Molecular Biotechnology: Principles and Applications - Glick B., Pasternak J. 2002

Fundamentals of Molecular Biotechnology
Optimization of Gene Expression Cloned in Prokaryotic Systems
Growth under Oxygen-Limiting Conditions

E. coli and many other microorganisms used for the expression of foreign Proteins typically grow only in the presence of oxygen. Unfortunately, the solubility of oxygen in aqueous media is limited, and as culture density increases, the dissolved oxygen content in the culture medium drops rapidly. Moreover, because oxygen dissolves very slowly, this problem cannot be solved simply by sparging air or oxygen through the medium, even with vigorous agitation. As the oxygen concentration decreases, exponential growth slows down, and the culture slowly transitions into a stationary phase characterized by a different metabolic status. One consequence of this is The formation of intracellular proteinases, which can degrade the target protein. Various approaches have been attempted to solve Structure/149.html">The problem of culture medium aeration, including modifying bioreactor design, increasing The rate of air sparging and agitation, and adding substances that increase oxygen solubility to the medium. However, none of these efforts have yielded any significant results.

Use of Protease-Deficient Host Strains

One potential approach to stabilizing foreign proteins synthesized by E. coli involves using host strains deficient in Proteolytic Enzymes. However, this approach presents certain challenges. E. coli Cells synthesize at least 25 different proteinases, and only some of them have been characterized at the genetic level. Furthermore, proteinases play a vital cellular function by degrading foreign or defective proteins, thereby ensuring Cell viability. In one study, strains carrying Mutations in one or even several proteinase genes were constructed; the more pronounced the cumulative proteinase deficiency, the poorer the strain grew. Thus, reducing proteinase activity leads to a depletion of cellular resources. Nevertheless, researchers successfully developed E. coli strains carrying mutations in the RNA polymerase sigma-factor Gene responsible for heat Shock Protein Synthesis (rpoH) and in the proteinase gene required for cell growth at high temperatures (degP), in which the specific activity of secreted proteins was 36-fold higher than that of wild-type strains. This apparent increase was due to a reduction in the rate of proteolytic protein degradation.

Bacterial "Hemoglobin"

Certain strains of Gram-negative obligate aerobic Bacteria of the genus Vitreoscilla inhabit oxygen-depleted, stagnant Water bodies. To obtain sufficient oxygen for growth and METABOLISM, they synthesize a hemoglobin-like substance that binds ambient oxygen and increases the concentration of available oxygen within The Cell. When the gene encoding this protein was introduced into E. coli cells, profound changes occurred immediately: the synthesis of cellular and recombinant proteins increased, the efficiency of proton pumps improved, and the yield and concentration of ATP increased, particularly under low-oxygen conditions in the medium. For this strategy to be applied to other host cells, these cells must not only efficiently express the Vitreoscilla "hemoglobin" gene but also synthesize heme, a component of the hemoglobin molecule. This will enhance the growth of commercially important bacteria such as E. coli, Streptomyces lividans, Corynebacterium glutamicum, and Xanthomonas maltophilia, as well as facilitate the expression of foreign genes within them.