Roles of the crotonyl-CoA carboxylase/reductase homologues in acetate assimilation and biosynthesis of immunosuppressant FK506 in Streptomyces tsukubaensis.

Marko Blažič,Kristina Gruden,Špela Baebler,Gregor Kosec,Hrvoje Petković

doi:10.1186/s12934-015-0352-z

Marko Blažič, Kristina Gruden + Show 3 more

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https://doi.org/10.1186/s12934-015-0352-z

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Abstract

BackgroundIn microorganisms lacking a functional glyoxylate cycle, acetate can be assimilated by alternative pathways of carbon metabolism such as the ethylmalonyl-CoA (EMC) pathway. Among the enzymes converting CoA-esters of the EMC pathway, there is a unique carboxylase that reductively carboxylates crotonyl-CoA, crotonyl-CoA carboxylase/reductase (Ccr). In addition to the EMC pathway, gene homologues of ccr can be found in secondary metabolite gene clusters that are involved in the provision of structurally diverse extender units used in the biosynthesis of polyketide natural products. The roles of multiple ccr homologues in the same genome and their potential interactions in primary and secondary metabolic pathways are poorly understood.ResultsIn the genome of S. tsukubaensis we have identified two ccr homologues; ccr1 is located in the putative ethylmalonyl-CoA (emc) operon and allR is located on the left fringe of the FK506 cluster. AllR provides an unusual extender unit allylmalonyl-CoA (ALL) for the biosynthesis of FK506 and potentially also ethylmalonyl-CoA for the related compound FK520. We have demonstrated that in S. tsukubaensis the ccr1 gene does not have a significant role in the biosynthesis of FK506 or FK520 when cultivated on carbohydrate-based media. However, when overexpressed under the control of a strong constitutive promoter, ccr1 can take part in the biosynthesis of ethylmalonyl-CoA and thereby FK520, but not FK506. In contrast, if ccr1 is inactivated, allR is not able to sustain a functional ethylmalonyl-CoA pathway (EMC) and cannot support growth on acetate as the sole carbon source, even when constitutively expressed in the chimeric emc operon. This is somewhat surprising considering that the same chimeric emc operon results in production of FK506 as well as FK520, consistent with the previously proposed relaxed specificity of AllR for C4 and C5 substrates.ConclusionsDifferent regulation of the expression of both ccr genes, ccr1 and allR, and their corresponding pathways EMC and ALL, respectively, in combination with the different enzymatic properties of the Ccr1 and AllR enzymes, determine an almost exclusive role of ccr1 in the EMC pathway in S. tsukubaensis, and an exclusive role of allR in the biosynthesis of FK506/FK520, thus separating the functional roles of these two genes between the primary and secondary metabolic pathways.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0352-z) contains supplementary material, which is available to authorized users.

Highlights

In microorganisms lacking a functional glyoxylate cycle, acetate can be assimilated by alternative pathways of carbon metabolism such as the ethylmalonyl-CoA (EMC) pathway
In the scope of this work, we have clearly demonstrated that the EMC pathway plays an important role in acetate assimilation in Streptomyces tsukubaensis NRRL 18488; it is not significantly expressed in standard carbohydrate-based growth media
Streptomyces tsukubaensis NRRL 18488 belongs to the group of microorganisms that contain the EMC pathway, as clearly demonstrated by genome analysis carried out in the scope of this work [12,13,14]

Summary

Introduction

In microorganisms lacking a functional glyoxylate cycle, acetate can be assimilated by alternative pathways of carbon metabolism such as the ethylmalonyl-CoA (EMC) pathway. In addition to the EMC pathway, gene homologues of ccr can be found in secondary metabolite gene clusters that are involved in the provision of structurally diverse extender units used in the biosynthesis of polyketide natural products. It has been demonstrated that in many secondary metabolite gene clusters ccr homologues are present and play essential roles in the biosynthesis of unusual extender units, such as ethylmalonyl-CoA. These homologues are involved in the reductive carboxylation of diverse 2-alkenoyl carboxylic acids, resulting in the formation of unusual alkylmalonyl thioester building blocks, used in the biosynthesis of the polyketide backbone (Fig. 1b), expanding the structural diversity of these biologically active metabolites [4]. While the pathway providing the allylmalonyl-CoA extender unit has been identified [5, 9], the biosynthetic origin of the ethylmalonyl-CoA extender unit in FK506-producing Streptomyces strains has

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