Oral Presentation Australian Microbial Ecology 2017

Forcing adaptation to reveal novel metabolic pathways: the malic acid bi-cycle of Geobacter sulfurreducens (#36)

Lucie Semenec 1 , Fumiko Watanabe 2 , Radhakrishnan Mahadevan 3 , Kengo Inoue 2 , Ashley Franks 1
  1. La Trobe University, Melbourne, VICTORIA, Australia
  2. Department of Biochemistry and Applied Biosciences, University of Miyazaki, Miyazaki, Japan
  3. Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Canada

While bacteria may contain a wide range of metabolic potential within their genome, specific conditions for the utilisation of carbon sources is often required.  Previously Geobacter sulfurreducens growth had not been observed when malate was the sole electron donor, acceptor and carbon source over a two-week timeframe. G. sulfurreducens contains malate utilising pathways which were thought to generally be energetically unfavorable under such conditions. In this study we grew G. sulfurreducens with malate as the sole electron donor, acceptor and carbon source but monitored growth for a longer time period than was done previously.  

 

In our study, G. sulfurreducens growth was observed in this strict medium after a period of 1.5 months. Cultures were transferred to fresh malate medium at late exponential growth to allow for adaptation to growth on malate. Growth significantly improved with successive generations, where the cultures reached exponential phase within one week by the 50th transfer compared to 1.5 months for the first generation. SWATH-MS proteomics revealed many biosynthetic and metabolic processes were downregulated in the malate-adapted cultures compared against non-adapted G. sulfurreducens grown in non-restrictive medium. Within the small subset of up-regulated proteins included a potential novel pathway of energy production and acetate biosynthesis. We call this pathway the malic acid bi-cycle (MAbiC) as it relies on connection of two cyclic metabolic pathways and is an extension of the citric acid cycle.

 

Re-sequencing of the adapted strain has allowed for genomic investigations of changes within the MAbiC at a genetic level. Any novel mutations revealed will be candidates for bioengineering strains proficient at this pathway which involves acetaldehyde production, a major commodity chemical in industrial applications. Furthermore, mutant strains of G. sulfurreducens are being constructed where the malate-utilization genes are deleted to confirm the role these genes play in the MAbiC. Lastly, high-performance liquid chromatography will quantify key intermediates in the pathway thus confirming its architecture.