Engineering the heterologous mevalonate pathway into E. coli exposed the host's metabolic network to a biochemical intermediate that the organism had not evolved to counteract. Given the complexity of the metabolic network and the extensive number of interactions that occur in the E. coli cytosol, it is not surprising that a deleterious interaction would arise. While it can be relatively simple to determine that an engineered synthetic biochemical pathway is not functioning in the heterologous host, it is often a far more challenging task to determine exactly what is causing the problem. Using microarray and metabolite analysis of just such a design problem, we have demonstrated that the growth inhibition associated with HMG-CoA accumulation in E. coli DP10 harboring pBAD33MevT is due to inhibition of one or more enzymes involved in the elongation or priming steps of FAB.
It has previously been reported that there is significant conversion of the acyl-CoA pool to malonyl-CoA in E. coli
when the initial steps of FAB are inhibited by chemicals, such as the antibiotics thiolactomycin (28
) and cerrulenin (17
), or genetic methods, such as disruption of protein function (58
) and mutation (36
). E. coli
coordinates FAB with lipid requirements (and hence growth), and the accumulation of long-chain acyl-ACPs is a key signal of slowing growth, which results in the coordinated down-regulation of fabB, fabA
, and accAB
) as well as the inhibition of enzymatic activity (27
). During this down-regulation of FAB, excess malonyl-CoA is recycled back to the acetyl-CoA pool through the actions of KAS1 (fabB
) and malonyl-CoA-ACP transacylase (fabD
). Thus, the impressive accumulation of malonyl-CoA observed in the mevalonate-producing cells is an indication that this complex regulation has been disrupted.
Supplementation of the growth medium with certain fatty acids abolished the HMG-CoA-induced growth inhibition in the active-pathway strain by removing the need for fatty acid anabolism and confirmed the hypothesis that FAB was inhibited, either directly or indirectly, by HMG-CoA accumulation. The results from the entire panel of fatty acid supplements offer insight as to where HMG-CoA may inhibit the FAB pathway. E. coli
requires both SFA and UFA to maintain proper membrane fluidity. Harder et al. observed that temperature-sensitive fabB
mutants were auxotrophic for trans
-UFA or a combination of SFA and cis
-UFA at the nonpermissive temperature (25
), while fabD
mutants grew at the nonpermissive temperature only when the medium was supplemented with 16:0 SFA or 18:1 UFA but not 16:1 UFA (26
). Additionally, when this fabD
mutant was grown in nonsupplemented medium at a temperature that only partially inhibited growth, the membrane lipids were enriched for 16:1 SFAs. Since the supplementation of UFA or a combination of SFA and UFA failed to relieve the HMG-CoA-associated toxicity, FabB does not appear to be the target of HMG-CoA inhibition in the FAB pathway. Instead, our results suggest that FabD was the target, since inhibited growth of the active-pathway strain in nonsupplemented medium enriched UFA and the addition of palmitic acid (16:0) (and, to a lesser extent, oleic acid [cis
-18:1]) markedly improved the growth of this strain.
FabD is the only enzyme in E. coli
that interacts with malonyl-CoA directly, and this occurs during the transfer of the malonyl moiety from CoA to ACP (10
). This reaction is critical to FAB since it provides the two-carbon units needed to extend fatty acids. The reaction mechanism is mediated by an active-site serine that attacks the thioester carbonyl and releases free CoA, leaving a malonyl-FabD complex. Next, the malonyl-FabD ester carbonyl is attacked by the phosphopantetheinyl thiol of ACP, which ultimately yields malonyl-ACP (47
). HMG-CoA is structurally very similar to malonyl-CoA and may interfere with substrate binding to the active site. Supporting this hypothesis is the evidence that another acyl-CoA, acetyl-CoA, has been shown to be a weak inhibitor of malonyl-CoA-ACP transacylase activity (38
The up-regulation of accBC, fabB, fabD
, and fabH
observed in the growth-inhibited, mevalonate-producing strain is consistent with the hypothesis that HMG-CoA inhibits FAB. This inhibition reduced the availability of long-chain acyl-ACPs, which was sensed by the heterologous host as a decoupling of the rates of FAB and growth. The resulting up-regulation of FAB genes was the host cell's attempt to maintain growth by up-regulating the flux of carbon into fatty acid anabolism. A similar transcriptional response to chemical inhibition of FAB has been documented for Mycobacterium tuberculosis
, where exposure to thiolactomycin elicited an up-regulation of the genes encoding β-ketoacyl-ACP synthase I (fabB
), malonyl-CoA-ACP transacylase (fabD
), and alkyl hydroperoxide reductase C (ahpC
), just as observed in the mevalonate-producing strain.
The up-regulation of FAB genes allowed the heterologous host to overcome the most deleterious effect of HMG-CoA, a complete cessation of growth, which is similar in principle to the resistance to thiolactomycin that E. coli
gains when fabB
is expressed on a multicopy plasmid (11
). Yet while the up-regulation of the FAB system was sufficient to allow slow growth in the presence of high levels of cytosolic HMG-CoA, there were consequences to the cell. Jackowski and Rock observed that inhibition of the initial steps of FAB increased the UFA/SFA ratio in the membrane (34
) because the inhibition reduced the availability of fatty acids, which, in turn, induced expression of the β-ketoacyl-ACP synthase I (fabB
). This enzyme has multiple roles in E. coli
's FAB pathway, including both SFA synthesis and the diversion of saturated long-chain acyl-ACPs into the desaturation pathway that produces UFA. Indeed, the increase in FAB gene expression observed in the HMG-CoA-inhibited cells resulted in a coordinate enrichment of UFA in the cell membrane and an overall decrease in fatty acids as a percentage of DCW.
The altered UFA/SFA ratio in the membrane that resulted from HMG-CoA inhibition of fatty acid anabolism in the mevalonate-producing strain could explain the cascade of osmotic, oxidative, and heat shock stress responses observed in that strain's transcriptional profile. Recent studies of the osmosensor ProP in E. coli
have discovered that membrane composition is a key signal governing the long-term response to alterations in medium osmolality (59
). The oxidative stress response in the active-pathway strain was also an indication of membrane-associated stress, since H2
is mostly a by-product of the respiratory electron transport chain in E. coli
), and both degradation of membrane integrity and osmotic stress have been reported to induce transcription of H2
defense genes (14
). Finally, the coordinated heat shock response to H2
is well established (15
), and the sum of all these stress responses appeared ultimately to result in a down-regulation of the translational machinery in the mevalonate-producing strain. Thus, the HMG-CoA-induced changes in membrane composition resulted in a coordinated modulation of many stress response regulons, which may be the ultimate cause for the slow growth observed in HMG-CoA-stressed cells.
In summary, the model for the growth inhibition observed in E. coli
expressing the MevT operon is one where the cell's type II fatty acid anabolism was impeded, most likely by inhibition of FabD activity, which in turn limited the availability of long-chain acyl-ACPs. This reduced availability increased the transcription of genes in the initial steps of fatty acid synthesis, which overcame the HMG-CoA inhibition enough to allow slow growth. As a consequence of low levels of acyl-ACP and increased expression of FabB activity, however, the membrane lipids were enriched in UFA. This in turn altered the membrane structural properties and induced the transcription of genes associated with osmotic, oxidative, and heat shock stress. The sum of these responses ultimately limited the growth rate in the mevalonate-producing culture. It should be noted that a similar deleterious interaction does not appear to occur during accumulation of HMG-CoA in S. cerevisiae
), which has a nondisassociated, type I FAB pathway, and interestingly, the type II pathway antibiotic thiolactomycin is not active against the S. cerevisiae
type I FAB pathway. This report highlights the unexpected interactions that can occur when a novel, heterologous biochemical pathway is engineered into a host organism as well as the utility of a systems biology approach to the design problems that inevitably arise in metabolic engineering.