DATOS DEMOGRÁFICOS DE LA POBLACIÓN INVESTIGADA

The main focus of study of lipid synthesis in microorganisms has been the production of membranes, since they comprise the main lipidic components of the cells and are involved in immunological reactions. However, many species of microorganisms can be described as oleaginous, that is they produce lipids as storage bodies as well as structural components of the cell envelope. This process is being seen as increasingly important, due to recent discoveries that have found storage lipids to be more involved in key

metabolic pathways than had been previously thought. Studies on cellular lipid

metabolism has therefore widened to look at the metabolism of both simple and complex lipids.

Lipid synthesis begins with the production of fatty acids. The principle enzyme complexes involved in the biosynthetic pathway are acetyl-CoA carboxylase and fatty acid synthase. The fatty acid synthetic pathway is highly conserved between species, with differences in the pathway arising in the structure of the enzymes rather than the steps occurring. The overall reaction is shown below:

acetyl-CoA + 7 malonyl-CoA + 1 4 NADPH —>

palmitoyl-CoA + 7 CO2 + 6 H2O + 14 NADP+ + 7 Co A

The acetyl-CoA carboxylase complex catalyses the reaction between acetyl-CoA and CO2

to form malonyl-CoA. The enzymic reaction requires the co-factors biotin and CO2 and

occurs in two steps, each catalysed by a separate protein or active site. The first reaction

is biotin carboxylase, which binds biotin and CO2, with the conversion of ATP to ADP.

The second is mediated by a carboxyltransferase, which converts acetyl-CoA to malonyl-

CoA, in an acetyl-CoA:C0 2 ligase reaction. Bramwell et a l (1996) found that propionyl-

CoA carboxylase activity was present in S. coelicolor, although no trace of acetyl-CoA

carboxylase activity could be found. Mycobacterium spp. were found to possess both

activities which were thought to be catalysed by a single enzyme complex (Wheeler et a l,

1992a).

For both acetyl-CoA carboxylase and fatty acid synthase, two forms of enzyme are found. Type I fatty acid synthase is found in animals, yeasts and a number of bacteria, including

Mycobacterium smegmatis and Corynebacterium diptheriae and consists of a multifunctional enzyme with separate activities on different sites. Type II, found in plants and most bacteria, is a system where each reaction is catalysed by a separate enzyme.

Evidence was obtained that S. coelicolor had a type I synthase (Flatman and Packter,

C h a p te r 1. In trodu ction

and S. erythrea that introduced synthetic intermediates could be metabolised by the pathway, indicating that the enzyme sites are not tightly linked and therefore of type II. Synthesis proceeds by the sequential addition of malonyl units on a growing acy 1-chain

until such time as it reaches a chain length of or C^g. The chain is then terminated by

an acyl-ACP thioesterase. The growing chain is linked to a separate acyl-carrier protein

(ACP) in type II systems, the levels of which appears to control fatty acid synthesis in E.

coli. The ACP binds to a primer which supplies the first two carbon units, usually acetyl-

CoA but also butyryl-CoA in Streptomyces spp. for the production of straight-chain fatty

acids (Wallace et a l, 1995). The S. coelicolor synthetase preferentially accepts longer

chain acyl-CoAs as primers, rather than acetyl-CoA or other short chain derivatives

(Packter et a l, 1985). Odd-numbered chains are created from propionyl-CoA starter

units and branched acyl-CoAs such as isobutyryl-CoA are used to construct branched- chain fatty acids. The synthetase reaction occurs in six steps:

acetyl transacylation

CH3CO-S CoA + HS-enzyme —> CHaCO-S-enzyme + CoA

malonyl transacylation

COOH.CH2CO-S CoA + HS-enzyme —> COOH.CH2 CO-S-enzyme + CoA

^-ketoacyl synthetase (condensing enzyme)

CHsCO-S-enzyme + C0 0 H.CH2C0 -S-enzyme —> CH3CO.CH2.CO-S-enzyme + CO2 + HS-enzyme

^-ketoacyl reductase

CH3.CO.CH2.CO-S-enzyme 4- NADPH -> CH3.CHOH,CH2.CO-S-enzyme 4- NAD?"*"

D-^-hydroxyacyl dehydrase

CH3 CH0H.CH2.C0-S-enzyme —> CHa.CHiCH.CO-S-enzyme 4- H2O

2,3 trans-enoyl reductase

CHaCHiCH.CO-S-enzyme 4- NADPH -> CH3.CH2.CH2 CO-S-enzyme 4- NADP+

The last product then re-enters the pathway at the (3-ketoacyl synthetase step. In E. coli

synthesis appears to continue until the chain contains 16 carbon units. It is removed from the fatty acid synthetase complex by hydrolysis, or by the action of palmitoyl transferase,

using H2O or CoA as co-factors respectively. The chain can be further lengthened by re-

introduction to the fatty acid synthase system, or can be acted on by a third type of fatty acid synthase (FAS III), also known as an elongase. These are found in animal, bacterial and plant systems and use malonyl-CoA as the 2 carbon unit donor (Ratledge, 1988). Research in plants has generated conflicting data as to whether the activated (acyl-CoA)

form of fatty acids or fatty acids themselves are acted upon by the elongase (Cassagne et

a l, 1994).

The synthesis of unsaturated fatty acids occurs by two mechanisms, which appear to be mutually exclusive. The anaerobic mechanism is so called because it occurs without the

C h a p te r 1. In trodu ction

need for oxygen as a co-factor and is found in some species of eubacteria. After the

synthesis of D-p-hydroxydecanoyl-ACP, the saturated fatty acid is converted to a cis-A^

monounsaturated form. The chain is then lengthened by distinct enzymes, which mirror the reactions occuring in the saturated fatty acid synthesis pathway. The end-products are cw-A^-palmitoleic acid or cw-A^i-vaccenic acid (Cig.j). The alternative system is found almost universally and is catalysed by desaturases. The double bond is introduced directly into the chain, usually at the A^ position. Bacteria appear to be unable to produce polyunsaturated fatty acids, unlike yeasts and other higher organisms.

Streptomycetes have a high level of branched-chain fatty acids and the S. coelicolor fatty

acid synthase was found to have a 1 0-fold preference for branched-chain primers over

acetyl-CoA (Packter et a l, 1985). Most effective were isovaleryl-CoA and isobutyryl-

CoA, the former responsible for generating Iso-C^^.q and the latter Iso-C^^.q.

Primers are derived in the ceU from branched chain amino acids and similar preferences in

vivo would lead to the high concentrations of these fatty acids seen in normal cells

(Section 1.3.2.1). Wallace and co-workers (1995) found similar results with three more

Streptomyces species. Other branched chain fatty acids are the product of methyl groups introduced into unsaturated fatty acid chains. Hydroxy fatty acids are the products of a- or cû-oxidation.

The acyl-ACPs, the final, water-soluble products of fatty acid synthesis, are used for simple and complex lipid biosynthesis. Triacylglycerols and phospholipids are synthesized

from a common precursor, l,2 -5«-diacylglycerol 3-phosphate (phosphotidic acid), which

is in turn a glycerol product of the glycolytic pathway by the reduction of dihyhroxyacetone phosphate. The synthetic pathway adds acyl moieties (fatty acids) to

the 3 -5«-glycerol phosphate, losing a phophate group with each attachment. The three

acylation reactions are catalysed by acyltransferases, which can be specific for each position on the glycerol moiety. In the plants, moulds and yeasts that have been studied,

the sn-2 position is occupied by an unsaturated fatty acid, unlike in animals where the

central fatty acid is predominantly a saturated fatty acid (Ratledge, 1982). In Yarrowia

lipolytica the level of control of triacylglycerol production operates with the availability of

long-chain acyl Co As (Kamiryo, 1983). As weU as fatty acids synthesized de novo,

externally imported fatty acids can be used in the manufacture of triacylglycerols (Ratledge, 1988).