Improvement of ansamitocin P-3 production by

actinosynnema mirum with fructose as the sole carbon

source

Y30141470
Tazhenova Lyailya
Q.Hua
 Plan
Introduction
Effect of Different Carbon Sources on the Growth and AP-3 Production of A.
mirum
Effects of Glucose or Fructose on Several Enzymes Activities of A. mirum
Expression Analysis of AP-3 Biosynthesis-Related Genes
Conclusions
INTRODUCTION
Actinomicin P-3 is the antitumor agent which usually produced by Actinosynnema spp. In this
study glucose, fructose, galactose, xylose, or sucrose was used as sole carbon sources and was
reported its effect to biomass and AP-3 production. In medium with fructose was obtained the
highest yield of AP- 3 production by Actinosynnema mirum.
Effect of different carbon sources on the growth and AP-3
production of A. Mirum

The maximal AP-3

concentration of 8.26 mg
L1 was obtained after 168
h in fructose medium
though A. mirum grew
poorly in this medium
comparatively to other
carbon sources.

Fig. 1 Cell growth and AP-3 production during shake flask batch cultivation of A.
mirum in minimal medium with different carbon sources. Glucose (black diamond),
fructose (white square), xylose (white triangle), sucrose (black inverted triangle ),
and galactose (white circle)
 In medium with glucose as sole carbon sources was
observed high accumulation of acetate (0.96 g L1 )
and succinic acid (0.55 g L 1) , whereas much
lower levels were detected in the case of fructose
medium.

Fig. 2 Fermentation profile in minimal medium with

glucose (black diamond) and fructose (white square) as
individual carbon sources. a Production of acetate. b
Production of succinate. c Sugar consumption. d pH
evolution
 Effects of Glucose or Fructose on Several Enzymes Activities of A.
mirum

Fig. 3 Comparison of several enzyme activities of central metabolism on glucose (black diamond)
and fructose (white square). G6PDH glucose-6-phosphate dehydrogenase, 6PGDH gluconate-6-
phosphate, FBPase fructose1,6-biophosphatase, GAPDH glyceraldehyde-3-phosphate
dehydrogenase, AKGDH -ketoglutarate dehydrogenase, MDH malate dehydrogenase
 Fig.4 Schematic representation of the central carbon metabolism and AP-3 pathway of A. mirum.
Enzymatic reactions/pathways are indicated for the uptake and activation of glucose and fructose, for
excretion acetate and succinate, for glycolysis, pentose phosphate pathway, and the tricarboxylic acid
cycle.
 The levels of AHBA synthase genes amir_3287 and
amir_3196 were significantly higher in cells grown on
fructose as the sole carbon source. Expressions of these
two regulator genes amir_3172 and amir_3202 were also
upregulated in fructose-grown cells, especially for
amir_3202, which showed a strong increase most of the
time. Gene amir_3186 is involved in the biosynthesis of
the unusual methylmalonyl-ACP extender unit. In fructose
medium, the transcription of amir_3186 was upregulated
as well, but its transcription level gradually decreased
with the cultivation time (Fig. 3). Regarding amir_2277
gene, which codes for methylmalonyl-CoA synthetase, its
expression was repressed by fructose. Overall, the results
indicated that the increased AP-3 production in fructose
medium was likely to be associated with the
overexpression of the AHBA synthase genes as well as
some regulator genes.
Fig. 5 Relative genes expression in A. mirum growing on
fructose and glucose media
Conclusion

In this study was proven that fructose is beneficial carbon source for
production ansamitocin by Actinosynnema mirum. The reason for the
significant enhancement of AP-3 production in fructose medium might be
attributed to the stimulation of the expression of AHBA synthase genes as well
as some regulator genes such as amir_3172 and amir_3202.