Assessment of intracellular pH and fermentation profile in xylose-fermenting Saccharomyces cerevisiae strains using ratiometric sensors

Abstract

The production of ethanol from second-generation feedstock like lignocellulose is limited by efficient growth of Saccharomyces cerevisiae on xylose. One of the reasons for that might be a lower ATP formation vs. proton formation flux in cells using the xylose reductase (XR)/xylose dehydrogenase (XDH) pathway compared to glucose as ideal substrate. The therefore reduced availability of ATP, as ATP is consumed to maintain ideal intracellular pH levels, might hinder sufficient growth. Heterologous H+-translocating pyrophosphatases might lead to an increase of ATP levels as they take over the role or co-contribute to proton pumping.
Therefore, the effect of heterologous expressed H+PPases on growth and fermentation profile of S. cerevisiae was analysed. To further investigate their effects on intracellular pH and ATP levels, biosensor strains expressing ratiometric sensors were created by chromosomal integration. Inserting a heterologous vacuolar H+-PPase led to a strong increase in maximum growth rate as well as ethanol productivity and reduced overall fermentation duration. In contrast, the addition of cytosolic H+-PPases did not have a clear effect on growth rate nor ethanol productivity. Further, our results show -against previous expectations- a reduction of intracellular pH on glucose and xylose when expressing heterologous H+-PPases. We suggest that the lower ATP vs. proton formation flux is not the main cause of inadequate growth for S. cerevisiae on xylose, but that it rather results from insufficient regulation and signalling of the native pH homeostasis system. This advance in xylose fermentation of S. cerevisiae using the XR/XDH pathway further opens the way for industrial use of pentose-fermenting strains and a more sustainable ethanol production process.

Popular Abstract

The yeast Saccharomyces cerevisiae is one of the most known microorganisms in the world as it has been used for thousands of years as a means to bake bread or brew beer or other beverages. In recent times its main fermentation product, ethanol, has become of increasing interest as it can be used as a basis for other chemicals or as a sustainable fuel – bioethanol. This can already be experienced when refilling the car and the option exists between “normal” unleaded petrol or petrol supplemented with bioethanol also known as E5 or E10. Biofuels can be made from various sugar sources containing glucose or fructose, but those have the issue that they compete with our own food production. Hence, to reduce negative environmental impacts, the use of other renewable feedstock like non-edible agricultural leftovers or forestry waste sounds appealing. Those so-called lignocellulosic wastes consist to a major degree of the sugar xylose. Using this carbon source can potentially reduce the competition for food and land resources, as well as the environmental impact associated with traditional ethanol production. Moreover, the fermentation of xylose, in addition to other sugars, into ethanol has high potential to improve the economic viability of biofuel production and thus help the development of a more sustainable economy. Unfortunately, our favoured yeast is not able to ferment this sugar naturally, so efforts to engineer yeast to efficiently ferment xylose have been the focus of many studies in recent years, unfortunately with still lower resulting yields and productivities than on glucose.
My Master thesis’ goal was to explore how xylose fermentation affects the cells energy and internal acidity and if the addition of artificial proton pumps can help the yeast to increase their energy level and make them feel like they are growing on their favourite sugar glucose. Our results showed that one of those artificial proton pumps leads to almost doubling the growth rate previously achieved on xylose thus reducing the time required to transform all the xylose into ethanol or other by-products. I also found that one of the modifications stabilised the cells acidity bringing it closer to the levels found on glucose. These results represent an important step towards the use of xylose as a major substrate in industrial yeast-based bioprocess.

• author: Eisentraut, Leon
• supervisor: Ed van Niel
• organization: Biotechnology; Applied Microbiology
• course: KMBM01 20231
• year: 2023
• type: H2 - Master's Degree (Two Years)
• subject: Technology and Engineering
• keywords: S. cerevisiae, metabolic Engineering, pyrophosphatases, xylose, ATPase, Applied Microbiology
• language: English
• id: 9126751
• date added to LUP: 2023-06-20 10:28:12
• date last changed: 2023-06-20 10:28:12