Engineered, highly productive biosynthesis of artificial, lactonized statin side-chain building blocks: The hidden potential of Escherichia coli unleashed.

We have recently reported the development of an efficient, whole-cell process for chemoenzymatic production of key chiral intermediates in statin synthesis by employing high-density Escherichia coli culture with the overexpressed deoxyribose-5-phosphate aldolase (DERA). The optically pure, 6-substituted cyclic hemiacetals can be used for the synthesis of atorvastatin, rosuvastatin and pitavastatin using further chemical steps. All of the synthetic routes established to date begin with a regiospecific oxidation of these lactol intermediates into the corresponding lactones, followed by several steps yielding 6-substituted, open-chain or lactonized derivatives which can be coupled by various approaches with the heterocyclic part of the statin molecule. Here we report for the first time the use of PQQ-dependent glucose dehydrogenases for a highly efficient, regioselective oxidation of artificial, derivatized aldohexoses, more specifically, the statin lactol intermediates. First, PQQ-dependent dehydrogenases of both soluble and membrane-bound type were characterized for their activity toward various DERA-derived lactols. Further, we describe a highly productive whole-cell system for oxidation of these 2,4-dideoxyaldopyranoses using a PQQ-dependent glucose dehydrogenase (Gcd) overexpressed in E. coli while taking advantage of the respiratory chain as the mediator of the electron transfer to oxygen. Finally, a two-step artificial biosynthetic pathway was developed by unleashing the intrinsic genetic potential of E. coli. The combined overexpression of the endogenous DERA and the membrane-bound, PQQ-dependent glucose dehydrogenase, the latter being coupled to the respiratory chain, allows direct biosynthesis of 6-substituted lactones in a highly productive, high-yield, cost-effective and industrially scalable process.

A highly productive, whole-cell DERA chemoenzymatic process for production of key lactonized side-chain intermediates in statin synthesis.

Employing DERA (2-deoxyribose-5-phosphate aldolase), we developed the first whole-cell biotransformation process for production of chiral lactol intermediates useful for synthesis of optically pure super-statins such as rosuvastatin and pitavastatin. Herein, we report the development of a fed-batch, high-density fermentation with Escherichia coli BL21 (DE3) overexpressing the native E. coli deoC gene. High activity of this biomass allows direct utilization of the fermentation broth as a whole-cell DERA biocatalyst. We further show a highly productive bioconversion processes with this biocatalyst for conversion of 2-substituted acetaldehydes to the corresponding lactols. The process is evaluated in detail for conversion of acetyloxy-acetaldehyde with the first insight into the dynamics of reaction intermediates, side products and enzyme activity, allowing optimization of the feeding strategy of the aldehyde substrates for improved productivities, yields and purities. The resulting process for production of ((2S,4R)-4,6-dihydroxytetrahydro-2H-pyran-2-yl)methyl acetate (acetyloxymethylene-lactol) has a volumetric productivity exceeding 40 g L(-1) h(-1) (up to 50 g L(-1) h(-1)) with >80% yield and >80% chromatographic purity with titers reaching 100 g L(-1). Stereochemical selectivity of DERA allows excellent enantiomeric purities (ee >99.9%), which were demonstrated on downstream advanced intermediates. The presented process is highly cost effective and environmentally friendly. To our knowledge, this is the first asymmetric aldol condensation process achieved with whole-cell DERA catalysis and it simplifies and extends previously developed DERA-catalyzed approaches based on the isolated enzyme. Finally, applicability of the presented process is demonstrated by efficient preparation of a key lactol precursor, which fits directly into the lactone pathway to optically pure super-statins.

FK506 biosynthesis is regulated by two positive regulatory elements in Streptomyces tsukubaensis.

BACKGROUND: FK506 (Tacrolimus) is an important immunosuppressant, produced by industrial biosynthetic processes using various Streptomyces species. Considering the complex structure of FK506, it is reasonable to expect complex regulatory networks controlling its biosynthesis. Regulatory elements, present in gene clusters can have a profound influence on the final yield of target product and can play an important role in development of industrial bioprocesses. RESULTS: Three putative regulatory elements, namely fkbR, belonging to the LysR-type family, fkbN, a large ATP-binding regulator of the LuxR family (LAL-type) and allN, a homologue of AsnC family regulatory proteins, were identified in the FK506 gene cluster from Streptomyces tsukubaensis NRRL 18488, a progenitor of industrial strains used for production of FK506. Inactivation of fkbN caused a complete disruption of FK506 biosynthesis, while inactivation of fkbR resulted in about 80% reduction of FK506 yield. No functional role in the regulation of the FK506 gene cluster has been observed for the allN gene. Using RT-PCR and a reporter system based on a chalcone synthase rppA, we demonstrated, that in the wild type as well as in fkbN- and fkbR-inactivated strains, fkbR is transcribed in all stages of cultivation, even before the onset of FK506 production, whereas fkbN expression is initiated approximately with the initiation of FK506 production. Surprisingly, inactivation of fkbN (or fkbR) does not abolish the transcription of the genes in the FK506 gene cluster in general, but may reduce expression of some of the tested biosynthetic genes. Finally, introduction of a second copy of the fkbR or fkbN genes under the control of the strong ermE* promoter into the wild type strain resulted in 30% and 55% of yield improvement, respectively. CONCLUSIONS: Our results clearly demonstrate the positive regulatory role of fkbR and fkbN genes in FK506 biosynthesis in S. tsukubaensis NRRL 18488. We have shown that regulatory mechanisms can differ substantially from other, even apparently closely similar FK506-producing strains, reported in literature. Finally, we have demonstrated the potential of these genetically modified strains of S. tsukubaensis for improving the yield of fermentative processes for production of FK506.

Novel chemobiosynthetic approach for exclusive production of FK506.

FK506, a widely used immunosuppressant, is produced by industrial fermentation processes using various Streptomyces species. Independently of the strain, structurally related compound FK520 is co-produced, resulting in complex and costly isolation procedures. In this paper, we report a chemobiosynthetic approach for exclusive biosynthesis of FK506. This approach is based on the Streptomyces tsukubaensis strain with inactivated allR gene, a homologue of crotonyl-CoA carboxylase/reductase, encoded in the FK506 biosynthetic cluster. This strain produces neither FK506 nor FK520; however, if allylmalonyl-S-N-acetylcysteamine precursor is added to cultivation broth, the production of FK506 is reestablished without FK506-related by-products. Using a combination of metabolic engineering and chemobiosynthetic approach, we achieved exclusive production of FK506, representing a significant step towards development of an advanced industrial bioprocess.

Origin of the allyl group in FK506 biosynthesis.

FK506 (tacrolimus) is a secondary metabolite with a potent immunosuppressive activity, currently registered for use as immunosuppressant after organ transplantation. FK506 and FK520 are biogenetically related natural products that are synthesized by combined polyketide synthase/nonribosomal peptide synthetase systems. The entire gene cluster for biosynthesis of FK520 from Streptomyces hygroscopicus var. ascomyceticus has been cloned and sequenced. On the other hand, the FK506 gene cluster from Streptomyces sp. MA6548 (ATCC55098) was sequenced only partially, and it was reasonable to expect that additional genes would be required for the provision of substrate supply. Here we report the identification of a previously unknown region of the FK506 gene cluster from Streptomyces tsukubaensis NRRL 18488 containing genes encoding the provision of unusual building blocks for FK506 biosynthesis as well as a regulatory gene. Among others, we identified a group of genes encoding biosynthesis of the extender unit that forms the allyl group at carbon 21 of FK506. Interestingly, we have identified a small independent diketide synthase system involved in the biosynthesis of the allyl group. Inactivation of one of these genes, encoding an unusual ketosynthase domain, resulted in an FK506 nonproducing strain, and the production was restored when a synthetic analog of the allylmalonyl-CoA extender unit was added to the cultivation medium. Based on our results, we propose a biosynthetic pathway for the provision of an unusual five-carbon extender unit, which is carried out by a novel diketide synthase complex.