Development of a Metabolic Pathway Transfer and Genomic Integration System for the Syngas-fermenting Bacterium

Overview

Journal Biotechnol Biofuels

Publisher Biomed Central

Specialty Biotechnology

Date 2019 May 16

PMID 31086564

Citations 13

Authors

Gabriele Philipps

Sebastian de Vries

Stefan Jennewein

Affiliations

Soon will be listed here.

Abstract

Background: spp. can synthesize valuable chemicals and fuels by utilizing diverse waste-stream substrates, including starchy biomass, lignocellulose, and industrial waste gases. However, metabolic engineering in spp. is challenging due to the low efficiency of gene transfer and genomic integration of entire biosynthetic pathways.

Results: We have developed a reliable gene transfer and genomic integration system for the syngas-fermenting bacterium based on the conjugal transfer of donor plasmids containing large transgene cassettes (> 5 kb) followed by the inducible activation of transposase to promote integration. We established a conjugation protocol for the efficient generation of transconjugants using the Gram-positive origins of replication and . We also investigated the impact of DNA methylation on conjugation efficiency by testing donor constructs with all possible combinations of Dam and Dcm methylation patterns, and used bisulfite conversion and PacBio sequencing to determine the DNA methylation profile of the genome, resulting in the detection of four sequence motifs with N-methyladenosine. As proof of concept, we demonstrated the transfer and genomic integration of a heterologous acetone biosynthesis pathway using a transposase system regulated by a xylose-inducible promoter. The functionality of the integrated pathway was confirmed by detecting enzyme proteotypic peptides and the formation of acetone and isopropanol by cultures utilizing syngas as a carbon and energy source.

Conclusions: The developed multi-gene delivery system offers a versatile tool to integrate and stably express large biosynthetic pathways in the industrial promising syngas-fermenting microorganism . The simple transfer and stable integration of large gene clusters (like entire biosynthetic pathways) is expanding the range of possible fermentation products of heterologously expressing recombinant strains. We also believe that the developed gene delivery system can be adapted to other clostridial strains as well.

Citing Articles

Engineered acetogenic bacteria as microbial cell factory for diversified biochemicals.

Zhang J, Li Y, Xi Z, Gao H, Zhang Q, Liu L Front Bioeng Biotechnol. 2024; 12:1395540.

PMID: 39055341 PMC: 11269201. DOI: 10.3389/fbioe.2024.1395540.

The Restriction-Modification Systems of P7.

Kottenhahn P, Philipps G, Bunk B, Sproer C, Jennewein S Microorganisms. 2023; 11(12).

PMID: 38138106 PMC: 10745947. DOI: 10.3390/microorganisms11122962.

Metabolic engineering of Clostridium ljungdahlii for the production of hexanol and butanol from CO and H.

Lauer I, Philipps G, Jennewein S Microb Cell Fact. 2022; 21(1):85.

PMID: 35568911 PMC: 9107641. DOI: 10.1186/s12934-022-01802-8.

Research progress of pathway and genome evolution in microbes.

Huang C, Wang C, Luo Y Synth Syst Biotechnol. 2022; 7(1):648-656.

PMID: 35224232 PMC: 8857405. DOI: 10.1016/j.synbio.2022.01.004.

Hexanol biosynthesis from syngas by P7 - product toxicity, temperature dependence and extraction.

Kottenhahn P, Philipps G, Jennewein S Heliyon. 2021; 7(8):e07732.

PMID: 34409191 PMC: 8361263. DOI: 10.1016/j.heliyon.2021.e07732.

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