Redistribution of Metabolic Fluxes in the Central Aerobic Metabolic Pathway of E. coli Mutant Strains with Deletion of the ackA-pta and poxB Pathways for the Synthesis of Isoamyl Acetate

Abstract

Although the bacterium E. coli is chosen as the host in many bioprocesses, products derived from the central aerobic metabolic pathway often compete with the acetate-producing pathways poxB and ackA-pta for glucose as the substrate. As such, a significant portion of the glucose may be excreted as acetate, wasting substrate that could have otherwise been used for the desired product. The production of the ester isoamyl acetate from acetyl-CoA by ATF2, a yeast alcohol acetyl transferase, was used as a model system to demonstrate the beneficial effects of reducing acetate production. All strains tested for ester production also overexpressed panK, a native E. coli gene that previous studies have shown to increase free intracellular CoA levels when fed with pantothenic acid. A recombinant E. coli strain with a deletion in ackA-pta produces less acetate and more isoamyl acetate than the wild-type E. coli strain. When both acetate-producing pathways were deleted, the acetate production was greatly reduced. However, pyruvate began to accumulate, so that the overall ester production remained largely unchanged. To produce more ester, a previously established strategy of increasing the flux from pyruvate to acetyl-CoA was adopted by overexpressing pyruvate dehydrogenase. The ester production was then 80% higher in the poxB, ackA-pta strain (0.18 mM) than that found in the single ackA-pta mutant (0.10 mM), which also overexpressed PDH.

Minimizing acetate formation in E. coli fermentations

“…In the wild type, PPC is more active than PCK, firstly, to compensate for the activity of PCK and secondly, to Overexpression of pck deregulates this system, causing an increase of the acetate production. Mutations in both pta and ackA have frequently been investigated [2,11,54,65]. All data report a strong reduction of acetate production, when ackA and/or pta are eliminated.…”

“…In addition to a loss of carbon and therefore an economic sink, acetate is also detrimental to recombinant protein production and inhibits cell growth [2]. For more information about overcoming acetate in E. coli recombinant protein fermentations see reference [3].…”

“…The second enzyme, pyruvate oxidase [EC 1.2.2.2], is a pheripheral membrane protein that converts pyruvate, ubiquinone and H 2 O to acetate, ubiquinol and CO 2 . It has been reported to be a nonessential aerobic enzyme active in the early stationary phase [8,9].…”

“…However, more recent studies has shown that pyruvate oxidase plays an important role in the aerobic growth efficiency of E. coli [10], perhaps to preserve the pool of free CoA-SH. Dittrich et al (2005) [2] confirm that the poxB pathway is more active during the late exponential and stationary phases, whereas the ackA-pta pathway is more active in the exponential stage of the cell growth. They also report that the two acetate producing pathways are affected by culture conditions such as pH.…”

Increasing Recombinant Protein Production in E. coli by an Alternative Method to Reduce Acetate

“…ackA (acetate kinase), pta (phosphate acetyltransferase) and poxB (pyruvate oxidase) (Diaz-Ricci et al, 1991;Yang et al, 1999;Contiero et al, 2000;Dittrich et al, 2005;De Mey et al, 2007a). These attempts resulted in a considerably decrease of acetate production but in return pyruvate, lactate or formate formation, which are also undesired by-products, increased to a large extent.…”