Optimization of the precursor supply for an enhanced FK506 production in Streptomyces tsukubaensis

Abstract

Tacrolimus (FK506) is a macrolide widely used as immunosuppressant to prevent transplant rejection. Synthetic production of FK506 is not efficient and costly, whereas the biosynthesis of FK506 is complex and the level produced by the wild type strain, Streptomyces tsukubaensis, is very low. We therefore engineered FK506 biosynthesis and the supply of the precursor L-lysine to generate strains with improved FK506 yield. To increase FK506 production, first the intracellular supply of the essential precursor lysine was improved in the native host S. tsukubaensis NRRL 18488 by engineering the lysine biosynthetic pathway. Therefore, a feedback deregulated aspartate kinase AskSt* of S. tsukubaensis was generated by site directed mutagenesis. Whereas overexpression of AskSt* resulted only in a 17% increase in FK506 yield, heterologous overexpression of a feedback deregulated AskCg* from Corynebacterium glutamicum was proven to be more efficient. Combined overexpression of AskCg* and DapASt, showed a strong enhancement of the intracellular lysine pool following increase in the yield by approximately 73% compared to the wild type. Lysine is coverted into the FK506 building block pipecolate by the lysine cyclodeaminase FkbL. Construction of a ∆fkbL mutant led to a complete abolishment of the FK506 production, confirming the indispensability of this enzyme for FK506 production. Chemical complementation of the ∆fkbL mutant by feeding pipecolic acid and genetic complementation with fkbL as well as with other lysine cyclodeaminase genes (pipAf, pipASt, originating from Actinoplanes friuliensis and Streptomyces pristinaespiralis, respectively) completely restored FK506 production. Subsequently, FK506 production was enchanced by heterologous overexpression of PipAf and PipASp in S. tsukubaensis. This resulted in a yield increase by 65% compared to the WT in the presence of PipAf from A. friuliensis. For further rational yield improvement, the crystal structure of PipAf from A. friuliensis was determined at 1.3 Å resolution with the cofactor NADH bound and at 1.4 Å with its substrate lysine. Based on the structure the Ile91 residue was replaced by Val91 in PipAf, which resulted in an overall increase of FK506 production by approx. 100% compared to the WT.

Genomic Insights and Synthetic Biology Applications of Marine Actinomycete Streptomyces griseoincarnatus HNS054

“…While overexpression of the aborycin BGC genes in HNS1451 did not lead to high aborycin production, we suspect that the supply of amino acids, especially noncanonical amino acids, might be a limiting factor. This aligns with the study by Schulz et al [ 28 ], who demonstrated a significant increase in FK506 production by optimizing pipecolate supply in S. tsukubaensis . Similarly, optimizing the noncanonical amino acid synthesis pathway in HNS1451 could potentially unlock further improvements in aborycin production.…”

Impact of Nitrogen-Containing Compounds on Secondary Metabolism in Streptomyces spp.—A Source of Metabolic Engineering Strategies

“…It had already been shown for all producers that the overexpression of the respective lysine cyclodeaminase genes resulted in an increase in the desired secondary metabolites. A positive correlation between increased lysine availability and increased secondary metabolite production was shown in cephamycin biosynthesis in S. clavuligerus [134] and tacrolimus (SK-506) biosynthesis in S. tsukubaensis [56] (Table 3).…”

“…One possibility is to induce BGC expression in a native Streptomyces host. An example of the application of this strategy is the titer optimization of KF-506 in S. tsukubaensis [56]. The advantage of this strategy is that such a host is genetically tractable and genetic manipulations of the host genome are possible, including the overexpression of regulatory genes and removal of competitive pathways with gene cluster knock-out, deactivation of negative regulatory genes, and replacement of native promoters with stronger promoters.…”

“…The combined overexpression of the feedback-deregulated aspartate kinase Ask Cg* and the dihydropicolinate synthase DapA St , showed a strong enhancement in the intracellular lysine pool in S. tsukubaensis and consequently increased the yield of FK-506 by approximately 73% compared with the wild type. Furthermore, FK506 production was enhanced by heterologous overexpression of the mutated version of lysine cyclodeaminase Pip Af [56] (Table 3).…”

Optimization of FK-506 production in Streptomyces tsukubaensis by modulation of Crp-mediated regulation

“…A lot of efforts have been reported for optimization of the biotechnological process of FK-506/FK-520 fermentation in S. tsukubaensis by employing different strategies including genetic approaches, strain improvement, media supplementation, metabolic and protein engineering, as well as combinational strategies (Xia et al 2013 ; Ban et al 2016 ; Wu et al 2021b ). Recently, a new optimization strategy has been presented that combines the enhancement of the precursor supply for L-lysine with the improvement of pipecolic acid formation in S. tsukubaensis (Schulz et al 2022 ). But the aspect of increasing the FK-506 yield by manipulation of regulatory steps at a global level remained uninvestigated, although the effects of phosphate and N-acetylglucosamine for FK-506 biosynthesis have been studied previously (Martín 2004 ; Martínez-Castro et al 2013 ; Ordóñez-Robles et al 2018 ).…”