Selective vanillate production from sugarcane bagasse-derived aromatic compounds using an engineered Pseudomonas sp....

Area of Science:

• Biotechnology
• Microbial Engineering
• Synthetic Biology

Background:

• Sugarcane bagasse is a rich source of aromatic compounds.
• Selective production of vanillate (VA) from complex biomass extracts is challenging.
• Metabolic engineering offers a pathway to valorize biomass components.

Purpose of the Study:

• To engineer Pseudomonas sp. NGC7 for selective vanillate (VA) production from sugarcane bagasse alkaline extract.
• To overcome limitations in VA production, including the accumulation of byproducts like syringate (SA) and 4-hydroxybenzoate (HBA).
• To optimize VA production using a concentrated extract and a flow-through-based medium.

Main Methods:

• Genetic engineering of Pseudomonas sp. NGC7, including vanA4B4 gene disruption.
• Solid-phase extraction using DIAION HP20 resin for aromatic compound concentration.
• Mutagenesis and screening for improved SA degradation capabilities.
• Heterologous expression of the HBA hydroxylase gene (praI).
• Fed-batch culture and evaluation in a mineral salt-supplemented medium.

Main Results:

• Engineered strain NGC7ΔvanA4B4 produced VA from saccharide-free extract, with growth promoted by organic acids.
• Concentration of aromatics and fractionation of organic acids improved VA production.
• Mutations in vanR2 enabled SA degradation during VA production.
• Expression of praI facilitated efficient HBA degradation.
• The engineered strain achieved selective VA production of 4.30 mM at a 77 mol% yield in a practical medium.

Conclusions:

• Genetic modification of Pseudomonas sp. NGC7, specifically vanR2 mutation and praI expression, are key strategies for selective VA production.
• Organic acids in the extract enhance microbial growth, supporting VA biosynthesis.
• The developed process demonstrates efficient and selective vanillate production from lignocellulosic biomass.