Engineering Anaerobic Amino Acid Production in Saccharomyces Cerevisiae: Alanine As Case of Study

H.F. Cueto-Rojas¹, A. Goel¹, N. Milne², J.M. Daran², J.J. Heijnen¹, S. A. Wahl¹

¹Cell Systems Engineering, Dept. of Biotechnology, TU Delft, Julianalaan 67, 2628BC Delft, The Netherlands

²Industrial Microbiology, Dept. of Biotechnology, TU Delft, Julianalaan 67, 2628BC Delft, The Netherlands

In an industrial setting, anaerobic conditions are especially attractive as costs related to energy input for mixing and oxygen transfer are drastically reduced. In addition, highest theoretical product yields can be achieved. Synthetic biology nowadays enables the introduction of extensive changes in the metabolic capabilities and regulation of microorganisms.

The budding yeast Saccharomyces cerevisiaeis well known for its high fermentative capacity and robustness in large-scale fermentation processes. However, it has not yet been used for industrial amino acid production. The aim of our project is to reprogram yeast metabolism to achieve efficient amino acid synthesis under anaerobic conditions.

Current processes rely on aerobic cultivations, using bacteria mainly C. glutamicum and E. coli. To reach anaerobic production of amino acids with sufficient yield, synthetic biology is applied to reprogram yeast central carbon metabolism, especially to obtain:

1)     Redox-balanced production pathways.

2)     Biosynthetic pathways including the export have to produce free energy in the form of ATP.

3)     The export mechanism has to be energy-efficient and thermodynamically favorable.

4)     Other fermentative pathways have to be eliminated.

5)     Intracellular accumulation in compartments has to be avoided for an efficient transport to the extracellular space.

To evaluate the success of genetic modifications, extra- and intracellular metabolite measurements are performed. Analytical protocols for the quantification of pathway intermediates have been developed (GC- and LC-MS). Thermodynamic analysis is performed to identify limiting reaction steps.

Homoalanine fermentation is a straightforward pathway that requires the expression of one gene: Alanine dehydrogenase NADH-dependent. The developed strain expresses the enzyme Alanine Dehydrogenase (ADH) of Bacillus subtilis. However, no difference in amino acid production could be observed between mutant and wild-type strain. No accumulation in the intra- and extracellular space was observed in glucose-limited anaerobic chemostats. Two hypotheses were formulated to explain these results: 1) alanine excretion is the limiting step of the production process and 2) as ammonium is used as N-source no ATP is produced in the homoalanine fermentation pathway, due to the fact that 1 mole ammonium requires 1 mole ATP for the excretion of the charge imported with the ammonium ion and only 1 ATP is produced per mole of alanine (zero in total).

Hess et al. (2006) observed excretion of amino acids when yeast cells are cultured at high ammonium and low potassium concentrations. Potassium (K⁺) and ammonium (NH₄⁺) have a similar ionic radius and the authors suggested that this leads to unspecific import of NH₄⁺ by K⁺ channels (especially at very low K⁺concentration).

Accumulation of NH₄⁺ in the intracellular space is toxic and cells have to export ammonia in order to maintain the intracellular homeostasis. Several mechanisms are possible: urea production, overproduction of amino acids or excretion of ammonium using active transport. To this date neither urea synthesis nor NH₄⁺ active transporters have been reported for S. cerevisiae (Hess et al., 2006); which supports the hypothesis of overproduction of amino acids as defence mechanism against high concentrations of ammonium in the intracellular space.

Using an anaerobic glucose-limited chemostat with 500 mmol/l ammonium and 2.5 mmol/l potassium (as suggested by the work of Hess et al.) the alanine excretion was increased from 8.2 mmol/Cmol X/h to 178.9 mmol/Cmol X/h in the WT strain and from 14 mmol/Cmol X/h to 274 mmol/Cmol X/h in the recombinant strain. Other differences were observed in biomass and glycerol production.

Other experiments using high-ammonium low potassium conditions showed intracellular alanine concentrations as high as 100 mmol/l_{intracellular}. These results suggest that the limiting process in L-alanine production seems to be the transport from the cytosol to the extracellular space.

It is assumed that the measured extracellular alanine is exported via the reversed activity of the alanine importer proteins (symporters of one molecule of alanine and one proton). Using this mechanism, the intracellular concentration is expected to be 1440 times higher than the extracellular concentration at equilibrium conditions. Experimental observations suggest that L-ala concentration in the intracellular space is around 20 mmol/l_{intracellular}; being at equilibrium with an extracellular concentration close to 14 µmol/l_{extracellular}. The unfavourable symport mechanism has to be engineered to achieve higher extracellular alanine concentrations.

Furthermore, bioprocess optimization requires a thorough characterization of transport processes of both product and substrate. Characterization of the ammonium transporters requires accurate data of the intra- and extracellular space. An enzymatic reaction kit (Roche) was modified a) to be used in 96-well format for high throughput measurements and b) to increase its sensitivity for low ammonium concentrations (between 5 µmol/l to 300 µmol/l). With the developed assay, it was possible to measure intra- and extracellular concentrations of ammonium in S. cerevisiae CENPK 113-7D under aerobic nitrogen-limited conditions. The intracellular concentration found was 1.36 +/- 0.07 mmol/l_{Intracellular} at an extracellular residual concentration of 61.0 ± 8.1 µmol/l. The measured ratio (22.95) is within the expected range (between 4.9 and 67.1) according to thermodynamic calculations assuming a pmf between -150 mV and -190mV, and intracellular pH_i between 6.5 and 6.8. To our knowledge, this is the first time that intracellular concentrations of ammonium are measured directly in vivo.

References:

Alberty RA (2003). Thermodynamics of biochemical reactions. Hoboken, NJ: Wiley Interscience.

Duarte N, et al. (2004). Genome Res. 14: pp. 1298-1309

Hädicke O, Klamt S (2010) J Biotechnol., 147 pp. 88-101

Hess DC, et al. (2006). PLoS Biology 4:11 pp. 2012-2023

Ikeda M, (2003) Adv. Biochem. Eng. Biotechnol. 79: pp.1-35

Ishimoto, M., et al. (2012).  Biosci. Biotechnol. Biochem., 76:9 pp.1802-1804

Marini et al. (1997) Mol. Cell. Biol., 17 pp.4282-4293

Nookaew I., et al.(2008). BMC Systems Biology 2: 7

Rocha M., et al. (2008). BMC Bioinformatics 9:499

Sekito T., et al. (2008). IUBMB Life 60:8 pp. 519-525