Development of a Propionibacterium-Escherichia coli shuttle vector for metabolic engineering of Propionibacterium jensenii, an efficient producer of propionic acid

Abstract

Propionic 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.

Fig 1

Batch fermentation kinetics of propionic acid production from glycerol with P. jensenii ATCC 4868 at pH 7.0 and 30°C for 144 h. The initial glycerol concentration was 30 g/liter; samples were taken from the fermentor every 12 h. △, propionic acid (PA); □, residual glycerol; ▲, dry cell weight (DCW); ■, acetic acid (AA); ♦, succinic acid (SA); *, specific activity of GDH.

Fig 2

The transcription level relative to 16S RNA of 11 predicted ORFs from pZGX01. The total RNA extracted from cells was reverse transcribed to cDNA. The obtained cDNA was subjected to quantitative PCR, and 16S RNA was set as the guide sample.

Fig 3

Scheme for vector pZGX04 construction. A large fragment containing orf2 to orf10 was obtained from pZGX01 by digestion with EcoRI and BamHI, and the fragment was ligated to EcoRI-BamHI-digested E. coli plasmid pUC18. The resulting plasmid was digested with NheI and self-ligated. Then, the resulting plasmid was digested with StuI (inside orf2) and ligated to a 1.5-kb PCR product carrying the cmx(A) and cml(A) genes, yielding the shuttle vector pZGX04. Cm^r, gene encoding chloramphenicol-resistant protein; Ap^r, gene encoding ampicillin-resistant protein; RepA, gene encoding Rep A protein; RepB, gene encoding Rep B protein; lacZ, β-galactosidase; ori, colE replication origin.

Fig 4

Synthesis pathways for propionic acid from glycerol, glucose, and lactate in Propionibacterium. The biosynthetic pathway from glycerol to propionic acid proceeds from glycerol to dihydroxyacetone (DHA) to dihydroxyacetone phosphate (DHAP) to phosphoenolpyruvate to pyruvate and oxaloacetate and from oxaloacetate to malate, fumarate, succinate, succinyl coenzyme A (CoA), methylmalonyl CoA, propionyl CoA, and propionate (see arrows). Acetate is the main byproduct in propionic acid production. Gray color indicates the biosynthetic pathway from glycerol to propionic acid.

Fig 5

Batch fermentation kinetics of propionic acid production from glycerol with P. jensenii ATCC 4868 (pZGX04-gldA) at pH 7.0 and 30°C for 144 h. Batch fermentations were performed under anaerobic conditions (nitrogen gassing) in a 3-liter Bioreactor containing 2 liters of culture medium supplemented with 30 g/liter glycerol. Samples were taken from the fermentor every 12 h. △, propionic acid (PA); □, residual glycerol; ▲, dry cell weight (DCW); ■, acetic acid (AA); ♦, succinic acid (SA); *, specific activity of GDH.