The fatty acid composition of the cells grown on triolein-containing media showed that the exogenous lipid was being incorporated into the cells. Given that the addition of lipids
to cultures promotes growth, one of the mechanisms by which that could occur is that de
novo lipid synthesis is repressed, since the external fatty acids can be incorporated and
used unmodified. This would free ATP and acetyl-CoA for other purposes. Therefore,
the level of lipid synthesis in S. lividans cells grown with and without lipids was examined
to confirm if this was happening.
3.3.4.1 The spectrophotometric acetyl-CoA carboxylase assay
Acetyl-CoA carboxylase is the first enzyme in the fatty acid biosynthetic pathway. The presence or lack of activity of the enzyme in a cell-free extract is therefore an indication of the sythesis of fatty acids within a particular culture. Acetyl-CoA carboxylase activity was
therefore assayed in S. lividans cells grown in Hpid and non-lipid media.
For the spectrophotometric assays (Section 2.5.19) the cells were grown as described in Section 3.3.1 and harvested in the rapid growth phase, when glucose in the media remained at a level above Ig/L (where appropriate). The assay trace had a typical profile as shown in Figure 3.3.11.
It is obvious from the trace that the background rate obtained before the addition of substrate (acetyl-CoA) constituted at least 50% of the final rate. Because of this, acetyl- CoA was used as the start reagent to enable the background rate to be discounted from the specific activity calculations.
C h a p te r 3. E ffects o f lip id s on g ro w th a n d m e ta b o lism o f S. lividans 2 . 0 E c o CO 4^ 1 .5 — CO CD CJ c CO ■e o CO < 1.0 - 0 . 5 P K / L D H a c e t y l - C o A T i m e ( m i n )
Figure 3.3,11. Typical trace o f the acyl-CoA carboxylase assay, using glucose-grown CFE. At the addition points (arrowed), Pyruvate kinase/Lactate dehydrogenase (PK/LDH) and acetyl-Co A were mixed with the rest o f the assay components.
33.4.2 Components of the spectrophotometric assay
A series of assays were carried out with glucose-grown cell free extract (CFE) omitting various components, since a large number of co-factors are needed in the assay mix. The figures given have had the background rate subtracted from them.
Component removed Specific activity (nmol min^mg^)
Acetyl-CoA (background rate) 58.1
None (whole assay) 23.8
PEP -55.0
KCl 79.7
GSH 24.3
sodium bicarbonate -1 1 . 2
ATP 17.3
Table 3.3.3 Specific activities o f glucose-grown cell-free extract (CFE) with omissions o f assay components. Activities are given as the actual rate minus the background rate, the mean o f duplicates.
C h a p te r 3. E ffects o f lip id s on g r o w th a n d m e ta b o lism o f S. liv id an s
The background rate, this time taken as that obtained without substrate, is in all the cases 40-100% of the total obtained. Even in the whole assay it comprised 70% of the final rate and as such compromises the validity of any actual rates obtained.
3.3.4.3 Addition of avidin to the assay
When 0.25U of avidin (Sigma) was added to the 1 ml assay mix, the activity of the CFE was stopped completely. Avidin binds to biotin which is a co-factor in the carboxylase, so an inhibition of activity indicates that a biotin-requiring reaction is taking place. Therefore the glucose-grown CFE probably contained some acetyl-CoA carboxylase activity.
3.3.4.4 Application of the assay to triolein- and mixed-grown CFEs
Assays were carried out with glucose-, mixed- and triolein-grown CFEs to compare the amount of acetyl-CoA carboxylase activity in each. The specific activities of the CFEs are shown in Table 3.3.4.
Cell Type Blank
(no acetyl-CoA) [as nmol min ^mg H
Average specific activity - blank [nmol min img^]
Percentage of actual activity of glucose CFE Glucose 58.1 23.9 100 Mixed 51.1 2.7 11.2 Triolein 5.8 0.2 0.7
Table 3.3.4 Comparison o f acetyl-Co A carboxylase activities o f different CFEs
The spectrophotometric assay was not conclusive, but provided evidence that a difference did exist in acyl-CoA carboxylase activities of different cells. The glucose-grown cells
had 1 0 and > 1 0 0 times the rate of activity of the mixed-and triolein-grown cells,
respectively. The low background rate of the triolein-grown CFE may indicate that the background activity is due to a high level of endogenous substrate in the other CFEs, but this should have been minimzed by ultracentrifugation and dialysis carried out in the preparation of the CFEs (Section 4.2).
3.3.4.5 Incorporation of Na2-[^"^C]0 ^ by acetyl-CoA carboxylase activity
A second acetyl-CoA carboxylase assay was used to see if the problems of background rate and sensitivity could be improved. This method used CFEs of the three types of cells (glucose, triolein and mixed) but the extracts were prepared without ultracentrifugation or dialysis since the monitoring of activity did not rely on an NAD+/NADH reaction. Using the method described in Section 2.5.18, the assay was run for 30 min at 30°C and labelled carbon assimilation was estimated using dpm values obtained after scintillation counting.
C h a p te r 3. E ffects o f lip id s on g ro w th a n d m e ta b o lism o f S. lividans
However, the activity from all the CFEs was very low and so near to levels obtained with controls that they were inconclusive.
It was considered that the bicarbonate was not being used due to competition with
dissolved C O2, so a second set of experiments were devised to eliminate dissolved H C O3'
ions from the assay mix. This was done by bubbling the assay components with nitrogen
that had been passed through IM CaDH)^to remove all disolved CO2 and adding the
substrate through a seal. However, this measure did not greatly improve the total radioactivity recovered after the time course.
CFE type mean dpm + (SEM) Specific activity (nmol min^mg i)
Glucose 155.5 (3.9) 0.84
Mixed 78.8 (1.53) 0.35
Triolein 72.1 (1.16) 0.28
Table 3.3.5 Specific activities affixation o f radiolabelled Na2C0^ by cell free extracts. Five samples were counted fo r each CFE, from which a mean and standard error o f the mean (SEM) were calculated. Specific activities were obtained using a dpm o f 87400for 10[Lmol substrate.
The low count gave a specific activity for the glucose-grown CFE of 0.84 nmol min-^mg \ This is 30 times less than the value obtained from the spectrophotometric assay. The
value is, however, consistent with rates obtained for other S. lividans enzymes (e.g. the
linked assay in Section 4.5; which was about 1.0 nmol min-^mg-^) but the disparity between the activities found by the two types of assays indicated that acetyl-CoA
carboxylase activity is difficult to monitor in S. lividans.
A recently published paper indicates that acetyl-CoA carboxylase may only be present at
very low levels, if at all, in S. coeiicolor. Bramwell et al. (1996) found the activity of
propionyl-CoA carboxylase to be 0.8 |Limol min-^mg-\ using crude cell free extracts. However, no acetyl-CoA carboxylase activity could be detected, despite this enzyme having direct applications to the production of polyketide compounds, unlike propionyl-
CoA carboxylase. The cells used were grown on complex media. The Mycobacterium
spp. tested by Wheeler and co-workers (1992), possess both acetyl-CoA and propionyl- CoA carboxylase activities that are catalysed by a single enzyme complex. Activity was found to be dependent on the growth substrate. It would be interesting to investigate
propionyl-CoA carboxylase levels in S. lividans, especially since some putative acetyl-
CoA carboxylase activity was present in the CFEs.
3.3.5 [U-'^^C] glucose assimilation by viable cells
Since the acetyl-CoA carboxylase assays were not conclusive, another assay had to be
C h a p te r 3. E ffects o f lip id s on g ro w th a n d m eta b o lism o f S. lividans
incorporation of labelled glucose into the membrane fraction of the S. lividans cell was a
second possibility. Although the labelled glucose could be incorporated as glycerol or as fatty acids (since the assay measures [^"^C]-fixation in total lipids) it gives an indication of
overall hpid turnover in the cehs. The S. lividans cells tested with this method (Section
2.6.2) were harvested in rapid growth phase from 0.5% (w/v) glucose minimal medium, 0.5% (w/v) triolein minimal medium and 0.25% (w/v) glucose plus 0.25% (w/v) triolein minimal medium, as described in Section 3.3.1. Glucose concentrations were determined in the broths to ensure that the supply was not lower than Ig/L before the harvesting stage.
Following the method in Section 2.6.2, the ceh pellets were resuspended in similar growth media to that which they were grown in and [U-^^C] glucose was added. A pellet of 0.15g wet weight was used for each ceh type, with an additional pellet of mixed cells of 0.5g wet weight. Blanks of boiled glucose-grown cells and omitting labelled substrate had a dpm ml-^ of 14.3 and 37.2, respectively. Cell Types 0.25ml dpm ml 1 : 1.0 ml dpm ml 1 3.0 ml dpm ml mean (dpm g-' cells) SEM Glucose 395.5 386.5 429.46 2962 71.2 Triolein 45.88 33.47 41.15 268 19.7 Mixed (0.15g) 33.22 41.1 24.8 2 2 0 25.6 Mixed (0.5g) 139.2 63.13 142.98 230 42.5
Table 3.3.6 Counts o f extracted cell lipids after incubation with radiolabelled glucose. Three different volumes o f each extracted lipid were counted, giving a mean dpm and standard error (SEM).
The rate of glucose carbon incorporated into the hpids did appear to be at least 10 times as much in the glucose cells as in the cells grown in the presence of triolein, in a similar way to the acetyl-CoA carboxylase activities. This would indicate that the usual level of fatty acid synthetic enzymes are depressed in the presence of fatty acids, even when glucose itself is also present, preventing the metabohsm of the hpids as an energy source. This result is supported by the protein content of CFEs grown on the three types of medium (Figure 4.16.1). In these, bands can clearly be seen in the glucose CFE that are not as intense in cells grown with glucose plus triolein together, indicating that some enzyme expression is repressed by the presence of hpids in the medium.
Kendrick and Ratledge (1996) found that in four species of fungi, elongation and
desaturation of fatty acids in the ceh were reduced to 1 0% of that in glucose-grown cells
when the fungi were grown in vegetable oils. This indicated that fatty acid modification
enzymes are repressed in oil-grown cehs. Caulobacter crescentus represses fatty acid
synthesis when exogenous fatty acids are present (Letts et a l, 1982). Deh'Angelica and
C h a p te r 5. E ffects o f lip id s on g ro w th a n d m eta b o lism o f S. lividans
finding it is inhibited by the presence of free fatty acids in the medium. Specifically, the enzyme (l,3)-(3-glucan synthase has greatly lowered levels in such media.
An alternative experiment to trace de novo fatty acid synthesis would be to track the
incorporation of [*^C]-acetate into cellular lipid, as utilized by Holdsworth and Ratledge (1988). This would remove the possible contributions of labelled glycerol to the lipids, allowing the level of fatty acid synthesis to be followed exclusively, although the uptake of acetate may be repressed by the presence of glucose in the medium.
3.4 Summary
The effect of exogenous lipids to an S. lividans shake flask fermentation is twofold.
Firstly, the growth of the organism is improved, with a faster rate of growth and a higher final biomass being seen in cultures that have a lipid supplement. This could be due to the lipid being a richer source of energy than simple sugars or amino acids, or because it
causes changes in the metabolism of the ceU. The studies carried out on de novo hpid
formation in the cell indicated that Hpid biosynthesis was restricted in cultures containing fatty acids. The improvement in growth could therefore be the result of the freeing of energy and raw materials from the fatty acid synthetic pathway into other processes in the
cell. Since the primary raw materials used in fatty acid synthesis are acetyl-CoA
molecules, it is possible these are simply re-routed into the biosynthetic pathway of antibiotics, where they are also the main constituent. An increase in the level of antibiotic
synthesis is the second effect of Hpids on the S. lividans fermentation. The addition of
exogenous Hpid causes the production of actinorhodin where otherwise there would be none. This could be from a simple relationship, where actinorhodin production increases
with higher acetyl-CoA levels, caused by the breakdown of fatty acids. A second
possibiHty is that the fatty acids trigger actinorhodin production by mfluencmg the expression of the biosynthetic genes, directly, or by interacting with an existing control mechanism. This is less likely, since such a mechanism would be of Httle use to the ceU.
However, given the compHcated nature of actinorhodin expression in S. lividans (Section
C h a p te r 4. E n zym o lo g y o f lip id d e g ra d a tio n in S. lividans
4. THE ENZYMOLOGY OF LIPID DEGRADATION IN STREPTOM YCES