Abstract
Genetic modification of Rhodococcus jostii RHA1 was carried out in order to optimise the production of pyridine-2,4-dicarboxylic acid and pyridine-2,5-dicarboxylic acid bioproducts from lignin or lignocellulose breakdown, via insertion of either the Sphingobium SYK-6 ligAB genes or Paenibacillus praA gene respectively. Insertion of inducible plasmid pTipQC2 expression vector containing either ligAB or praA genes into a ΔpcaHG R. jostii RHA1 gene deletion strain gave 2–threefold higher titres of PDCA production from lignocellulose (200–287 mg/L), compared to plasmid expression in wild-type R. jostii RHA1. The ligAB genes were inserted in place of the chromosomal pcaHG genes encoding protocatechuate 3,4-dioxygenase, under the control of inducible Picl or PnitA promoters, or a constitutive Ptpc5 promoter, producing 2,4-PDCA products using either wheat straw lignocellulose or commercial soda lignin as carbon source. Insertion of Amycolatopsis sp. 75iv2 dyp2 gene on a pTipQC2 expression plasmid led to enhanced titres of 2,4-PDCA products, due to enhanced rate of lignin degradation. Growth in minimal media containing wheat straw lignocellulose led to the production of 2,4-PDCA in 330 mg/L titre in 40 h, with > tenfold enhanced productivity, compared with plasmid-based expression of ligAB genes in wild-type R. jostii RHA1. Production of 2,4-PDCA was also observed using several different polymeric lignins as carbon sources, and a titre of 240 mg/L was observed using a commercially available soda lignin as feedstock.
Highlights
The aromatic heteropolymer lignin accounts for 15–25% of plant cell wall lignocellulose, and is the most abundant renewable source of aromatic carbon in the biosphere
We have previously reported that re-routing of protocatechuic acid via either protocatechuate 4,5-dioxygenase (Sphingobium SYK-6 ligAB genes) or protocatechuate 2,3-dioxygenase (Paenibacillus praA gene) in R. jostii RHA1, followed by ammonia cyclisation of the extradiol ring fission product, generates pyridine-2,4-dicarboxylic acid (2,4-PDCA) or pyridine-2,5-dicarboxylic acid (2,5PDCA) bioproducts respectively, in titres of 90–125 mg/L cell culture, in M9 minimal media containing either 0.1% vanillic acid or 1% wheat straw lignocellulose [8]
The ΔpcaHG gene deletion strain showed only slightly reduced growth on lysogeny broth, compared to wild-type R. jostii RHA1, but when grown on M9 minimal media containing 0.1% PCA as carbon source, showed little or no growth over 48 h, consistent with the β-ketoadipate pathway being the major pathway for catabolism of protocatechuic acid
Summary
The aromatic heteropolymer lignin accounts for 15–25% of plant cell wall lignocellulose, and is the most abundant renewable source of aromatic carbon in the biosphere. The conversion of lignocellulose from plant biomass into fuels and chemicals via the biorefinery concept requires the efficient conversion of cellulose, hemi-cellulose and lignin into high value products, but at present the majority of lignin produced by pulp/paper manufacture and biofuel production is burnt for energy. One strategy for biocatalytic valorisation of lignin that has shown promise is to engineer lignin-degrading micro-organisms to produce target chemicals [2]. Metabolic funnelling of monocyclic lignin breakdown products via protocatechuic acid and subsequent metabolism via the β-ketoadipate pathway has facilitated the metabolic engineering of Pseudomonas putida. Metabolic engineering of Corynebacterium glutamicum to produce cis,cis-muconic acid has been reported [7]
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