title: Roles of the crotonyl-CoA carboxylase/reductase homologues in acetate assimilation and biosynthesis of immunosuppressant FK506 in Streptomyces tsukubaensis.
abstract: BACKGROUND
  In 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.
  
  
  RESULTS
  In 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.
  
  
  CONCLUSIONS
  Different 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.
authors.affiliation: Department of Food Science and Technology, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 1000, Ljubljana, Slovenia. marko.blazic@aciesbio.com.|Acies Bio, d.o.o., Tehnološki park 21, 1000, Ljubljana, Slovenia. marko.blazic@aciesbio.com.
authors.forename: Marko
authors.initials: M
authors.lastname: Blažič
authors.affiliation: Acies Bio, d.o.o., Tehnološki park 21, 1000, Ljubljana, Slovenia. gregor.kosec@aciesbio.com.
authors.forename: Gregor
authors.initials: G
authors.lastname: Kosec
authors.affiliation: Department of Biotechnology and Systems Biology, National Institute of Biology, Večna pot 111, 1000, Ljubljana, Slovenia. spela.baebler@nib.si.
authors.forename: Špela
authors.initials: Š
authors.lastname: Baebler
authors.affiliation: Department of Biotechnology and Systems Biology, National Institute of Biology, Večna pot 111, 1000, Ljubljana, Slovenia. kristina.gruden@nib.si.
authors.forename: Kristina
authors.initials: K
authors.lastname: Gruden
authors.affiliation: Department of Food Science and Technology, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 1000, Ljubljana, Slovenia. hrvoje.petkovic@bf.uni-lj.si.|Acies Bio, d.o.o., Tehnološki park 21, 1000, Ljubljana, Slovenia. hrvoje.petkovic@bf.uni-lj.si.
authors.forename: Hrvoje
authors.initials: H
authors.lastname: Petković
mesh_terms: D000085:Acetates / Q000378:metabolism; D000214:Acyl Coenzyme A / Q000235:genetics / Q000378:metabolism; D044944:Acyl-CoA Dehydrogenases / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D007166:Immunosuppressive Agents / Q000737:chemistry / Q000378:metabolism*; D005810:Multigene Family; D011401:Promoter Regions, Genetic; D060888:Real-Time Polymerase Chain Reaction; D013302:Streptomyces / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016559:Tacrolimus / Q000737:chemistry / Q000378:metabolism*; D059467:Transcriptome
chemical_list: D000085:Acetates; D000214:Acyl Coenzyme A; D001426:Bacterial Proteins; D007166:Immunosuppressive Agents; C102829:ethylmalonyl-coenzyme A; D044944:Acyl-CoA Dehydrogenases; C100154:acyl-CoA dehydrogenase (NADP+); D016559:Tacrolimus