Chapter 17 Flashcards
What are the differences between batch, fed-batch and continuous fermentations?
Batch fermentation is when sterile growth medium is inoculated with microorganisms and no fresh growth medium is added.
Fed-batch is when nutrients are added incrementally during the fermentation process without removing any of the medium
Continuous fermentation is when fresh growth medium is added continuously and a continuous removal of medium.
How has fed-batch fermentation been used to improve the production of insulin, IFN-y and Fab fragment?
For insulin- at high levels of tryptophan, synthesis of target protein was repressed. After consumption of trptophan, synthesis of insulin begins. With each addition of tryophan to the medium increased biomass and target protein produced.
Expression of y-IFN under control of pL promoter, addition of growth medium was carried out simultaneously with temperature induction of pL promoter at late exponential phase use of fed-batch strategy to increase the length of the cell growth phase following induction.
Fab – lactose used to induce the gene which is under the control of lac promoter. Signal sequences inserted upstream of antibody (light and heavy) genes. Lactose is inducer and but is also metabolized providing additional carbon source for Fab producing E. coli cells supporting both cell growth and project accumulation.
What parameters must be monitored and controlled in an optimized fermentation process?
Must monitor and control dissolved oxygen concentration, pH, temperature, degree of mixing. Air bubbles cannot be too big and dispersed evenly. Must add acid or base to maintain a constant pH in the media. Must maintain optimal temperature growth, if too low the organism doesn’t grow fast enough, if too hot may cause premature expression of target protein or produce heat shock proteases.
What is the effect of a recombinant plasmid on the growth of microbial cells?
Plasmid instability is caused by unequal distribution of plasmids to daughter cells during growth and cell division. Once cells have lost plasmid, they grow faster and eventually dominate the cell culture. Recombinant bacterial cell partitions its resources between production of foreign protein and cell growth and therefore it is difficult to engineer a bacteria to both produce foreign protein and grow to a high cell density.
How does the mixing of a growing microbial culture affect the transfer of oxygen from the growth medium to the cells
Adequate mixing ensure that the cells in the media have access to the oxygen. Mixing helps to adequately disperse bubbles and prevent them from getting too large which decrease the rate of transfer of oxygen to the cells.
What are the advantages of a high cell density during a large-scale fermentation? What conditions lead to a high cell density during a large-scale fermentation?
Increasing the density allows the maximization of volumetric productivity. The greater the density, the greater the product formed. Optimize growth medium, inhibit acetate formation and monitor dissolved oxygen to obtain high cell density.
What strategies can be employed to prevent acetate inhibition of the growth of recombinant E. coli strains?
Instead of using glucose, use fructose or mannose as a carbon source
Can also introduce acetolactate synthase gene into E. coli host cell, which converts pyruvate into acetolactate.
Can also redirect carbon flow to TCA cycle by adding pyruvate carboxylase or aspartase (using aspartate as carbon source) enzyme into host E. coli genome.
What are the relative advantages and disadvantages of using an STR or an airlift fermenter?
Advantages of the STR: highly flexible operating conditions, readily available commercially, provides efficient gas transfer to growing microbial cells, used extensively by fermentation engineers and microbiologists.
Disadvantage: can cause shearing because of mechanical mixing.
Advantages of airlift: more energy efficient especially for dense suspensions, elimination of potential site of entry for contaminating organism with removal of mechanical stirrer, can be readily adapted for pilot or large scale fermentation process.
Disadvantage: can cause uneven gas distribution. May cause excessive foaming which restricts flexibility or effective range of operating conditions and potential size
Compare the growth and induction of a recombinant microbial culture using two reactors in tandem and a single reactor?
In tandem reactor, each fermenter is optimized for specific function. The first stage involves the ideal conditions for maximizing cell density and the suspension from the first stage is then transferred to the second stage which maximizes the foreign protein production.
Two stage system not easy to implement on single reactor because difficult to raise temperature quickly or mix chemical inducer rapidly or evenly. Some inducers are too expensive to be used on a large scale.
How can quiescent E. coli cells be engineered to produce large amounts of foreign protein?
In quiescent state, cell overexpresses Rcd, a regulatory protein in hns mutant E. coli host cell. When Rcd gene in induced, the cell will gradually stop synthesizing host protein but continue to produce foreign proteins. Place the rcd under the control of pL or pR promoters which are repressed by temperature sensitive cI repressor. At 30C, the promoters are inactivated because of the cI repressor. At 42C, the cI repressor is inactivated. Thus, the rcd gene can be expressed on one plasmid and on another plasmid, the foreign protein can be expressed. (fig. 17.5)
What are some of the advantages of secreting a recombinant protein into the growth medium?
High-level cytoplasmic expression in E. coli of many different foreign proteins results in the formation of inclusion bodies consisting of insoluble
improperly folded protein
What strategy would you use to ensure that a plasmid encoding a target protein is not lost during large-scale growth of a recombinant bacterium?
Delete an essential gene from chromosomal DNA of the bacteria and place gene on plasmid
