Porting the synthetic D‐glucaric acid pathway from Escherichia coli to Saccharomyces cerevisiae

Abstract

D-Glucaric acid can be produced as a value-added chemical from biomass through a de novo pathway in Escherichia coli. However, previous studies have identified pH-mediated toxicity at product concentrations of 5 g/L and have also found the eukaryotic myo-inositol oxygenase (MIOX) enzyme to be rate-limiting. We ported this pathway to Saccaromyces cerevisiae, which is naturally acid-tolerant and evaluate a codon-optimized MIOX homologue. We constructed two engineered yeast strains that were distinguished solely by their MIOX gene - either the previous version from Mus musculus or a homologue from Arabidopsis thaliana codon-optimized for expression in S. cerevisiae - in order to identify the rate-limiting steps for D-glucaric acid production both from a fermentative and non-fermentative carbon source. myo-Inositol availability was found to be rate-limiting from glucose in both strains and demonstrated to be dependent on growth rate, whereas the previously used M. musculus MIOX activity was found to be rate-limiting from glycerol. Maximum titers were 0.56 g/L from glucose in batch mode, 0.98 g/L from glucose in fed-batch mode, and 1.6 g/L from glucose supplemented with myo-inositol. Future work focusing on the MIOX enzyme, the interplay between growth and production modes, and promoting aerobic respiration should further improve this pathway.

Avicennia marina biomass characterization towards bioproducts

“…Glucan and xylan are the precursors for the production of high value chemicals, e.g. furfural, hydroxymethylfurfural (HMF), and levulinic acid, by thermochemical processes (Alvira et al 2010) or glucaric acid by fermentation processes (Gupta et al 2016a). Characterization of other arid region lignocellulosic biomass, Phoenix dactylifera and Salicornia bigelovii, has reported glucan and xylan values of 41.2 g_glucan/100_ gTS, 21.5 g_xylan/100_gTS and 9.1 g_glucan/100_gTS, 7.7 g_xylan/100_gTS, respectively (Ashraf et al 2016;Cybulska et al 2014).…”

Consolidated bioprocessing of lignocellulose for production of glucaric acid by an artificial microbial consortium

“…D-Glucaric acid, identified as “top value-added chemical from biomass” by US Department of Energy in 2004 1 , is an important platform chemical with a wide variety of applications such as therapeutic uses and biopolymer production 2, 3, 4 . Conventionally, D-glucaric acid was produced via nitric acid oxidation of D-glucose, a nonselective and expensive process associated with a large exotherm, low yields and toxic byproducts 4, 5, 6 . Biological production of D-glucaric acid, therefore, has been attracted increasing interest due to the potential for a cheaper and more environmentally friendly process by avoiding costly catalysts and harsh reaction conditions 3, 4 .…”

“…Moreover, an N-terminal SUMO fusion to MIOX gave rise to a 75% increase in D-glucaric acid production from myo -inositol where up to 4.85 g/L of D-glucaric acid was produced from 10.8 g/L myo -inositol in recombinant E. coli 4 . However, E. coli seems not an excellent candidate for D-glucaric acid production at high titer because D-glucaric acid concentrations above 5 g/L appears to inhibit its further production by E. coli through a pH-mediated effect 2, 3, 5 .…”

“…Thus, Gupta et al 5 ported the synthetic D-glucaric acid pathway from E. coli to S. cerevisiae , another model strain widely used industry that has better acid tolerance 3 , and found that MIOX4 from Arabidopsis thaliana outperformed MIOX from M. musculus 5 . The maximal titer in the S. cerevisiae strain containing MIOX4 was 1.6 g/L D-glucaric acid from glucose supplemented with myo -inositol 5 . Chen et al 3 used delta-sequence-based integrative expression to increase MIOX4 activity and stability, successfully increasing glucaric acid titer about eight times over that of episomal expression.…”

“…Here we used the same genes and strategy as Chen et al described 3 to construct the biosynthetic route for D-glucaric acid production in a different baker’s yeast strain, S. cerevisiae INVSc1. In light of myo -inositol availability was rate-limiting in the S. cerevisiae strain containing miox4 gene from A. thaliana 3, 5 , the gene of opi1 in S. cerevisiae INVSc1 was also knocked out accordingly to remove its negative regulation on myo -inositol synthesis 3, 8. As a result, a more robust engineered strain of S. cerevisiae INVSc1 was obtained, producing the record-breaking titer of D-glucaric acid in S. cerevisiae .…”