Enfoque constructivista

Saccharopolyspora erythraea is put in the group of nocardioform actinomycetes in subgroup 2 (pseudocardia and related genera) in genus Saccharopolyspora.

Morphologically and physiologically diverse actinomycetes that form extensively branched vegetative and aerial hyphae. Smooth, spiny, or hairy spores are produced

singly, in pairs, or in chains of variable length on aerial hyphae. Saccharopolyspora erythraea was transferred from Streptomyces erythraeus. Labeda (1987) reported that the transfer causes from the chemotaxonomic analysis of cells of Streptomyces erythraeus NRRL 2338, the type strain of the species, which revealed that it is not a representative of the genus Streptomyces because the cell walls contain meso- diaminopimelic acid, arabinose, and galactose. Thus, the new species Saccharopolyspora erythraea is described, and strain NRRL 2338 (equivalent to ATCC 11635 and ISP 5517) is designated as the strain type or wild type strain.

1.1.3.1

Characterisation and genetics

The specific character of S. erythraea are less thermotolerant than the other species and growing between 20 and 42°C. It is characterised by orange to red colonies with

mainly pink to brownish-grey aerial mycelium bearing short spore chains in imperfect spirals. This strain can utilise arabinose, melibiose, and raffrnose and there are not

any reports for lactose, melezitose or a-methyl-D-glucoside utilisation. The spore

surface is spiny. This strain can be isolated from soil. The type strain, NRRL 2338, is

widely used in research and development, where most of them produce erythromycin A and B. On the other hand, industry prefers to use such a mutant strains for

erythromycin production to reach the highest yield. Mutation is an important method

in improving strain to produce high yield of erythromycin on an industrial scale.

The acquisition of knowledge about the genetics of S. erythraea is progressing in two directions : (1) location of the structural and regulatory gene for antibiotic production

in relation to other genetic markers and (2 ) development of vector that will be useful

for cloning the erythromycin production genes.

INTRODUCTION Actinomycetes

As information in these two areas is acquired, it will facilitate efforts to clone

structural genes of erythromycin biosynthesis for the study of their properties both in vivo and in vitro (Seno and Hutchinson, 1986).

Generally, the strains that produce erythromycin A can be placed in two groups

according to their growth, sporulation, and pigment production on different types of

media as shown in Table 1.2. Strains of type A produced grey spores abundantly and

a brown pigment when grown on a medium containing cornstarch and com steep liquor and buffered with CaCOj while, strain NRRL 2338 and other strain type B

produced a moderate amount of pinkish spores and the “Russet Vinaceous (reddish-

brown)” pigment (Bunch and McGuire, 1953 and Seno and Hutchinson, 1986) when grown on the R2T protoplast regeneration medium. These variants were of the B type mostly and produced 2 to 1 0 fold less erythromycin but did not have altered resistance

to antibiotic. The reverted the parental phenotype (type A) at a frequency of about 1 in 10^ but the resulting erythromycin production increased only 2-3 fold.

Now the development of suitable gene cloning vectors is the focus of the studies of the genetics of antibiotic production in S. erythraea. The successful application of recombinant DNA technology to yield improvement or the production of hybrid antibiotics required the development of methods to maintain a cloned gene in single or

multiple copies in the host organism with minimal deleterious effect on the antibiotic- producing physiology.

The mutagenic treatments, by using N-methyl-N'-nitro-N-nitroguanidine, ethyl

methanosulfate and ultraviolet light, of spores rather than mycelia provide stable

progenies. These spores should be grown to express fully acquired phenotypes and to eliminate segregating clones. Then the cells grown from mutagenised spores are again

spomlated for storage and for further mutational treatment.

INTRODUCTION Actinomycetes

Table 1.2 The grey (type A) and red (type B) S. erythreus strains. (Bunch and McGuire, 1953)

Type A TypeB

M5 NRRL 2338

CA119 Red variants of type A CA 340

ER598 ER620

The need for strain improvement arises from various requirement. These include :

increases in total potential and the titre of a desired component ; change in the ability to assimilate carbon sources and nitrogen sources, growth rate ; enhancement of tolerance to higher concentration of carbon and nitrogen sources ; increase in

sporulation ability etc. (Omura and Tanaka, 1986).

1.1.3.2

Cultivation, morphology and product formation

As previously mentioned, hyphal morphology of actinomycetes in liquid culture had some effects to growth and product formation. There is no exception for the genera Saccharopolyspora. Lilly et a l (1992) reported that the physical environment in fermenters could have an effect on antibiotic production, for instance rheological

property. The cultures of S. erythraea displayed non-Newtonian rheological property which correlated mainly with changes in morphology during the fermentation.

However, its rheological behaviour during the initial growth phase was found fitted with the power law. Moreover, the consistency index was also found risen with

biomass concentration.

Techniques used for measuring the morphology of filamentous cultures. Image

analysis, revealed that the culture consisted of short hyphae with a few branches. The mean main hyphal length of the culture remained in the range of 15-25 |im throughout

the fermentation and no sign of fragmentation has occurred. (Adams and Thomas,

1988; Packer and Thomas, 1990),

INTRODUCTION Secondary Metabolites

Heydarian et al. (1996) indicated that the limitation for growth and antibiotic production of S. erythraea CA340 at dissolved oxygen tension (DOT) level were different. The growth was found inhibited when the culture was grown at DOT of

1 0 % air saturation whereas the specific erythromycin production was virtually

identical to that of a culture where the DOT did not drop below 65%. Furthermore,

during the observation of growth limitation at 10% DOT, it revealed that 750 rpm

stirrer speed of a 7 L fermenter could cause the mechanical damage to the mycelia in

comparison with that of 500 rpm. In contrast with Heydarian et a l some reports revealed that erythromycin could be produced in both oxygen-limited and oxygen

sufficient cultures (Clark et al, 1995).