 subspecies of P. freudenreichii. Finally, the glycerol dehydrogenase gene (gldA) from Klebsiella pneumoniae was expressed in P. jensenii ATCC 4868 with the constructed shuttle vector. In a 3-liter batch culture, the PA production by the engineered P. jensenii ATCC 4868 strain reached 28.23 ؎ 1.0 g/liter, which was 26.07% higher than that produced by the wild-type strain (22.06 ؎ 1.2 g/liter). This result indicated that the constructed vector can be used a useful tool for metabolic engineering of P. jensenii.

Improved Production of Propionic Acid in Propionibacterium jensenii via Combinational Overexpression of Glycerol Dehydrogenase and Malate Dehydrogenase from Klebsiella pneumoniae

“…The constructed vectors were transformed into E. coli JM110, a methylation-deficient strain, and the demethylated vectors were then transformed into P. jensenii ATCC 4868. The preparation of competent cells and transformation into P. jensenii were performed as described previously (16). …”

“…In previous studies, we constructed a novel endogenous plasmid, named pZGX01, and a shuttle vector, designated pZGX04, between Propionibacterium and Escherichia coli as tools for engineering P. jensenii to produce PA efficiently (16). The present study aimed to identify the key pathways for PA synthesis in P. jensenii and then overexpress the relevant enzymes to improve the PA titer.…”

“…The metabolic engineering of Propionibacterium is of great interest, particularly because dairy propionibacteria have been granted generally recognized as safe status by the U.S. Food and Drug Administration (22). To date, only three studies have undertaken improved PA production via metabolic engineering, namely, acetate kinase gene knockout in P. acidipropionici (11), glycerol dehydrogenase (GDH) overexpression in P. jensenii (16), and phosphoenolpyruvate carboxylase expression in P. freudenreichii (23). Figure 1 shows the metabolic pathway of PA from glycerol in Propionibacterium under anaerobic conditions.…”

“…The genus Propionibacterium is a class of Gram-positive organisms and is divided into dairy propionibacteria (isolated mainly from dairy products) and cutaneous propionibacteria (typically found on skin and known human pathogens). Propionibacterium acidipropionici (5-13), P. freudenreichii (14), P. thoenii (15), and P. jensenii (16) are dairy propionibacteria and have been used for PA production, and research has focused mainly on process optimization for improved PA titers and productivity.…”

“…Although several endogenous plasmids have been found in Propionibacterium (16)(17)(18)(19)(20), very little metabolic engineering of Propionibacterium has been carried out. Metabolic engineering in these bacteria progresses slowly because of their thick cell walls, strong restriction-modification systems, and high GC content and a lack of detailed genome information (21).…”