#FEMSmicroBlog: Citric acid transporter and 'a matter of honor'

05-10-2020

A new paper in FEMS Yeast Research identified the trasporter of citric acid in the yeast Yarrowia lipolytica. Optimisation of industrial production of citric acid using yeasts is one step ahead. In this #BehindThePaper interview for the #FEMSmicroBlog, we spoke with senior author Michael Sauer who presents the significance of the paper to different audiences and tells why, sometimes, finding the right protein is a matter of honor. #FascinatingMicrobes 

 

Can you summarize the significance of the paper for microbiologists in a different field?

Membrane proteins are decisively underrepresented in the list of biochemically characterized proteins. However, the exchange of molecules across membranes is a fundamental process for life in general. Here, we identified the citric acid exporter from the yeast Yarrowia lipolytica. This yeast accumulates high amounts of citric acid under specific conditions. It was unclear up to now which transport protein(s) are involved in the export of the acid.

Astonishingly, the yeast strains which we analyzed seem to have only one type of protein capable to fulfill this task. Deletion of the respective gene, which we named Cex1, entirely abolishes citric acid accumulation. This is quite astonishing as most of the metabolic enzymes which we looked at had several counterparts with similar activities. The metabolic network of Y. lipolytica is remarkably robust.

Knockout studies of single genes have very often no phenotype at all, so we were surprised to see that the important function allowing the export of citric acid has no safety copy. It is furthermore noteworthy that the here identified protein has no similarity to the only other known citric acid exporter CexA from Aspergillus niger, but both genes can functionally complement each other in both species.

Growth curve and metabolite production by Yarrowia lipolytica. Closed circles is Cex1 mutant, open circles is the wildtype (see the paper for more details)

 

Can you convey the fascination of the findings to kids and young adults?

Membranes are like the skin of cells. They separate the “inside” of cells from the potentially dangerous outside world. It is essential for life to keep all precious things inside of the cells together, and to shield them from the outside, which could cause harm.

Therefore, membranes are very fundamental structures for every living organism. However, just as separation is fundamental, a proper exchange of substances across those membranes is at least as important to sustain life. Carbon sources need to be taken up (food) and products need to be exported (the cell needs also to use the toilet, so to say).

Here we look at a yeast – Yarrowia lipolytica – which produces high amounts of citric acid. Citric acid is industrially very interesting as it is used for food and beverage production. We wondered, ‘How does citric acid, which is produced inside of the cell, leaves the cell?’. We found a protein spanning the membrane fulfilling this task. It took us some time and effort to find it and then we were quite surprised to see that only one type of protein which can do the trick exists in this yeast.

 

Can you summarize the relevance of the paper for policymakers?

Industrial microbiology is a key factor for the development of the bioeconomy. One major biotech product on the market is citric acid, which is used for the food and beverage industry, but also for pharmaceutical applications and in cleaning products. Since many decades, citric acid sold on the market is not produced from lemons anymore, but by microbial fermentation from sugar.

Citric acid is by volume one of the major biotech products. About 2 mio. t per year are produced by microbial fermentation. Increasing the efficiency of microbial fermentation processes is one of the tasks of industrial microbiologists. Metabolic engineering and synthetic biology are the tools used.

However, most approaches focus on metabolism itself and overlook the importance of transport processes into the cell and out of the cell. Very often these transport processes are rate limiting, therefore it would be worthwhile to look at them in more detail.

Since transport processes are technically difficult to analyze they are often neglected. Here, we identified the citric acid exporting protein from the yeast Yarrowia lipoplytica. The identification is a first step for a better characterization of this process and finally an exploitation to improve our microbial cell factories.

In general, we should strive to close the lack of knowledge about transport proteins, because they are essential for the understanding of life. Being of utmost importance for all microbial fermentation processes, transport proteins are directly connected to value generation.

 

Is there a (personal) story behind the paper? I.e. what lead you to pursue these results, something unexpected, your personal/scientific journey,…

The story started some time ago with the industrial citric acid production organism Aspergillus niger. The fungus used for this fermentation is very well characterized and we know a lot about the biochemical processes behind. However, how the citric acid is exported from the cell – against a strong gradient – has curiously not been found. Recently, we identified the transport protein from A. niger, enabling detailed biochemical studies.

A second organism, which produces citric acid quite efficiently, is the yeast Yarrowia lipolytica. Also here, the transport protein was the missing link for a proper description of the molecular processes. Having identified the transporter from A. niger, we searched by sequence similarity and found one single protein which is very similar. Testing this gene, it turned out that it has nothing to do with citric acid export, indeed a knockout has no phenotype at all.

So we started to look for other genes and knocked one out after the other, with no success. Finally, it became a question of honor. Transcriptome analysis comparing yeasts under citric acid production conditions with yeasts not producing this acid pointed to a gene in Yarrowia lipolytica which has absolutely no similarity to the A. niger transporter.

We could identify it as the citric acid exporter, which is quite interesting as we have now two transport proteins in our hand, which are structurally quite different, but both are the only functional citric acid exporters in their species and both are very efficient.

About the author of this blog

Michael Sauer (@michl_wien) is Assistant Professor at the Institute of Microbiology and Microbial Biotechnology at BOKU – University of Natural Resources and Life Sciences, Vienna. He is an industrial microbiologist focused on sustainable microbial chemical production. His research is based on exploring biodiversity to find nature’s answers to industrial problems, combined with synthetic biology to augment the capabilities of microorganisms, when required. After receiving a diploma in biotechnology at the Swiss Federal Institute of Technology (ETHZ) in Zürich, he pursued his doctoral studies in biochemistry at the University of Vienna. A postdoctoral experience at the University of Milano-Bicocca brought him in close contact to industry – a contact, which he never lost since then. He obtained the venia-docendi in industrial microbiology at BOKU. Michael is section editor for FEMS Microbiology Letters and FEMS delegate for the Austrian Association of Molecular Life Sciences and Biotechnology.

About this blog section

The section #FascinatingMicrobes for the #FEMSmicroBlog explains the science behind a paper and highlights the significance and broader context of a recent finding. One of the main goals is to share the fascinating spectrum of microbes across all fields of microbiology. #BehindThePaper interviews aim to bring the science closer to different audiences, and to tell more about the scientific or personal journey to come to the results.

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