Pathway engineering of Propionibacterium jensenii for improved production of propionic acid

Abstract

Propionic acid (PA) is an important chemical building block widely used in the food, pharmaceutical, and chemical industries. In our previous study, a shuttle vector was developed as a useful tool for engineering Propionibacterium jensenii, and two key enzymes—glycerol dehydrogenase and malate dehydrogenase—were overexpressed to improve PA titer. Here, we aimed to improve PA production further via the pathway engineering of P. jensenii. First, the phosphoenolpyruvate carboxylase gene (ppc) from Klebsiella pneumoniae was overexpressed to access the one-step synthesis of oxaloacetate directly from phosphoenolpyruvate without pyruvate as intermediate. Next, genes encoding lactate dehydrogenase (ldh) and pyruvate oxidase (poxB) were deleted to block the synthesis of the by-products lactic acid and acetic acid, respectively. Overexpression of ppc and deleting ldh improved PA titer from 26.95 ± 1.21 g·L−1 to 33.21 ± 1.92 g·L−1 and 30.50 ± 1.63 g·L−1, whereas poxB deletion decreased it. The influence of this pathway engineering on gene transcription, enzyme expression, NADH/NAD+ ratio, and metabolite concentration was also investigated. Finally, PA production in P. jensenii with ppc overexpression as well as ldh deletion was investigated, which resulted in further increases in PA titer to 34.93 ± 2.99 g·L−1 in a fed-batch culture.

Microbial Propionic Acid Production

“…Acetate and lactate by-products were targeted in a gene knockout study in P. jensenii [65]. In this work, knock-out of the lactate dehydrogenase improved the final propionate titre slightly in fed-batch cultures but improvements in the productivity could not be observed.…”

“…13% improvement of propionate production. [64] Genome editing, overexpression P. jensenii poxB or ldh knock-out and ppc overexpression Maximum 30% improvement in titre and 24% improvement productivity [65] Improve acid tolerance Overexpression P. acidipropionici otsA overexpression strain Propionic acid yield 11% higher. [66] Overexpression P. jensenii strains overexpressing gadB, arcA, arc, gdh or ybaS Up to a 1.5-fold increase in yield and 5.4-fold increase in titre, in shake flasks [67] Increase of metabolic flux towards propionate production Overexpression P. shermanii CoAT overexpression strain Increase yield and productivity, maximum 10% and 46%, respectively.…”

“…It is difficult to extrapolate the potential implications of the failed acetate knock-out on glycerol [65] to the ability to use this strategy to overcome the yield challenges facing sugar fermentations. The over-expression of pathways that provide reduced cofactors, such as the pentose phosphate pathway, may be a more promising alternative as shown in our recent work [79], particularly since it may be impossible to eliminate acetate production by gene knock-out due to the promiscuity of the propionyl-CoA: succinate CoA-transferase (CoAT).…”

Bio-produced Propionic Acid: A Review

“…Propionic acid titres have been improved by overexpressing enzymes such as glycerol dehydrogenase, malate dehydrogenase [2] and fumarate hydratase in Propionibacterium jensenii, phosphoenolpyruvate carboxylase (PPC) in Propionibacterium freudenreichii, and propionyl-CoA:succinate CoA transferase in Propionibacterium shermanii [8]. The yield of propionic acid was also increased by the inactivation of the acetate kinase gene [23].…”

“…Various carbon and nitrogen sources have been examined and several cultivation techniques tested [8]. Since both the availability of genomic information and tools of synthetic biology with regard to Propionibacteria have improved recently, it has become a reality to increase the production of propionic acid with metabolically engineered Propionibacteria [8].…”