Identification of parallel and divergent optimization solutions for homologous metabolic enzymes

Abstract

Metabolic pathway assembly typically involves the expression of enzymes from multiple organisms in a single heterologous host. Ensuring that each enzyme functions effectively can be challenging, since many potential factors can disrupt proper pathway flux. Here, we compared the performance of two enzyme homologs in a pathway engineered to allow Escherichia coli to grow on 4-hydroxybenzoate (4-HB), a byproduct of lignocellulosic biomass deconstruction. Single chromosomal copies of the 4-HB 3-monooxygenase genes pobA and praI, from Pseudomonas putida KT2440 and Paenibacillus sp. JJ-1B, respectively, were introduced into a strain able to metabolize protocatechuate (PCA), the oxidation product of 4-HB. Neither enzyme initially supported consistent growth on 4-HB. Experimental evolution was used to identify mutations that improved pathway activity. For both enzymes, silent mRNA mutations were identified that increased enzyme expression. With pobA, duplication of the genes for PCA metabolism allowed growth on 4-HB. However, with praI, growth required a mutation in the 4-HB/PCA transporter pcaK that increased intracellular concentrations of 4-HB, suggesting that flux through PraI was limiting. These findings demonstrate the value of directed evolution strategies to rapidly identify and overcome diverse factors limiting enzyme activity.

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

“…We designed and synthesized two pathways for phenylpropanoid import and degradation, each of which converts coumarate into 4-hydroxybenzoate (4-HB) (Figure 1 and Figure S1). Each pathway was introduced into E. coli strains, JME38 and JME50, that had previously been engineered to grow with 4-HB (Standaert et al, 2018). None of the engineered strains acquired the immediate ability to grow with coumarate as the sole source of carbon and energy (Figure S2).…”

“…The genomes of the selected isolates were resequenced to identify new mutations (Table S1). Several of the mutations have previously been described for their effects on catabolism of 4-HB, such as silent mutations to the gene encoding the 4-hydroxybenzoate monooxygenase pobA (Standaert et al, 2018). Among the strains with the hca pathway, five of the six isolates had additional mutations to the native gene guaB , encoding inosine monophosphate (IMP) dehydrogenase (IMPDH), and to the intergenic region between hcaB and hcaC in the engineered pathway.…”

“…The pobA mutation has previously been shown to increase expression of PobA by destabilizing secondary structures in the mRNA (Standaert et al, 2018). To understand the effect of the intergenic mutations upstream of hcaC , we measured protein expression levels in the engineered strains.…”

“…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).…”

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

“…We previously constructed strains of E. coli that grow with the model lignin‐derived compounds protocatechuate (PCA) and 4‐hydroxybenzoate (4‐HB) as sole sources of carbon and energy using the 3,4‐cleavage pathway for PCA catabolism from Pseudomonas putida and a 4‐HB 3‐monooxygenase from P. putida or Paenibacillus sp. JJ‐1b (Clarkson et al , ; Standaert et al , ). Introduction of the relevant catabolic pathways was not sufficient to enable rapid growth with either carbon source.…”