An Escherichia coli FdrA Variant Derived from Syntrophic Coculture with a Methanogen Increases Succinate Production Due to Changes in Allantoin Degradation.

Ji Won Park,Yeon Joo Lee,Nam Yeun Kim,Gary Sawers,Su Nyung Kim,Yuseob Kim,Ok Bin Kim,E Young Kim

doi:10.1128/msphere.00654-21

Abstract

ABSTRACTWild-type Escherichia coli was adapted to syntrophic growth with Methanobacterium formicicum for glycerol fermentation over 44 weeks. Succinate production by E. coli started to increase in the early stages of syntrophic growth. Genetic analysis of the cultured E. coli population by pooled sequencing at eight time points suggests that (i) rapid evolution occurred through repeated emergence of mutators that introduced a large number of nucleotide variants and (ii) many mutators increased to high frequencies but remained polymorphic throughout the continuous cultivation. The evolved E. coli populations exhibited gains both in fitness and succinate production, but only for growth under glycerol fermentation with M. formicicum (the condition for this laboratory evolution) and not under other growth conditions. The mutant alleles of the 69 single nucleotide polymorphisms (SNPs) identified in the adapted E. coli populations were constructed individually in the ancestral wild-type E. coli. We analyzed the phenotypic changes caused by 84 variants, including 15 nonsense variants, and found that FdrAD296Y was the most significant variant leading to increased succinate production. Transcription of fdrA was induced under anaerobic allantoin degradation conditions, and FdrA was shown to play a crucial role in oxamate production. The FdrAD296Y variant increased glyoxylate conversion to malate by accelerating oxamate production, which promotes carbon flow through the C4 branch, leading to increased succinate production.IMPORTANCE Here, we demonstrate the ability of E. coli to perform glycerol fermentation in coculture with the methanogen M. formicicum to produce succinate. We found that the production of succinate by E. coli significantly increased during successive cocultivation. Genomic DNA sequencing, evaluation of relative fitness, and construction of SNPs were performed, from which FdrAD296Y was identified as the most significant variant to enable increased succinate production by E. coli. The function of FdrA is uncertain. In this study, experiments with gene expression assays and metabolic analysis showed for the first time that FdrA could be the “orphan enzyme” oxamate:carbamoyltransferase in anaerobic allantoin degradation. Furthermore, we demonstrate that the anaerobic allantoin degradation pathway is linked to succinate production via the glyoxylate pathway during glycerol fermentation.

Highlights

IMPORTANCE Here, we demonstrate the ability of E. coli to perform glycerol fermentation in coculture with the methanogen M. formicicum to produce succinate
We showed that long-term cocultivation of E. coli with the formate degrader Methanobacterium formicicum led to increased succinate production from glycerol fermentation by E. coli [18]
We reported that long-term (273-day) cocultivation of E. coli and M. formicicum showed increased succinate production under glycerol fermentation [18]

Summary

Introduction

IMPORTANCE Here, we demonstrate the ability of E. coli to perform glycerol fermentation in coculture with the methanogen M. formicicum to produce succinate. Prokaryotes have evolved to utilize amazingly diverse energy and carbon sources in nature, as they are capable of quickly generating adaptive variants due to rapid reproduction, which accumulates mutations in a large population. The applications of experimental evolution can be categorized into five general areas: (i) growth rate optimization, (ii) increase in tolerance, (iii) change in substrate utilization, (iv) increase in product yield or titer, and (v) general discovery [4] These studies predominantly used E. coli (78% among bacteria) and Saccharomyces cerevisiae (44% among yeast) [4]. We showed that long-term cocultivation of E. coli with the formate degrader Methanobacterium formicicum led to increased succinate production from glycerol fermentation by E. coli [18]. We tracked the genetic variation in the selected E. coli strains responsible for the increased succinate production

Methods

Results

Conclusion