In vivoinstability of chorismate causes substrate loss during fermentative production of aromatics

Abstract

Metabolic engineering of microbial strains to produce aromatic compounds deriving from the shikimate pathway is of great interest to the chemical industry as a more sustainable alternative for feedstock production. Chorismate is a significant intermediate in the shikimate pathway. In this study, the formation of phenylalanine and phenylpyruvate as by-products in strains engineered downstream of the chorismate node for increased aromatic production was explored in yeast fermentations. Tracer experiments showed that these compounds are synthesized de novo during fermentation, under conditions in which their synthesis was genetically blocked. Chorismate stability evaluation, as well as deletion mutation analysis throughout the phenylalanine biosynthesis pathway, suggested that this synthesis was a result of intracellular, non-enzymatic rearrangement of chorismate to phenylpyruvate via prephenate, which was followed by enzymatic transamination of phenylpyruvate to form phenylalanine. These results not only aid in the development of strain-engineering strategies to avoid the accumulation of by-products during fermentations aimed at increased aromatics production, but also deepen our understanding of yeast metabolism.

Engineering yeast shikimate pathway towards production of aromatics: rational design of a chassis cell using systems and synthetic biology

“…However, this step appears to proceed also spontaneously, as a study on the deletion of ARO7 shows (Winter et al, 2014). The next step to tyrosine is catalysed by prephenate dehydrogenase, encoded by TYR1.…”

“…In earlier studies the knock-out of ARO7 (encoding chorismate mutase) and TRP3 (encoding anthranilate synthase) has been described as a way to increase chorismate availability for pHBA formation (Krömer et al, 2013). However, this strategy also causes phenylalanine, tyrosine and tryptophan auxotrophy and the associated growth defect, even when cells are fed these amino acids (Winter et al, 2014).…”

“…Yields (in percent C-mol/C-mol) were calculated from the evaporation corrected titers while only the metabolised fraction of the carbon-source was respected. and analysed using gas chromatography -mass spectrometry (GC-MS) described previously (Winter et al, 2014). For this 0.2 mL of supernatant sample or 0.05 mL of cellhydrolysate were dried under a stream of N 2 and subsequently derivatized with N-methyltert-butyldimethylsilyl-trifluoroacetamide (MBDSTFA; Macherey-Nagel, Düren, Germany)…”

“…as described before (Krömer et al, 2013, Winter et al, 2014. For pABA the ion cluster m/z = 308-315 was analysed.…”

“…It was recently demonstrated that the conversion of chorismate to prephenate / phenylpyruvate in a ∆aro7 strain proceeds also spontaneously (Winter et al, 2014), the reaction rate is merely a matter of substrate and product concentrations, temperature and pH. Given that pABA production is kinetically limited, in a situation of high flux into the shikimate pathway during faster glucose metabolism, chorismate could be accumulating, leading to an overflow into PEA (a similar flux overflow resulting in an accumulation of PHE and TYR has been reported when producing pABA in E. coli (Koma et al, 2014)).…”