In fed-batch microbial fermentations, O2 is not only important as a nutrient, but also it has important effects on metabolism and physiology. There are a number of potential oxygen-sensitive steps which could affect recombinant expression: (i) the results of low oxygen, (ii) the results of high oxygen and (iii) the results of oxygen shifts.
- A shift to anaerobic metabolism can produce accumulation of by-products such as acetate and ethanol which have a negative impact on process performance. For example, acetate can limit productivity of recombinant E. coli MC1016 harbouring the plasmid for hGH production (Jensen and Carlsen, 1990). On the contrary, this partial anaerobiosis, often referred as “microaerobic growth” sometimes is beneficial for expression of certain heterologous genes in E. coli (Tseng et al., 1996). There are several effects under anoxic conditions:
- Regulation of amino acid synthesis by oxygen
- Effects in amino acids depletion
- Plasmid replication
- Hemoglobin expression to improve productivity
- Exposure to elevated oxygen partial pressures (due to OTR limitation or in large bioreactors) can lead to: (Konz et al, 1998).
- An oxidative damage to proteins in five different classes: metal-catalyzed oxidation, disulfide formation, methionine sulfoxide formation, oxidation of iron-sulfur centers and glycation and PUFA conjugation
- Degradation of oxidized proteins
- DNA oxidation and mutation
- Oxidation of free aminoacids
- Oxygen fluctuations can be also detrimental to protein expression. If we shift to oxygen-enriched air during expression of our heterologous gene, our product quality may initially decrease due to the oxidative conditions and afterwards the quality may increase to a new state as tress regulons are induced.
To conclude: fluctuating DO in fed-batch microbial fermentations is not a trivial process which can affect not only the amplification of plasmids in E. coli, but also the quality of our final recombinant protein, sometimes with dramatic consequences.
- Jensen, E. B and S. Carlsen. 1990. Biotechnol. Bioeng. 36: 1-11.
- Tseng, C. P., J. Albretch, and L. P. Gunsalus. 1996. J. of Bacteriol. 178: 1094-1098.
- Konz, J. O., J. King, and C. L. Cooney. 1998. Biotechnol. Prog. 14: 393-409.