Biofuels and Commodities
With the advent of genetic engineering, a revolution occurred within the sector of biofuel and commodity production. Suddenly, microbes were being utilised as scaffolding structures upon which to produce commercially valuable chemicals, for use in a wide range of industries. However, while gene expression analysis tools, such as RNA-Seq, were able to be used to identify changes at the level of transcription, for many years a detailed analysis of the dynamics of translation was missing. Such an analysis is crucial, with protein synthesis largely being the final step of gene expression, and similarly, biofuel and commodity production. The technique of ribosome profiling provided the breakthrough this sector needed and has to date provided a huge number of valuable insights into the translational processes that occur within many key model organisms used in the production of these chemicals.
The below papers exemplify the power that ribosome profiling has to reveal these key insights, thus unlocking the potential of these cellular factories to optimise chemical production.
Genome-scale analysis of syngas fermenting acetogenic bacteria reveals the translational regulation for its autotrophic growth
BMC Genomics, 2018; 19(1), pp.1-15.
Song, Y., Shin, J., Jin, S., Lee, J.K., Kim, D.R., Kim, S.C., Cho, S. and Cho, B.K.,
This paper looks at the bacterium Eubacterium limosum, an anaerobic acetogenic microbe which utilises CO2 and H2 as energy sources and can produce acetate, butyrate and ethanol. As such, it is under significant investigation as a model organism for commodity production in the biochemicals industry. However, a deep understanding of transcriptional and translational regulation has not yet been obtained. Here, researchers analyse the mechanisms of this regulation under heterotrophic and autotrophic conditions.
Key Findings
- Under autotrophic conditions, there was transcriptional upregulation of genes involved in the Woods-Ljungdahl pathway (WLP), gluconeogenesis, and reduction of electron carriers. Genes involved in electron bifurcation and cellular respiration were upregulated at the translational level.
- Genes involved in the carbonyl branch of the WLP, ATP synthase complex in the energy conservation system, and the ion-translocating complex, while being expressed at high levels transcriptionally, were translationally downregulated under autotrophic conditions resulting in lower translation efficiencies for the same genes when compared under heterotrophic conditions.
- Differences in translational efficiencies for the same genes under autotrophic conditions compared to heterotrophic conditions was linked to changes in levels of free energy associated with potential regulatory structural features in 5’ UTRs.
Implications
These results suggest that under conditions of autotrophic growth, there is a switch in gene expression in E. limosum with genes involved in the generation of reduced electron carriers via electron bifurcations pathways being expressed, as opposed to genes involved in carbon metabolism. Such knowledge may prove useful in constructing the ideal environmental conditions for this organism, and potentially generate improved yields.
Ribosome Profiling of Synechocystis Reveals Altered Ribosome Allocation at Carbon Starvation
mSystems, 2018; 3(5), pp.e00126-18.
Karlsen, J., Asplund-Samuelsson, J., Thomas, Q., Jahn, M. and Hudson, E.P.
Cyanobacteria are an ancient bacterial phylum that utilise sunlight to drive energy synthesis, and as such have evolved to adapt to frequent periodic fluctuations in light levels. This has led to interest in their use as biological chassis, to convert light and CO2 into useful chemicals and biofuels. In this paper, researchers utilise ribosome profiling to undertake the first genome-wide analysis of protein synthesis of any cyanobacterium. The specific organism analysed, Synechocystis 6803, has already been used as a model for isoprene, ethanol, sucrose, and hydrogen production, and has been extensively studied. Here, they examine the effects of carbon starvation on ribosome allocation, a key indicator of translational efficiency.
Key Findings
- Upon carbon starvation, there was an 80% decrease in overall translation, with increased ribosome pausing at start sites, stop sites, and untranslated regions.
- While most genes fell in tandem at the transcriptional and translational level upon carbon starvation, a number underwent significant translational upregulation, compared to their transcriptional levels. These included glutamine synthetase-inactivating factor IF7, thioredoxin M (sll1057), protease HtrA (slr1204), and heat shock protein HspA (sll1514), thought to be involved in stress response.
- Genes that displayed high ribosome pausing upon carbon starvation within the 5’ UTR were typically overrepresented in the functional categories of photosynthesis, respiration, and translation. These genes were also enriched for an AG-rich Shine-Dalgarno sequence in their 5’ UTRs.
- There was also significantly reduced level of correlation in ribosome pausing for four ribosome biogenesis proteins upon carbon starvation compared to high carbon availability, indicating translation is significantly affected under these conditions.
Implications
This paper has shown that cyanobacteria actively sequester ribosomes in untranslated regions of mRNA during carbon starvation. It also found that the ribosome distribution is specifically altered on certain stress response related transcripts. Furthermore, it has displayed the ability of ribosome profiling to discover mechanisms of adaptive control to varying external conditions.
Optimization of carbon and energy utilization through differential translational efficiency
Nature communications, 2018, 9(1), pp.1-13.
Al-Bassam, M.M., Kim, J.N., Zaramela, L.S., Kellman, B.P., Zuniga, C., Wozniak, J.M., Gonzalez, D.J. and Zengler, K.
The microbe Clostridium Ijungdahlii is a key organism in the biofuels synthesis industry, with the ability to produce ethanol and acetate from a variety of source materials. However, it’s transcriptional and translation regulation are poorly understood. Here, the authors presented an analysis of carbon and energy metabolism under a range of conditions, specifically looking in detail at the effects of these conditions upon the translational efficiencies of certain genes involved in these processes.
Key Findings
- Under heterotrophic conditions, the processes of carbon metabolism and de novo macromolecule synthesis and maintenance were upregulated, particularly glycolysis, the pentose phosphate pathway, and chorismate synthesis subsystem.
- Under autotrophic conditions, the processes of carbon fixation, fermentation, and motility were upregulated, with particular emphasis on the carbon monoxide dehydrogenase pathway, the 2,3-butanediol dehydrogenase pathway, flagellum production, and the Rnf pathway.
- The Rnf complex, responsible for energy conservation, is repressed specifically at the translational level under heterotrophic conditions (in particular the rnfC and rseC genes), thus providing for energy utilisation.
- Genes which displayed a high translational efficiency possessed strong ribosome binding site (RBS) motif conservation, and also displayed an increased AU content in the upper region of the RBS sequences.
Implications
This work laid the foundational groundwork for gene expression exploration in this industrially important microorganism. It revealed the level at which various pathways, in particular a number of metabolic systems, are regulated. Such insights may prove vitally important for advanced strain design and engineering, ultimately paving the way for more efficient commodity production.
Correcting direct effects of ethanol on translation and transcription machinery confers ethanol tolerance in bacteria
Proceedings of the National Academy of Sciences, 2014, 111(25), pp.E2576-E2585.
Haft, R.J., Keating, D.H., Schwaegler, T., Schwalbach, M.S., Vinokur, J., Tremaine, M., Peters, J.M., Kotlajich, M.V., Pohlmann, E.L., Ong, I.M. and Grass, J.A.
Microbial susceptibility to ethanol toxicity is an important factor in a range of industries, but particularly in the bio-commodities sector, where varying alcohol productions by these bacteria are of paramount importance. Consequences of such toxicity can result in lower microbial growth and thus lower ethanol production. While ethanol is hypothesised to have particular cellular targets, the exact mechanisms are less defined. Utilising spontaneously evolved ethanol-tolerant E. coli, researchers characterised mechanisms of resistance using a variety of genome-based techniques, including ribosome profiling.
Key Findings
- Serial-passage evolution experiments resulted in a number of mutations which resulted in significant resistance to ethanol toxicity, including mutations in the Rho (a transcription termination factor), MetJ (repressor of methionine synthesis), and RpsQ (a ribosome protein) genes.
- Ethanol was found to increase the miscoding error rate. However, this error rate was dramatically reduced in the mutant E. coli strain containing the 3 mutations noted above.
- Ribosome profiling revealed that, under treatment with ethanol, there was a distinct reduction in ribosome occupancy towards the 3’ end of mRNA transcripts. The translation termination appears linked to a Rho-mediated increase in transcription termination concurrent with ethanol treatment. There was also a significant increase in ribosome occupancy at non-start AUG codons under these conditions, although this was reduced in the mutant strain.
Implications
Here, the authors uncovered a number of mechanisms whereby ethanol exerts toxicity upon E. coli, information which may lay the groundwork for targeted strategies to combat such toxicity. Importantly, they also discovered a raft of mutations which confer a resistance to this toxicity, and as such may be particularly useful in designing ideal strains for commodity production.