Download Chem*4570 Applied Biochemistry Lecture 7 Overproduction of lysine

Chem*4570 Applied Biochemistry Lecture 7
Overproduction of lysine
A better system allowing lysine overproduction in a single organism and single fermentation
process has been obtained in strains of Corynebacterium glutamicum, and also in Brevibacterium
species. This takes advantage of the simpler regulation of the lysine pathway in this organism, in which
single enzymes catalyze aspartate kinase and homoserine dehydrogenase/ASA reductase steps instead
of the multiple isozymes found in E.coli.
The parent strain chosen is a moderate glutamate overproducer; however the glutamate is consumed
internally by acting as N-donor in lysine synthesis. The α-ketoglutarate so produced can be
converted into the aspartate needed to start the lysine pathway.
Lysine overproducing strains are genetically defecting at three stages:
1) Aspartate kinase is insensitive to lysine
2) DHP synthase is insensitive to lysine
3) Homoserine dehydrogenase is either absent or supersensitive to threonine
1) and 2) allow for overproduction of lysine because lysine accumulation does not down-regulate
synthesis of aspartyl phosphate or dihydropicolinate. Both direct enzyme inhibition and expression
of the enzymes lose sensitivity to lysine.
3) prevents diversion of substrate to the Met/Thr/Ile branch of the pathway. If homoserine is not made,
the medium must be supplemented to sustain growth of the culture, either with homoserine or the
missing amino acids. More recently, the Thr supersensitive strain was isolated, and this makes just
enough of Thr Met and Ile to sustain its own protein synthesis needs without diverting too much
substrate or requiring supplementation.
Other genetic and metabolic factors aiding overproduction
The overproduced amino acid must be secreted to the medium.
Microorganisms do not usually export amino acids, so the cell membranes must be made leaky to
promote transfer across the cell membrane. Two strategies are used:
1) A sublethal dose of penicillin, an inhibitor of cell wall proteoglycan assembly, causes cell wall
weakening.
2) The cell strains are deficient in biotin biosynthesis. Biotin is a required factor for fatty acid
biosynthesis, at the acetyl-CoA carboxylase step, so the cells are not manufacturing their own fatty
acids including the unsaturated fatty acids needed to maintain membrane fluidity. At the same time, cells
are fed only saturated fatty acids in the culture medium.
biotin:acetyl CoA carboxylase
Acetyl-CoA + ATP + CO2 → ADP + Pi + malonyl-CoA (required for elongation
step in FA synthase).
This results in brittle, fragile and leaky membranes due to the lack of fluidity. Leakiness also benefits
glutamate production, since proton motive force is dissipated and ATP production by oxidation
phosphorylation is inefficient. Since the glutamate pathway is the major energy yielding pathway, more
glutamate is produced.
Origin of overproducing species and strains
1) The overproduced compound is a normal terminal product of anaerobic energy metabolism
Ethanol
Butyrate and butanol
- yeasts
- Clostridium species
In these cases, the pathway arose as a process of natural adaptation for survival under anaerobic
conditions. Before the development of our O2 -rich atmosphere, pathways such as butanol production
or other reactions involving hydrogenases for oxidation reactions may well have been more common,
but were discarded by cells which adopted the high ATP-yielding oxidative phosphorylation system.
Yeast also had the benefit of several thousand years of humans selecting for better yield.
2) The overproduced compound is a terminal product of a modified pathway of energy
metabolism that arose in the wild.
Glutamate
Corynebacterium glutamicum
In this case, human intervention involved artificially maintaining a strain, thereby selecting for the desired
metabolic route.
3) The overproduced compound is the product of a metabolically costly biosynthetic pathway.
Lysine
E. coli, Corynebacterium, Brevibacterium
similarly for producers of Met, Trp
Direct human manipulation of genes important in the pathway was required.
Methods for metabolic manipulation
Natural selection under modified growth conditions worked for yeast and C. glutamicum, but
maybe we are not willing to wait hundreds of years anymore.
Induced mutation and selection
Classical approach: application of general mutagen and selection of desired mutants. from a large
number of irrelevant alterations.
Advantages: often needs no in depth knowledge of the biochemical system is necessary;
method relies on having a suitable selection process for the desired outcome.
Disadvantages: an extremely large number of totally irrelevant alterations will be made; the
approach relies on having selection methods that are efficient enough to screen large numbers of
trials.
Genetic engineering approach: includes knockouts (via gene disruption) or knock-downs (via RNA
interference in higher organisms) of specifically targetted genes; site directed mutagenesis of specific
enzymes.
Advantages: changes can be very precise and tailor-made to the system. Selection methods
can be built into the same vectors that induce the changes
Disadvantages: may require precise knowledge and depth of understanding of the
biochemistry of the metabolic system. Functional mutations may require detailed knowledge of
the structure of the targetted protein.
For example, tryptophan biosynthesis also involves a long a complex pathway, starting from
deoxyheptonate aldolase
phosphoenolpyruvate + erythrose-4-phosphate → 7-carbon precursor → first cyclic product
+ 12 more steps
The end-product tryptophan controls the “first” reaction at the specific aldolase that condenses PEP and
erythrose-4-P, and also at the branch point that separates the Phe,Tyr pathway from Trp.
PEP is a product of glycolysis, and erythrose-4-P is a product of the pentose phosphate pathway.
In exploring potential tryptophan overproducers, Trp sensitivity of the key isozymes was eliminated, but
the results were disappointing. Little improvement was obtained by enhancing expression of enzymes
within the pathway. Some improvement was obtained by enhanced expression of the transketolase that
makes erythrose-4-P, but the best results required enhanced expression of several pentose phosphate
pathway enzymes. Thus the limiting factor in this case was not the pathway itself, but the entry of
substrate to the pathway.