Manipulation of cell control systems to improve industrial yield

Microorganisms and over-production

Microorganisms have created a system where they only create what they need to survive. these systems prevet wasteful over-production of enzymes and metabolites.

Industrial microbiology and over-production

We want microorganisms to over-produce a desired product at levels above what the cell needs. Cellular mechanisms must be overcome, modified or removed to achieve commercially viable yields

control of enzyme activity

this is a rapid form of regulation as it acts directly on enzymes that already exist. this commonly occurs via feedback (end-product) inhibition where the final product of a pathway inhibits an earlier enzyme to prevent over-production

Control of synthesis

slower form of regulation as it involves changes in gene expression and therefore requires transcription and translations. this type of control regulates whether enzymes are produced or not. it occurs through repression, where enzyme synthesis is switched off or induction where its switched on

What is feedback inhibition

this occurs when the final product inhibits and early, rate-limiting enzyme and prevents further synthesis once sufficient product has accumulated. Efficient for cell but undesirable in industrial fermentation as accumulation shits down further production

Overcomeing feedback inhibition

to increase yield we must remove or reduce sensitivity of enzymes to inhibition, use mutants lacking functional regulatory enzymes or block competing branches in metabolic pathways

What does overcoming inhibition mean

this ensure carbon flux is directed toward the desired product even when accumulations are high

What is repression

occurs when the presence of an end product prevents transcription of genes encoding enzymes in a biosynthetic process. as product builds up enzyme synthesis stops. This commonly affects anabolic pathways such as amino acid biosynthesis.

overcoming repression

Can be overcome by: selecting repression-defective mutants, blocking synthesis of repressing molecules or using auxotrophic mutants which lack parts of a pathway and therefore it cannot accumulate end products to switch off the pathway

Induction

some enzymes only produce in the presence of specific enzyme producers this prevents unnecessary enzyme production. this can delay production and require costly inducers

constitutive mutants

these produce enzymes continuously, regardless of the presence or absence of an inducer this means there is no lag phase, continuous production and higher productivity. These mutants are selected using chemostats or sequential batch cultures

why lysine

It is an essential amino acid and is used in lots of animal feed and food suplement meaning it has a very high demand but is not excreted by wild type organisms

the lysine biosynthetic pathway

L-lysine is produced from aspartate a branched metabolic pathway that also produces theronine, methionine and isoleucine

key regulatory features of lysine

Aspartokinase catalyses the first committed step and is subject to concerted feedback inhibition by lysin and threonine. Homoserine dehydrogenase also diverts intermediates away from lysine and toward threonine and methionine. in wild-type cells accumulation of lysine shuts down the pathway

Strategy to overcome control systems

industrial lysine production uses mutant strains of C. glutamicum that are auxotrophic for homoserine.

what do mutant C.glutamicum lack

functional homoserine dehydrogenase

cannot synthesise threonine or methioine

do not accumulate repressing end products

what is the result of using mutant strains of C. glutamicum

Feedback inhibition is removed

carbon flux is redirected entirely toward lysine

aspartate semialdehyde accumulates and is converted to lysine

Role of auxotrophy

auxotrophic mutants require supplementation of the missing compound in limited amounts. this allows the cell to grow, control systems to remain inactive and sustained lysine overproduction

Process considerations

L-lysine production is typically carried out as batch fermentation in aerated stirred-tank reactors using cheap carbon sources such as molasses. careful control of carbon, pH and biotin concentration is required to maintain high yields and avoid by-product formation.