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- Sreenivas, Krishnan, et al.
(författare)
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Evaluation of Pyrophosphate‐Driven Proton Pumps in Saccharomyces cerevisiae under Stress Conditions
- 2024
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Ingår i: Microorganisms. - 2076-2607. ; 12:3
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Tidskriftsartikel (refereegranskat)abstract
- In Saccharomyces cerevisiae, pH homeostasis is reliant on ATP due to the use of proton-translocating ATPase (H+-ATPase) which constitutes a major drain within cellular ATP supply. Here, an exogenous proton-translocating pyrophosphatase (H+-PPase) from Arabidopsis thaliana, which uses inorganic pyrophosphate (PPi) rather than ATP, was evaluated for its effect on reducing the ATP burden. The H+-Ppase was localized to the vacuolar membrane or to the cell membrane, and their impact was studied under acetate stress at a low pH. Biosensors (pHluorin and mQueen-2m) were used to observe changes in intracellular pH (pHi) and ATP levels during growth on either glucose or xylose. A significant improvement of 35% in the growth rate at a pH of 3.7 and 6 g·L−1 acetic acid stress was observed in the vacuolar membrane H+-PPase strain compared to the parent strain. ATP levels were elevated in the same strain during anaerobic glucose and xylose fermentations. During anaerobic xylose fermentations, co-expression of pHluorin and a vacuolar membrane H+-PPase improved the growth characteristics by means of an improved growth rate (11.4%) and elongated logarithmic growth duration. Our study identified a potential method for improving productivity in the use of S. cerevisiae as a cell factory under the harsh conditions present in industry.
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| 2. |
- Sreenivas, Krishnan
(författare)
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Explorations of interlinked energy and redox metabolism in two industrially applied microorganisms
- 2024
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Anaerobic fermentation, compared to aerobic processes, remains the most scalable method for bioproduction of compounds. This is due to that aerobic microbial processes require large amounts of energy and are unable to satisfy the oxygen demand of a high-density microbial biomass. Thus, anaerobic microbes are readily scalable to large volumes. It was found that most anaerobic microbes have evolved with the Embden-Meyerhof-Parnas pathway for efficient and luxurious growth under these conditions due to adequate energy production provided by the pathway. It is also found that most of these microbes are able to establish a balanced redox and energy metabolism either by the use of anaerobic respiration or other forms of ATP synthesis. Some microbes, however, are of industrial relevance but are not capable of luxurious growth without the use of external electron acceptors or other external conditions. Among these microbes, the suspected issues are usually attributed to redox or energy imbalances. Thus, the thesis expands on our understanding of the regulatory mechanisms and explores possible engineering methods to improve their growth rates by focusing on two specific microbes, namely the bacterium Limosilactobacillus reuteri and the yeast Saccharomyces cerevisiae.Oxygen tolerance of Lb. reuteri is important as it is one of the mechanisms to alleviate the redox imbalance. This study expands on the variations of oxygen tolerance between strains and shows that Lb. reuteri DSM 17938 does not necessarily produce more peroxide per biomass but has greater resistance than its counterparts. In parallel the various lactate dehydrogenases present in Lb. reuteri DSM 17938 were enzymatically characterised to explore the presence of alternative control mechanisms that may be present due to the simultaneous utilisation of two different central carbon pathways. The impact of overexpression of the native phosphofructokinase candidates which are predicted to be from a minor family revealed issues related to protein burden in lean media.The introduction of a proton pumping pyrophosphatase (H+-PPase) to supplement the proton pumping ATPase (H+-ATPases) in S. cerevisiae was also explored. Under stressful conditions, the study revealed that the H+-PPase could improve the growth rate and successfully act in restoring pH homeostasis. The H+-PPase improved growth of S. cerevisiae in high acetic acid concentrations and showed that there may be more limiting factors in xylose engineered S. cerevisiae. The study also revealed new avenues for improving productivity for ethanol production using lignocellulosic biomass as well as possible alternative methods that could be implemented to increase production of existing compounds that are currently ATP limited.
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