Construction and Optimization of a Heterologous Pathway for Protocatechuate Catabolism in Escherichia coli Enables Bioconversion of Model Aromatic Compounds

Abstract

The production of biofuels from lignocellulose yields a substantial lignin by-product stream that currently has few applications. Biological conversion of lignin-derived compounds into chemicals and fuels has the potential to improve the economics of lignocellulose-derived biofuels, but few microbes are able both to catabolize lignin-derived aromatic compounds and to generate valuable products. While Escherichia coli has been engineered to produce a variety of fuels and chemicals, it is incapable of catabolizing most aromatic compounds. Therefore, we engineered E. coli to catabolize protocatechuate, a common intermediate in lignin degradation, as the sole source of carbon and energy via heterologous expression of a nine-gene pathway from Pseudomonas putida KT2440. We next used experimental evolution to select for mutations that increased growth with protocatechuate more than 2-fold. Increasing the strength of a single ribosome binding site in the heterologous pathway was sufficient to recapitulate the increased growth. After optimization of the core pathway, we extended the pathway to enable catabolism of a second model compound, 4-hydroxybenzoate. These engineered strains will be useful platforms to discover, characterize, and optimize pathways for conversions of ligninderived aromatics.IMPORTANCE Lignin is a challenging substrate for microbial catabolism due to its polymeric and heterogeneous chemical structure. Therefore, engineering microbes for improved catabolism of lignin-derived aromatic compounds will require the assembly of an entire network of catabolic reactions, including pathways from genetically intractable strains. Constructing defined pathways for aromatic compound degradation in a model host would allow rapid identification, characterization, and optimization of novel pathways. We constructed and optimized one such pathway in E. coli to enable catabolism of a model aromatic compound, protocatechuate, and then extended the pathway to a related compound, 4-hydroxybenzoate. This optimized strain can now be used as the basis for the characterization of novel pathways.KEYWORDS lignin, protocatechuic acid, ortho-cleavage pathway, experimental evolution, synthetic biology, ligninolysis, metabolic engineering, ortho-cleavage B iofuels derived from lignocellulose will likely play an important role in the transition to a sustainable and carbon-neutral economy (1). In a typical biotransformation, carbohydrate-rich cellulose and hemicellulose are extracted and fermented to yield the desired biofuel, such as bioethanol. The lignin, comprising up to 25% of the dry

Horizontal transfer of a pathway for coumarate catabolism unexpectedly inhibits purine nucleotide biosynthesis

“…There is no a priori reason to expect that a pathway for degradation of an aromatic compound would interact with a native pathway for nucleotide biosynthesis. Phenolic amides such as feruloyl amide have been shown to inhibit a different step in nucleotide biosynthesis (Pisithkul et al, 2015), but neither the substrate nor products of coumarate degradation are toxic at the relevant concentrations (Clarkson et al, 2017; Standaert et al, 2018). These types of inhibitory cross-talk are likely to be common with introduced metabolic pathways, though they are rarely identified and alleviated (Kim and Copley, 2012; Kizer et al, 2008; Michener et al, 2012).…”

“…As described previously, the pathway design used synthetic promoters, terminators, and custom ribosome binding sites (Chen et al, 2013; Espah Borujeni et al, 2014; Kosuri et al, 2013; Salis et al, 2009). Plasmids expressing sgRNA for chromosomal modifications were constructed as described previously, using an inverse PCR to linearize the expression vector followed by assembly with synthesized oligonucleotides (Clarkson et al, 2017). Plasmid pJM303, expressing the D243G mutant of the E. coli IMPDH, was constructed by amplifying the mutant guaB allele from JME89 and cloning it into pMCSG7 under the control of a T7 promoter.…”

“…Genome modifications were performed as described previously, using the lambda-red recombineering system in combination with Cas9-mediated selection (Clarkson et al, 2017; Jiang et al, 2015). Integration cassettes were amplified from synthesized plasmids or the chromosomal DNA of mutant strains, as needed.…”

“…Growth rates were measured as described previously (Clarkson et al, 2017). Briefly, cultures were grown overnight to saturation in M9 + 1 mM IPTG + 2 g/L glucose.…”

Biotransformation of lignin: Mechanisms, applications and future work

“…,128 For example, Sonoki et al116 and vanB encoding αand β-subunits respectively of vanillate demethylase, and catA encoding catechol dioxygenase) into the genome of E. coli so that the modified E. coli can produce muconic acid from vanillin. However, the decarboxylation reaction of protocatechuate remained a bottleneck and rate-limiting step as stated in previous studies 127.…”