Download 1 2 Resp iratio n : Gly co lysis: TC A -cy cle

Reprint from: Enfors-Häggström, Bioprocess technology- Fundamentals and Applications,
KTH, Stockholm, 2000
Chapter 4. METABOLIC BASIS OF PRODUCT FORMATION
1
The cellular metabolism is made up of a large number of reactions co-ordinated by enzymes
subjected to various control mechanisms. Some of these reactions may lead to a desired
product. Knowledge of metabolism and physiology constitutes the basis for developing
control strategies in biotechnical processes. This chapter provides a framework of metabolic
and physiological features relevant to microbial processes. Textbooks on biochemistry and
microbial metabolism should be consulted for further details. Metabolism of cultured animal
cells will be treated separately (chapter 17).
4.1 Metabolic organisation
A simplified overview of the primary metabolism is shown in Fig. 4.1. Metabolism can be
visualised as composed of three compartments as indicated by the boxes for catabolism,
anabolism and synthesis of macromolecules.
Fig. 4.1. Primary metabolism - an overview.
The catabolic and anabolic compartments contain biochemical pathways whereas the
synthesis of macromolecules rather is the assembly of pre-made building blocks. A flow of
material runs through the three compartments, eventually resulting in the production of new
cells i.e. growth. Environmental changes such as changes in substrate concentration, pH and
temperature and the accumulation of waste products follow from this process.
Glycolysis:
Glucose
Pyruvate
Acetyl-CoA
TCA -cycle:
2
NADH
ATP
CO
Respiration:
H O
ATP
NAD+
O2
2
NADH
ADP
2
Fig 4.2. Aerobic energy metabolism- a simplified overview. The reduced energy substrate
(glucose) is oxidised to pyruvate by NAD + in a series of reactions called glycolysis. this
generates a small amount of the energy carrier ATP. Pyruvate is further oxidised to CO2 by
co-enzymes (NAD +) in the TCA-cycle. The reduced coenzymes (NADH) are re-oxidised (
NAD + ) in the respiratory chain, where molecular oxygen is the ultimate receiver of the
electron. This oxidation is coupled to generation of a relatively large amount of ATP.
In anaerobic respiration, nitrate, sulphate or some other oxidised specie is used as electron
acceptor instead of molecular oxygen. In fermentative energy metabolism, the NADH from
the glycolysis is reoxidised by reduction of pyruvate to fermentation products like ethanol,
lactate etc. (Fig 4.6-4.7)
The entrance of substrates into the catabolic compartment is the starting point for all these
activities. The catabolic compartment contains the central metabolic pathways i.e. glycolysis,
the hexose monophosphate shunt (HMS) and the pentose cycle, the tricarboxylic acid cycle
(TCA) as well as respiration and the generation of the energy carrier ATP (Fig. 4.2). (In
addition to the aerobic heterotrophic metabolism - other types of energy metabolism like
aerobic autotrophic, anaerobic fermentation, anaerobic respiration and photosynthesis occur
in microorganisms.) NAD+/NADH are, with few exceptions, recirculated within the
catabolic compartment. Intermediate metabolites from the catabolic pathways constitute the
precursors for biosynthesis of building blocks in the anabolic compartment. Catabolism also
furnishes the reducing power (NADPH) required for biosynthesis.
Anabolism is the synthesis of building blocks used for macromolecule synthesis. Here, the
well known pathways for amino acid and nucleotide synthesis are harboured as well as the
synthesis of fatty acids and sugar moieties. Finally, these building blocks are assembled into
macromolecular constituents of the cell like DNA, RNA, protein, membrane lipids and cell
walls.
Products produced in biotechnical processes may originate from any of the three metabolic
compartments in Fig. 4.1, or constitute the result of them all as in the production of bakers
yeast or clean water in a sewage plant. The characteristics of product formation in the
metabolic compartments will be described later in sections 4.6, 4.7 and 4.8. However, the
metabolism in the three individual compartments is regulated according to different
principles. It is important to understand the metabolic regulatory mechanisms since i) these
mechanisms must sometimes be circumvented to achieve overproduction of certain
metabolites, and ii) they furnish the basis for the microbial physiology which, in various
indirect ways, influence the performance of the whole process.
3
4
A number of carbohydrate carbon and energy sources including glucose are transported into
the E. coli cell by the phosphoenolpyruvate: sugar phosphotransferase system (PTS), a
so called "group translocation" transport system (Fig. 4.4). Phosphorylation of glucose to
glucose-6-P occurs during transport through the inner cell membrane. The phosphate group is
derived from phosphoenolpyruvate (PEP) which in turn is converted to pyruvate (PYR). A
number of enzymes participate in the transfer of phosphate from PEP to glucose or to other
carbohydrates. Two of these enzymes (Enz I and Hpr) are common to all PTS-sugars while
4.3.2 The phosphotransferase system of E. coli.
Microorganisms can use many different substrates which feed into the central metabolism at
various points. Each substrate has a specific transport system and specific initial catabolic
enzymes that convert it to an intermediate of the central pathways. The various substrates are
transported into the cell and enter the metabolism with different easiness and are not equally
efficient for energy production. A microorganism in Nature is often simultaneously exposed
to a variety of substrates that can be used as carbon and energy sources. To guaranty that the
most efficient substrate is metabolised first, intricate mechanisms exist that regulate transport
and initial catabolism. E. coli will be used as an example to illustrate such features.
The central metabolic pathways (HMS, glycolysis, TCA-cycle) are made up of constitutive
enzymes which always are present in the cell. (In facultative organisms like E. coli or S.
cerevisiae transition from aerobic to anaerobic conditions shuts off respiration and the TCAcycle.) Regulation of the activities of individual enzymes in these pathways, leading to coordination of rates between them and in response to the cellular energy demand are well
known and will not be considered here.
4.3.1 Aerobic energy metabolism in aerobic and facultative organisms.
4.3 Catabolic regulation.
Which are then the metabolic bottlenecks? Generally, the transport of a key substrate into the
cell is regarded as the growth rate limiting reaction. This is clearly recognised during
microbial growth in a minimal medium with glucose as the sole carbon and energy source.
On the other hand, if the same microbe is cultivated in a complex medium containing premade building blocks (amino acids, nucleotides) and reaches a much higher growth rate it is
no longer obvious which is the growth rate limiting reaction. In fact, during such conditions
the growth rate may approach the maximum DNA replication rate, or rather the minimum
time between two consecutive initiations of replication.
mechanisms that compensate for unbalances in the flow of materials. To avoid wasteful
overproduction of building blocks the synthesis is regulated in relation to the consumption
for macromolecular synthesis. These mechanisms are well understood and it is also easy to
imagine that overall protein synthesis rate is limited by the availability of amino acids. But
how is then the synthesis of the various macromolecular species adjusted to fit each other and
the growth rate? Recent research give by hand that a mechanism, since long known as the
"stringent response" and originally thought of as a shock response to amino acid starvation,
has a much broader range of action and, in fact, might be a growth rate regulating signal, as
further described in section 4.5.
4.2 Metabolic regulation
Figure 4.3 indicates the different regulatory principles within each metabolic compartment.
These principles will be described in sections 4.3, 4.4 and 4.5. During growth, the metabolic
activities are regulated in a highly co-ordinated manner to ensure maximum economical use
of substrates for maximum growth rates during the prevailing conditions. As visualised in
Fig. 4.3, it is the rate with which energy, precursors and building blocks flow through the
system that determines the overall reaction rate (growth rate), which in turn is determined by
the growth rate supporting ability of the medium. Nature has evolved metabolic systems that
appear to have over capacity, in terms of enzyme capacity relative to substrate concentration.
This means that the metabolic network often is subsaturated with substrate and responds to
increases in substrate concentration and substrate quality with an increase in reaction rate.
Fig. 4.3. Principles of regulation of metabolism.
The input rate of components into the anabolic compartment limits the biosynthesis rate.
There is, however, not a very strong feed-back from anabolism to catabolism. Even if growth
ceases and anabolism is shut off, the catabolism, although reduced, is never shut off.
Catabolism must always be running in order to furnish the cell with energy for maintenance
purposes. If the energy metabolism ceases or the energy charge falls below a critical value
the cell will die. One exception to this is endospore formation or other forms of resting stage
formation.
The rate of macromolecule synthesis is also limited by the input rate of building blocks,
already determined by the events in the catabolic compartment. Although evolution has
adjusted the capacities in various metabolic sequences to each other there must also exist
5
each sugar has its specific membrane bound permease (Enz II) and some sugars also a
specific soluble enzyme which is the last link between Enz II and PEP. For glucose, this
soluble enzyme is called Enz IIIglc. Enz IIIglc has turned out to have a central regulatory role
for the metabolism of non-PTS-sugars i.e. sugar substrates that are transported by other
transport systems.
Fig. 4.4 The phosphoenolpyruvate: sugar phosphotransferase system of E. coli. Enz I
= Enzyme I; Hpr = Heat stable protein; Enz IIIglc = Enzyme III, soluble enzyme,
specific for glucose; Enz IIglc = Enzyme II, membrane bound glucose specific
permease.
In E. coli the PTS-system transports among others glucose, fructose, mannose, mannitol. The
PTS-sugars are superior to other sugar substrates in sustaining rapid growth. In other
bacterial species other sugars can be transported by the PTS-system. The PTS-system is
abundant in anaerobic as well as facultative bacteria. Strictly aerobic bacteria use
predominantly hexokinase for phosphorylation of hexoses in combination with other types of
transport systems (e.g. symports or active transport).
The metabolic consequence of the PTS-system is indicated in Fig.4.5. For every molecule of
glucose (or another PTS-sugar) transported into the cell - one molecule of PEP is inevitably
converted to PYR and thus withdrawn from glycolysis and HMS.
If a product produced in a biotechnical process requires precursors from that part of the
metabolism it means that the maximum theoretical yield from glucose may be less than from
other substrates, not transported by the PTS system. This is the case for certain amino acids
like the aromatic ones and those originating from oxaloacetate. (In E. coli the anaplerotic
reaction supplying oxaloacetate uses PEP, not PYR as is the case in eukaryotic cells.) In such
cases it might be worthwhile to investigate whether non-PTS-sugars (e.g. glycerol or xylose)
can be used in the process.
Fig. 4.5 Interaction between PTS and initial metabolism.
4.3.3 Regulation of transport and initial catabolism by PTS.
6
All carbohydrate catabolic operons in E. coli, including those of the PTS-system, are
regulated by induction of the substrate and by cAMP via its receptor protein. These operons
usually contain genes for a membrane bound permease and the initial catabolic enzymes
necessary for conversion of the substrate to an intermediate of the central metabolic
pathways. For maximum transcription intracellular inducer and a certain concentration of
cAMP is required.
The PTS system regulates the transcription of catabolic operons by two mechanisms. Firstly,
it influences the formation of cAMP by activating the adenylate cyclase when PTS sugars are
exhausted. This is thought to be brought about by Enz IIIglc in its phosphorylated form.
Adenylate cyclase activity is also regulated by the membrane potential. When glucose or any
other PTS-sugar is present cAMP levels are low because adenylate cyclase activity is very
low. Secondly, Enz IIIglc, in its non-phosphorylated form, hinders substrates to enter the cell
by binding to the permeases, making them inactive. This phenomenon is called "inducer
exclusion" and has been shown for lactose, maltose, melibiose and glycerol in E. coli.
By these mechanisms (and others not described here) a hierarchy of substrates exist, the
purpose of which is to ensure consumption in the most favourable order. Knowledge of such
relationships are important e.g. in processes where complex media are used and for
understanding waste water treatment processes.
4.3.4 Overflow metabolism.
Acetaldehyde
+
NAD NADH
A DH
Ald DH
ADP
1/2 Glucose
NAD+
ATP
Pyruvate
PDH
AcetylCoA
NADH
CO 2
Pyruvate NAD+
cytosol
mitosol
NADH
CO2
PDC
ATP
ACoAS
TCA
ATP
2 CO
2
7
In E. coli and S. cerevisiae, the aerobic metabolism of glucose leads to excretion of the
partially oxidised products acetic acid and ethanol respectively. This is called overflow
metabolism. It occurs when the glucose concentration exceeds a critical value and is typical
for facultative organisms that have part of their anaerobic enzyme set up active under aerobic
conditions. Thus, many but not all microorganisms exhibit some sort of overflow
metabolism. This behaviour is often detrimental to a production process because production
of ethanol and acetic acid reduce the yield of biomass and the desired product and the
compounds are toxic to the organism. It can, however, be avoided by keeping the glucose
concentration low with the fed-batch technique (chapter 7).
Ethanol
+
NAD
NADH
Acetate
3NADH
+FADH
ATP ADP
+
NAD
+FAD
H2O 3O 2
Fig 4.6. Energy metabolism of S. cerevisiae. At low rate of glycolysis all pyruvate is
completely oxidised to CO2 in the TCA cycle. When the glycolysis rate exceeds a critical
value, part of the pyruvate is reduced to ethanol. If ethanol is present and the rate of
glycolysis is below the critical value, ethanol is consumed and converted to acetylCoA via
acetate.
PDC: pyruvate decarboxylase, ADH : alcohol dehydrogenase, AldDH: aldehyde
dehydrogenase, ACoAS: Acetylcoenzyme A synthase, PDH: pyruvate dehydrogenase.
As phenomena, the overflow metabolism of E. coli and S. cerevisiae is very similar although
from a metabolic regulation point they may be very different. In none of the cases is the
regulatory mechanisms behind the overflow metabolism known.
In S. cerevisiae, overflow metabolism, i.e. ethanol formation under aerobic conditions, starts
when the rate of glycolysis becomes higher than corresponding to an observed maximum rate
of respiration. This overflow metabolism sets on when the glucose concentration exceeds
8
about 30 mg/L and it corresponds to a specific growth rate of about 0.3 h-1 which is about
60% of the maximal growth rate. The so called bottleneck for the electron flow from
pyruvate to the molecular oxygen is unknown. It is not the capacity of the respiration per se,
but may be some reaction in the TCA-cycle (see Fig 4.6).
Ethanol production is a means to dispose of the surplus carbon when the metabolism
downstream the pyruvate can not keep up with the high rate of pyruvate formation forced on
the cell by a high glucose concentration. Concomitantly some of the pyruvate (about 10% of
ethanol amount) is secreted as acetaldehyde and acetate. The additional glycolysis
contributed by the overflow metabolism provides extra ATP in addition to that obtained from
glucose going through to respiration. This extra energy supply is used for increasing the
growth rate. A mathematical description of these reactions are given in the section of fedbatch technique ( chapter 7).
The overflow metabolism of yeast is sometimes referred to as the glucose effect. However, it
must be distinguished from the original meaning of the glucose effect, that was used as a
synonym to the Crabtree effect, which is catabolite repression (i.e. on enzyme synthesis
level) of the respiration, that takes place during long-term exposure of the cell to high glucose
concentration. The overflow metabolism is observed within seconds after exposure of the cell
to high glucose concentration and it is therefore sometimes also referred to as the short-term
glucose effect.
In E. coli the overflow metabolism is observed as conversion of pyruvate by pyruvate
dehydrogenase to acetylCoA and further to actetate that is secreted to the medium (see Fig
4.6, though observe that bacteria do not have mitochondria !). A maximum respiratory rate,
that is reached before the maximum glucose uptake is reached, is characteristic also for E.
coli. When the rate of glycolysis is low, acetate is resorbed by the cells, if present in the
medium. The regulatory explanation to acetic acid formation by E. coli is even less clear than
the ethanol formation by yeast. It may, in fact, have several causes. Firstly, there appears to
be a lack of regulation of the glucose uptake rate at high glucose concentrations. Enz IIglc
(Fig 4.4) is believed not to respond to the normal regulatory signal (the membrane potential)
when saturated with glucose. Secondly, at high glucose concentrations the α-ketoglutarate
dehydrogenase activity is decreased, as it is under anaerobic conditions, thus interrupting the
TCA-cycle. In analogy with the situation in yeast, acetic acid production may, in terms of
energy production, be beneficial to the bacterium. Though, different strains of E. coli vary in
their ability to form acetic acid.
4.3.5 Summary.
Catabolism is regulated at the point of entrance of various substrates into the central
metabolic pathways. These pathways are made up of constitutive enzymes whose activities
are regulated in relation to the energy demand and the properties of the substrate. In
facultative aerobic organisms overflow metabolism results in production of partially oxidised
products like ethanol and acetic acid. Understanding the regulation of catabolism and energy
formation is especially important in biotechnical processes because these activities furnish
the basis for the rest of the metabolism (Figs. 4.1 and 4.3).
4.4 Regulation of anabolism
9
Biosynthetic pathways leading to the formation of building blocks (amino acids, nucleotides,
constituents of cell walls) for macromolecule synthesis have a defined starting point and end
in the final product. The maximum flux of material through these pathways is determined by
the rate with which precursors, energy and reducing power are supplied from the catabolism.
To avoid wasteful overproduction of certain metabolites, accumulated end products inhibit
their own synthesis. Such phenomena are known as feed-back inhibition of enzyme activities
and repression of enzyme synthesis. This type of regulation is typical for the biosynthetic
pathways. To achieve overproduction of metabolites from the anabolic compartment it is
necessary to overcome the regulatory mechanisms that normally limit their production.
Examples of such processes will be described below (section 4.7).
4.5 Regulation of synthesis of macromolecules
During growth in sufficient media (e.g. in a laboratory fermenter) the capacity of the protein
synthesising machinery as well as the synthesis of individual proteins and other
macromolecules (membrane lipids, cell walls) is co-ordinated and adjusted to the growth rate
as will be explained below. Microorganisms living in their natural habitats seldom experience
such situations but for short periods of time. The "normal" situation is rather characterised by
a deficiency of essential nutrients. To survive transitions from feast to fast microorganisms
exhibit additional mechanisms that adjust metabolism to special stress situations that may
arise. Regulatory systems with such properties affect several genes or groups of genes and
are called "global regulatory systems" (Table 4.1).
Table 4.1 Global regulatory systems
Multigene system
Environmental stimulus
Nitrogen utilisation
Ammonia limitation
Carbon utilisation
Carbon/energy limitation
Phosphate utilisation
Phosphate limitation
Stringent response
Amino acid/energy limitation
Heat shock response
Heat, certain toxic agents
SOS response
UV and other DNA damaging agents
Translation apparatus
Growth rate supporting ability of medium
Osmotic stress response
High osmolarity
Anaerobic respiration
Presence of electron acceptors other than oxygen
Anaerobic fermentation
Absence of electron acceptors
Aerobic response
Addition of oxygen
The regulation of genes encoding catabolic and anabolic enzymes, already discussed, are,
together with all other protein encoding genes, under control of a general growth rate
regulating signal that adjusts the overall protein synthesis rate to the availability of nutrients.
This is effected by an increase in the cellular content of RNA polymerase, tRNA, ribosomes
and protein elongation factors in relation to growth rate.
10
What are then the molecular mechanisms behind these phenomena? This is unfortunately not
yet fully understood. However, the well known response to amino acid starvation, shown to
exist in E. coli, S. typhimurium, B. subtilis (and exists probably in many other prokaryotes as
well), and named "the stringent response", may play a role in this context. Two small
molecules, guanosine pentaphosphate (pppGpp) and guanosine tetraphosphate (ppGpp) are
formed in response to amino acid deficiency. The pppGpp forming enzyme - pppGpp
synthetase I (gene designation: relA) - is attached to ribosomes and the synthesis of pppGpp
is triggered when an uncharged tRNA binds to the complementary codon in the ribosomal
site A. The primary effects of the stringent response are inhibition of synthesis (transcription)
of rRNA and tRNA. How (p)ppGpp mediates this response is still under debate. (p)ppGpp
can however also be synthesised in response to carbon and energy deprivation. This response
is also well documented but not well characterised. It is clear though, that this route of ppGpp
synthesis is independent of relA.
The physiological effects of the stringent response mediated through (p)ppGpp include
reduction of the protein synthesis rate as a consequence of the reduced number of ribosomes
and tRNA molecules in the cell. Furthermore, synthesis of phospholipids and cell walls are
arrested concomitantly with a reduced flux in glycolysis. A number of genes are, on the other
hand, under positive stringent control i.e. the relative rate of the synthesis of these proteins
increase. This holds for operons controlled by attenuation encoding amino acid biosynthetic
enzymes. Some of the heat shock proteins including protease La (chapter 16) are also under
positive stringent control. This appears logical from a physiological point of view in that the
proteases diminish unnecessary activities and at the same time supply amino acids but may
be an obstacle in processes for the production of recombinant proteins.
The stringent response functions as a shock response when the organism is deprived of an
amino acid immediately adjusting the metabolism to the new situation. The stringent
response functions also as a general mechanism that continuously senses the availability of
amino acids during different growth conditions and respond to these differences by adjusting
the basal level of (p)ppGpp in the cell. The basal level of (p)ppGpp is 10-20 times lower than
during amino acid deficiency but it varies inversely to the growth rate. Thus, it is believed
that (p)ppGpp can act as a fine regulator of the macromolecule synthesis rate during normal
growth conditions, but also as a complete inhibitor of the synthesis of stable RNA species
(tRNA, rRNA) when an amino acid is totally unavailable.
4.6 Products from catabolism
4.6.1 Aerobic heterotrophic energy metabolism.
Products from the catabolic compartment in aerobic organisms include intermediate
metabolites from the central pathways or derivates thereof. An important example is citric
acid production by Aspergillus niger. Other TCA-related substances have also been
considered for production in biotechnical processes. Normally such products are not
overproduced unless they are end products in overflow metabolism but are used up as
precursors for biosynthesis or converted to carbon dioxide. During citric acid production
growth cannot occur because this process consumes all the carbon source and yields very
11
little energy. Another important product is acetic acid (vinegar) obtained from Acetobacter
sp. Strictly aerobic acetic acid producing bacteria obtain energy from the partial oxidation of
ethanol yielding acetic acid as an end product. This means that the product is spontaneously
produced as is the end products of anaerobic fermentations described below.
4.6.2 Anaerobic fermentative energy metabolism.
A number of products that are, or have been produced in biotechnical processes are end
products of anaerobic fermentative energy metabolism. Examples of such products include
ethanol, lactic acid, butanediol and acetone-butanol.
Products of this type are spontaneously produced both in growing and non-growing cells. The
rate of production increases with growth rate but the yield coefficient varies inversely to the
growth rate. The principles of these fermentations are outlined in Fig. 4.7. The mixed acid
fermentation of E. coli is also shown (Fig 4.8) because it will be triggered if oxygen
limitation occur in any process utilising E. coli (e.g. as host for a plasmid encoded product).
Anaerobic fermentative energy metabolism gives a very low energy yield compared to
aerobic. ATP is produced only by substrate level phosphorylation. Fermentative organisms
obtain 2 ATP/glucose in glycolysis and 1-2 ATP further in other reactions associated with
acid production (e.g. acetic or butyric acid). A high flux through glycolysis is required to
obtain sufficient energy for growth resulting in excretion of large amounts of end products. In
facultative organisms the rate of glycolysis increases during a shift from aerobic to anaerobic
conditions. This was originally observed in yeast by Pasteur and has since then been called
the Pasteur effect. Today this phenomenon can be explained in terms of regulation of the
phosphofructokinase, the rate limiting enzyme of glycolysis. This enzyme responds to
changes in the ATP concentration, or rather to the energy charge. During anaerobic
conditions the energy charge tends to fall resulting in an increased enzyme activity.
The second point of importance in fermentative energy metabolism is the cofactor balance. In
glycolysis two moles of NADH are produced per mole of glucose. The cofactors must be
regenerated to maintain the flux in glycolysis. In yeast and homofermentative lactic acid
bacteria this is accomplished by the reduction of acetaldehyde to ethanol and pyruvate to
lactate respectively (Fig. 4.7). In other cases it may be more complicated as in the mixed acid
fermentation of E. coli (Fig 4.8).
12
Fig. 4.7 Left: Fermentation of glucose to ethanol. Right: Fermentation of glucose to lactic
acid. Lactic acid bacteria (Lactobacillus spp; Lactococcus spp.) use the PTS system to
transport lactose. Intracellular lactose-P is hydrolysed into galactose-P and glucose which
subsequently is phosphorylated by hexokinase as depicted above.
Fig. 4.8 Outline of the mixed acid fermentation used by, among others, E. coli.
4.6.3 Anaerobic respiration.
13
A number of microorganisms that normally derive energy from aerobic respiration can use
nitrate as electron acceptor when oxygen is lacking (see Fig 4.2). Thus, they are facultatively
anaerobic. The most common type of anaerobic respiration is denitrification that is common
among Pseudomonas and many other bacteria. The denitrification includes a stepwise
reduction of nitrate to gaseous nitrogen :
NO3- ----> NO2- ----> NO ----> N2O ----> N2
Each step in the denitrification is coupled to ATP synthesis. The different enzymes (nitrate
reductase, nitrite reductase, nitric oxide reductase and nitrous oxide reductase, respectively)
are differently sensitive to oxygen, which may result in incomplete denitrification and release
of the intermediates, especially nitrite and nitrous oxide. Nitrate reduction to nitrogen gas is
an important reaction in the removal of nitrogen from waste water (Chapter 19).
A small group of facultative and obligate anaerobic bacteria can use sulphate as electron
acceptor and obtain energy by electron transport phosphorylation. Sulphate is reduced to
hydrogen sulphide. This reaction is important because i) it contributes to the corrosion of iron
and ii) hydrogen sulphide may accumulate in anaerobic digesters because sulphate is reduced
in preference to carbonate, which otherwise would have yielded methane gas.
4.7 Products from anabolism
14
Classical examples are amino acids like lysine and glutamic acid. Vitamins and nucleotides
belong also to this group. The aromatic amino acids tyrosine, phenylalanine and tryptophan
have long been extremely difficult to produce directly from glucose but genetic methods have
eventually made this possible.
Anabolic metabolites are never overproduced by normal microorganisms. To obtain
production worthwhile for an industrial process the metabolic regulatory mechanisms must
be overcome. The strong desire to produce amino acids forced research to understand these
mechanisms and to circumvent them. The principles of this regulation and the methods
developed which made economical production possible will be illustrated by using the
aromatic amino acids as an example.
Figure 4.9 outlines the biosynthetic pathway for tyrosine, phenylalanine and tryptophan. The
pathway starts with the condensation of erythrose-4-P and phosphoenolpyruvate to DAHP.
This step is catalysed by three isoenzymes, the DAHP-synthases, which all contribute to the
flux through the pathway. A series of successive reactions lead to chorismate, the last
common intermediate. Three individual branches then lead to the respective amino acid. The
synthesis is regulated at the points indicated in the figure by feed-back inhibition of enzyme
activity (allosteric regulation) and repression of enzyme synthesis (transcriptional regulation).
Each amino acid regulates its own branch and one of the three isoenzymes in the first step of
the common pathway. This mechanism ensures that the common pathway will not be turned
off as long as there is a need for one of the amino acids. Similar mechanisms exist in other
branched biosynthetic pathways.
Classical and newer methods for abolishing the regulatory mechanisms involve: i)
Auxotrophic mutants, i.e. a mutant with a block (an enzyme is deleted or otherwise
unfunctional) somewhere in a biosynthetic pathway. The regulatory enzymes are not
inhibited in the auxotrophic mutant because the end product is not synthesised. Such a mutant
may overproduce the intermediate in the pathway before the metabolic block or increase the
flux to other end products of a branched pathway. ii) Feed-back resistant enzymes, i.e.
mutant regulatory enzymes that are insensitive to allosteric inhibition by the end product or to
repression of synthesis (mutation in the repressor gene) or to both. Such a mutant will
overproduce the end product of that pathway because there are no longer any mechanisms
that recognises the cells real demand for it. iii) Gene amplification, i.e. genes encoding rate
limiting enzymes, usually the regulatory ones, are inserted on a multicopy plasmid to ensure
that enzyme levels are not rate limiting.
15
Fig. 4.9 Biosynthesis of the aromatic amino acids (shikimic acid pathway). Feed-back
inhibition is indicated by backward arrows from the end products. PEP =
phosphoenolpyruvate; E4P = erythrose-4-phosphate; DAHP = 3-deoxy-D-arabinoheptulosonate 7-phosphate; DHQ = 3-dehydroquinate; DHS = 3-dehydroshikimate; EPSP =
5-enolpyruvoylshikimate-3-phosphate. aroG = DAHP synthase (phe); aroF = DAHP synthase
(tyr); aroH = DAHP synthase (trp); pheA = the bifunctional enzyme chorismate mutase
prephenate dehydratase; tyr A = the bifunctional enzyme chorismate mutase prephenate
dehydrogenase; trpE = anthranilate synthase.
16
As an example, a combination of these methods can yield an organism with the following
genetic properties that overproduces the aromatic amino acid phenylalanine: ∆tyr, ∆trp,
pXX/aroF, pheAFBR (FBR = feed back resistant). This organism is auxotrophic for tyrosine
and tryptophan (deletions of the trp E operon and tyr A operon) a property which increases
the flux of metabolites to phenylalanine. The strain must however be supplied with these
amino acids in order to grow. Furthermore this microorganism harbours a multicopy plasmid
which contain genes for the tyrosine inhibited DAHP-synthase and a feed-back resistant
(both allosterically and transcriptionally) prephenate dehydratase. During growth with added
tyrosine and tryptophan there will be no real overproduction of phenylalanine because
tyrosine inhibits synthesis of the plasmid encoded DAHP-synthase(tyr) and phenylalanine
regulates the chromosomal DAHP-synthase(phe). When the added tyrosine (and tryptophan)
is exhausted from the medium growth stops but DAHP-synthase(tyr) will be produced as a
result of relief of inhibition. This leads to enhanced production of phenylalanine since
phenylalanine itself cannot inhibit DAHP-synthase(tyr) and the chorismate mutase
prephenate dehydratase is a mutant enzyme, insensitive to regulation by phenylalanine.
4.8 Macromolecular products
Macromolecules like proteins (e.g. hydrolytic enzymes or recombinant proteins) or
polysaccharides are important products in the biotechnical industry. It is not possible to give
any general rules regarding the conditions for their production because of the different nature
and metabolic affiliation of the various macromolecular species. This circumstance
distinguishes macromolecular products from metabolites derived from catabolic or anabolic
pathways. For example, extracellular hydrolytic enzymes like amylases or proteases are
spontaneously produced by microorganisms in response to environmental stimuli like
presence or absence of certain nutrients. Such enzymes are in fact part of the catabolic
machinery as is the intracellular enzyme β-galactosidase. The latter is of course under control
of induction and cAMP. To obtain overproduction of such an enzyme it is necessary to
genetically modify the regulatory mechanisms e.g. by creating a constitutive mutant that is
independent of induction. This may also be combined with the use of a multicopy plasmid
that harbours the gene as is common practise in production of recombinant proteins. The
production of recombinant proteins is further governed by the chosen promoter ruling out
normal regulatory mechanisms. It is important to consider the effects of a too high copy
number or a too strong promoter on the cellular physiology because it may lead to exhaustion
of precursors and energy which in turn may trigger some of the starvation responses (Table
4.1). Production of recombinant proteins will further be described in chapter 16.
4.9 Secondary metabolism
The preceding sections of this chapter has dealt with the primary metabolism and products
thereof. A great number of industrially important products such as antibiotics are, however,
derived from the secondary metabolism. Secondary metabolism, common in plants and
microorganisms, is characterised by not being essential for growth. The purpose of it or
benefit from it for the organism is still under debate. Usually, secondary metabolites are
produced during the stationary phase. This depends on their formation being repressed during
growth by different mechanisms. These include: 1) Carbon catabolite inhibition and
repression i.e. good carbon end energy sources repress formation of enzymes in the
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secondary metabolite pathways. 2) Nitrogen metabolite repression i.e. easily metabolised
nitrogen sources like ammonium ions likewise repress secondary metabolite producing
enzymes. 3) Repression of pathways or individual enzymes by inorganic phosphate.
Metabolic regulatory mechanisms like these necessitate the use of special cultivation
conditions like fed batch techniques to obtain reasonable production. The synthesis of
secondary metabolites is further regulated by feed-back inhibition by the end product or by
regulation of the synthesis of precursors used for its formation. To overcome such obstacles it
is necessary to improve strains by mutation.
Secondary metabolism is not separated from the primary metabolism but, on the contrary,
intimately connected to it because precursor molecules and building blocks are derived both
from catabolic and anabolic pathways. This network is illustrated in Fig. 4.10, which at the
same time provides a rationale for classification of antibiotics according to their origin in the
primary metabolism.
Fig. 4.10 Connection between primary and secondary metabolism. Classification of secondary
metabolites.