Abstract
Transfer of nitrogen fixation (nif) genes from diazotrophs to amenable heterologous hosts is of increasing interest to genetically engineer nitrogen fixation. However, how the non-diazotrophic host maximizes opportunities to fine-tune the acquired capacity for nitrogen fixation has not been fully explored. In this study, a global investigation of an engineered nitrogen-fixing Escherichia coli strain EN-01 harboring a heterologous nif island from Pseudomonas stutzeri was performed via transcriptomics and proteomics analyses. A total of 1156 genes and 206 discriminative proteins were found to be significantly altered when cells were incubated under nitrogen-fixation conditions. Pathways for regulation, metabolic flux and oxygen protection to nitrogenase were particularly discussed. An NtrC-dependent regulatory coupling between E. coli nitrogen regulation system and nif genes was established. Additionally, pentose phosphate pathway was proposed to serve as the primary route for glucose catabolism and energy supply to nitrogenase. Meanwhile, HPLC analysis indicated that organic acids produced by EN-01 might have negative effects on nitrogenase activity. This study provides a global view of the complex network underlying the acquired nif genes in the recombinant E. coli and also provides clues for the optimization and redesign of robust nitrogen-fixing organisms to improve nitrogenase efficiency by overcoming regulatory or metabolic obstacles.
Highlights
In nature, a variety of genes and islands can be rapidly and frequently horizontally transferred among bacteria, resulting in the acquisition of certain properties such as nitrogen fixation, antimicrobial resistance and pathogenesis, which help bacteria to succeed in altered habitats or new niches[1,2,3]
Following the pioneering work on nitrogen-fixation engineering in 1970s4,5, several groups have reported successful gene transfer of nif genes to E. coli in the past five years[11,12,18,19]. These recombinant E. coli stains showed much lower nitrogenase activity compared with the original host11,12, and the horizontally acquired ability was insufficient to enable diazotrophic growth on nitrogen-free medium, implying the presence of (i) regulatory coupling between the host and heterologous nitrogen-fixation systems, as well as (ii) a regulatory/or metabolic barrier that results in reduced nitrogenase activity in the engineered cells
The up-regulated genes mainly belonged to three major functional categories: nitrogen metabolism, transport or membrane protein and unknown function, while the down-regulated genes were mainly involved in energy synthesis, transport, protein synthesis and regulation
Summary
A variety of genes and islands can be rapidly and frequently horizontally transferred among bacteria, resulting in the acquisition of certain properties such as nitrogen fixation, antimicrobial resistance and pathogenesis, which help bacteria to succeed in altered habitats or new niches[1,2,3]. Following the pioneering work on nitrogen-fixation engineering in 1970s4,5, several groups have reported successful gene transfer of nif genes to E. coli in the past five years[11,12,18,19]. These recombinant E. coli stains showed much lower nitrogenase activity compared with the original host11,12,, and the horizontally acquired ability was insufficient to enable diazotrophic growth on nitrogen-free medium, implying the presence of (i) regulatory coupling between the host and heterologous nitrogen-fixation systems, as well as (ii) a regulatory/or metabolic barrier that results in reduced nitrogenase activity in the engineered cells. Given its natural integrity and well-studied regulation, the A1501 NFI is a promising model for studying the synthetic biology of nitrogen fixation systems
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