https://research.chalmers.se/publication/506706/file/506706_Fulltext.pdf

Biobased adipic acid - The challenge of developing the production host.

Glucaric acid is a high-value-added chemical that can be used in various fields. Because chemical oxidation of glucose to produce glucaric acid is not environmentally friendly, microbial production has attracted increasing interest recently. Biological pathways to synthesize glucaric acid from glucose in both Escherichia coli and Saccharomyces cerevisiae by co-expression of genes encoding myo-inositol-1-phosphate synthase (Ino1), myo-inositol oxygenase (MIOX), and uronate dehydrogenase (Udh) have been constructed. However, low activity and instability of MIOX from Mus musculus was proved to be the bottleneck in this pathway. A more stable miox4 from Arabidopsis thaliana was chosen in the present study. In addition, high copy delta-sequence integration of miox4 into the S. cerevisiae genome was performed to increase its expression level further. Enzymatic assay and quantitative real-time PCR analysis revealed that delta-sequence-based integrative expression increased MIOX4 activity and stability, thus increasing glucaric acid titer about eight times over that of episomal expression. By fed-batch fermentation supplemented with 60 mM (10.8 g/L) inositol, the multi-copy integrative expression S. cerevisiae strain produced 6 g/L (28.6 mM) glucaric acid from myo-inositol, the highest titer that had been ever reported in S. cerevisiae. In this study, glucaric acid titer was increased to 6 g/L in S. cerevisiae by integrating the miox4 gene from A. thaliana and the udh gene from Pseudomonas syringae into the delta sequence of genomes. Delta-sequence-based integrative expression increased both the number of target gene copies and their stabilities. This approach could be used for a wide range of metabolic pathway engineering applications with S. cerevisiae.

/pdf/metabolic-engineering-of-saccharomyces-cerevisiae-for-1ud1ptkw3o.pdf

Metabolic engineering of Saccharomyces cerevisiae for efficient production of glucaric acid at high titer

Characterization of genetic circuits and biosynthetic pathways in different hosts always requires promoter substitution and redesigning. Here, a strong, broad-spectrum promoter, Pbs, for Escherichia coli, Bacillus subtilis, and Saccharomyces cerevisiae was constructed, and it was incorporated into the minimal E. coli–B. subtilis–S. cerevisiae shuttle plasmid pEBS (5.8 kb). By applying a random mutation strategy, three broad-spectrum promoters Pbs1, Pbs2, and Pbs3, with different strengths were generated and characterized. These broad-spectrum promoters will expand the synthetic biology toolbox for E. coli, B. subtilis, and S. cerevisiae.

Construction and Characterization of Broad-Spectrum Promoters for Synthetic Biology

A critical review on the progress and challenges to a more sustainable, cost competitive synthesis of adipic acid

Metabolic regulation of gene expression for the microbial production of fine chemicals, such as organic acids, is an important research topic in post-genomic metabolic engineering. In particular, the ability of transcription factors (TFs) to respond precisely in time and space to various small molecules, signals and stimuli from the internal and external environment is essential for metabolic pathway engineering and strain development. As a key component, TFs are used to construct many biosensors in vivo using synthetic biology methods, which can be used to monitor the concentration of intracellular metabolites in organic acid production that would otherwise remain "invisible" within the intracellular environment. TF-based biosensors also provide a high-throughput screening method for rapid strain evolution. Furthermore, TFs are important global regulators that control the expression levels of key enzymes in organic acid biosynthesis pathways, therefore determining the outcome of metabolic networks. Here we review recent advances in TF identification, engineering, and applications for metabolic engineering, with an emphasis on metabolite monitoring and high-throughput strain evolution for the organic acid bioproduction.

/pdf/transcription-factor-engineering-for-high-throughput-strain-2hccsq4snw.pdf

Transcription Factor Engineering for High-Throughput Strain Evolution and Organic Acid Bioproduction: A Review

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.

Amita Gupta

Papers

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