Development of a Plasmid-Free Biosynthetic Pathway for Enhanced Muconic Acid Production in Pseudomonas chlororaphis HT66

Abstract

Muconic acid is a platform chemical and an important intermediate in the degradation process of a series of aromatic compounds. Herein, a plasmid-free synthetic pathway in Pseudomonas chlororaphis HT66 is constructed for the enhanced biosynthesis of muconic acid by connecting endogenous ubiquinone biosynthesis pathway with protocatechuate degradation pathway using chromosomal integration. Instead of being plasmid and inducer dependent, the engineered strains could steadily produce the high muconic acid using glycerol as a carbon source. The engineered strain HT66-MA6 achieved a 3376 mg/L muconic acid production with a yield of 187.56 mg/g glycerol via the following strategies: (1) block muconic acid conversion and enhance muconic acid efflux pumping with phenazine biosynthesis cluster; (2) increase the muconic acid precursors supply through overexpressing the rate-limiting step, and (3) coexpress the "3-dehydroshikimate-derived" route in parallel with the "4-hydroxybenzoic acid-derived" route to create a synthetic "metabolic funnel". Finally, on the basis of the glycerol feeding strategies, the muconic acid yield reached 0.122 mol/mol glycerol. The results suggest that the construction of synthetic pathway with a plasmid-free strategy in P. chlororaphis displays a high biotechnological perspective.

Enhanced biosynthesis of arbutin by engineering shikimate pathway in Pseudomonas chlororaphis P3

“…All these genes were individually cloned into pBBR1MCS and introduced into P3-Ar3 by electrotransformation. Figure 3 b clearly depicts that the growth of cells was delayed when introduced pBBR1MCS, which was in accordance with our earlier study [ 17 ]. To evaluate the effects of these candidates, each of the transformants was cultured in KB medium, and the production of arbutin was detected by HPLC.…”

“…chlororaphis P3Δ pobA to construct P3Δ pobA -pBBR-MNX1–AS. Figure 2 a displayed the cell growth and arbutin production when cultivation of P3Δ pobA -pBBR-MNX1–AS in KB medium supplemented with 0.5 g/L 4-HBA for 72 h. Evidently, the cell growth was impaired when introducing exogenous plasmid, and the arbutin was successfully produced but at a later stage of fermentation than that to other products reported in our earlier study [ 15 , 17 , 18 ] in the presence of inducer and antibiotics. Chromosomal integration is advocated as the preferred strategy to overcome this drawback.…”

“…Chromosomal integration is advocated as the preferred strategy to overcome this drawback. As demonstrated previously, phenazine biosynthesis genes are expressed under the strong phenazine synthesis promoter ( P phz ) and the native promoter is a powerful tool for the construction of new pathway [ 17 ]. On this basis, the phenazine biosynthetic genes are driven by stronger promoter that could be candidates for substitution with target genes under the control of native strong promoter P phz .…”

“…In our earlier study, several chorismatases were expressed in P. chlororaphis on the different chromosomal locus, when XanB2 from X. oryzae pv. oryzae was expressed on phzA locus under the control of P phz , more than 1.5 g/L 4-HBA was produced using glycerol as a carbon source [ 17 ]. XanB2 is an ideal enzyme for designing arbutin biosynthesis pathway, and therefore it was employed to generate 4-HBA for arbutin synthesis.…”

“…To further enhance the arbutin production, we attempted to identify and improve the rate-limiting step in the synthetic pathway of arbutin production. On the basis of previous study for enhanced shikimate pathway [ 17 – 19 ], four categories of genes were selected from the gluconeogenesis pathway, pentose phosphate pathway, shikimate pathway and “chorismate-arbutin” pathway. The first group, PEP synthase encoded by ppsA was generally used to improve the precursor PEP.…”