Redox buffering, respiration, and fermentation

1. Redox buffering, respiration, and fermentation

2. Too much NADH can be a bad thing • NADH/NAD is one of the most “connected” molecules in the cell • As a result, changes in the reduced/oxidized ratio can severely impact cell physiology • “over” reduced cells (too much NADH) die

3. Mechanisms for dealing with NADH • In class you learned: • Increase in NADH/NAD ratio and ATP concentrations shut down glycolysis and citric acid cycle to limit additional production of NADH • Lack of oxygen leads to production of fermentative products such as lactate or ethanol • NADH is oxidized by mitochondrial NADH dehydrogenase (NDI1)to initiate the electron transport chain; and cytosolic NADH can be transported into the mitochondria by glycerol-3-phosphate dehydrogenase (soluble: GPD1 and GPD2; membrane bound: GUT2) and malate-aspartate shuttle (not present in yeast – instead an acetaldehyde/ethanol shuttle) Need to know: • What about the “Crabtree effect”? What about external, cytosolic-facing NADH dehydrogenases? What about other NADH shuttles? • Glycerol can be produced from dihydroxyacetone via (soluble) GPD1 and/or GPD2 using NADH followed by Glycerol Kinase in a substrate level phosphorylation reaction

4. Crabtree effect and redundancy • Production of reduced molecules such as ethanol, glycerol, and lactate under aerobic conditions • Offers another mechanism to deal with “overflow” reducing power generated by metabolic processes such as glycolysis to maintain homeostasis • Redundancy is often seen in metabolic and regulatory networks to ensure survival • There’s more than one way to deal with a “problem” • Yeast also produce two external, cytosolic facing NADH dehydrogenases (NDE1 and NDE2) as a way of handling reducing power (NADH) and have an ethanol shuttle

5. NADH in yeast

6. But, why Crabtree? • Hypothesis 1: respiratory pathway is limiting; cannot keep up with glucose metabolism and build up NADH • Hypothesis 2: Glucose repression of respiratory metabolism – this yeast likes to ferment glucose at high [glucose] even if oxygen present – glucose affects protein kinases that control gene expression and glucose transport • Hypothesis 3: overflow metabolism at pyruvate – accumulation of pyruvate leads to production of ethanol, glycerol, etc.

7. How to address hypothesis 1 • Construct yeast strains with the following attributes: • Wild type; Control; CON • Express water forming NADH oxidase in CON; pulls NADH away from electron transport, limiting respiration and reduces cytosolic [NADH]; directly reduces oxygen to water using NADH; NOX • Express alternative terminal oxidase; takes electrons from ubiquinol directly to oxgen to make water without proton translocation; AOX • Triple deletion yeast strain lacking external NADH oxidases (NDE1 and NDE2) and glycerol-3-phosphate dehydrogenase (GUT2) • Express NOX in triple deletion strain lacking NDE1, NDE2 and GUT2

8. AOX and NOX affect different pathways • Most notable differences observed in byproduct formation – lower ethanol in AOX; lower glycerol in NOX Reduced glycerol production in NOX due to cytosolic NADH oxidation as observed in deletion strain (without NOX more glycerol than CON; with NOX low glycerol)

9. Looking at identical growth (dilution) rates using chemostat • Limitation – carbon limited (no carbon overflow) or nitrogen limited (allows for glucose repression of respiration; high [glucose]) • r = rates of uptake, Dcrit = all rates below reflect respiratory growth

10. NOX and AOX effects • NOX and AOX expression increased rate of glucose and oxygen take up…increased glucose oxidation (also see increased carbon dioxide production) • At low growth rates, NOX and AOX expression affect ethanol and glycerol production • Just below Dcrit, uptake rates are 3-3.5X higher

11. Growth rate, AOX and NOX influence enzyme activity differently • Brown = Dcrit • Yellow = 0.1 h-1

12. AOX and NOX expressing cells exhibit higher NADH oxidation capacity and lower NADH/NAD ratio • Low growth rate = white • Dcrit = gray • N-limited = black

13. Analyzing Global Gene Expression

14. GTTCGA....The gene CAAGCT....cDNA Via reverse transcription GUUCGA....mRNA

15. Spotted arrays are usually treated with samples from two different tissues, each labeled with a different “color” of dye (Red and Green) Highly expressed in tissue A Highly expressed in tissue B

16. Microarray animation • http://www.bio.davidson.edu/courses/genomics/chip/chip.html

17. NOX expression influences expression of several genes • Red – increased mRNA • Green – decreased mRNA (relative to CON)

18. So does AOX expression

19. Changes in expression linked to metabolites

20. Why differences between NOX and AOX expression? • Cytosolic NADH (from glycolysis in yeast) is predominantly reoxidized by cytosolic-side NADH dehydrogenases with overflow going to glycerol • NOX reduces cytosolic NADH, therefore reduces glycerol production • Mitochondrial NADH (from TCA) is used by NDI1 for respiration, overflow here (high mitochondrial NADH prevents entry of pyruvate) leads to ethanol production • AOX reduces mitochondrial NADH levels by providing additional electron transport path – note TCA enzyme expression increases in response to AOX expression so more NADH generated

21. Paper requirements • 5 page minimum, 10 page max, double-spaced, type-written, cite references (reference page does not count toward page minimum), beware of overdoing it with figures (glycolysis, citric acid, regulatory cascades etc. figures do not count towards page minimum, but you may include) • You may wish to address one or more following topics in your paper (but make it a coherent, related story):

22. Assess transcriptional regulatory data in comparison with course info regarding regulation of glycolysis and TCA, does it make sense? • Describe affected metabolic pathways and provide suitable rationales for effects. • Respiro-fermentative behavior, how about that? • Glucose repression in yeast • How do ATP and NADH levels correspond to growth rate/biomass yield? • Metabolic homeostasis via allostery and gene regulation