Enhancement of FK506 production by engineering secondary pathways of Streptomyces tsukubaensis and exogenous feeding strategies

Abstract

FK506 is a clinically important macrocyclic polyketide with immunosuppressive activity produced by Streptomyces tsukubaensis. However, the low titer at which it is produced is a bottleneck to its application and use in industrial processes. We have overexpressed five potential targets associated with FK506 production (fkbO, fkbL, fkbP, fkbM, fkbD) which were identified in our previous study, with the aim to improve FK506 production. The results of the analysis showed that the constructed strains with an additional copy of each gene increased FK506 production by approximately 10-40 % compared with the wild-type strain D852. The results of the gene expression analysis indicated that each gene was upregulated. Combinatorial overexpression of the five genes resulted in a 146 % increase in the FK506 titer to 353.2 mg/L, in comparison with the titer produced by D852. To further improve the production of FK506 by the engineered strain HT-FKBOPLMD, we supplemented the medium with various nutrients, including soybean oil, lactate, succinate, shikimate, chorismate, lysine, pipecolate, isoleucine and valine. Optimization of feeding concentrations and times resulted in HT-FKBOPLMD being able to produce approximately 70 % more FK506, thereby reaching the maximal titer of 457.5 mg/L, with lower amounts of by-products (FK520 and 37,38-dihydro-FK506). These results demonstrate that the combination of the metabolically engineered secondary pathways and the exogenous feeding strategies developed here was able to be successfully applied to improve the production of industrially and clinically important compounds.

Biosynthetic Modularity Rules in the Bisintercalator Family of Antitumor Compounds

“…Homologues to this enzyme have been also described in FK-506 (FkbL, Streptomyces sp. kMA6548 ), virginiamycin S (VisC, Streptomyces virginiae ) and tubulysin (TubZ, Angiococcus disciformis ) pathways [56,57,58]. Sandramycin and quinaldopeptin gene clusters, must therefore contain rapL homologues, as these bisintercalators contain l -pipecolic acid.…”

Enhanced production of individual ganoderic acids by integrating Vitreoscilla haemoglobin expression and calcium ion induction in liquid static cultures of Ganoderma lingzhi

“…Similar results have also been reported for Streptomyces graminearus , in which combined gene cluster engineering and glycine feeding increase gougerotin production by 2.5‐, 1.25‐,and 1.3‐fold higher compared to the WT strain, the engineered strain and the WT strain by glycine feeding respectively (Jiang et al ., ). Exogenous feeding of nutrients to cultures of the engineered S. tsukubaensis leads to 3.2‐, 1.3‐ and 2.6‐fold improvement in FK506 production compared with those in the WT strain, the engineered strain and the WT strain fed with nutrients respectively (Huang et al ., ).…”

Integrating multi-omics analyses of Nonomuraea dietziae to reveal the role of soybean oil in [(4′-OH)MeLeu]4-CsA overproduction

“…Since soybean oil is a natural lipid mixture and can be hydrolyzed into fatty acids and glycerol, both of which will subsequently participate in the metabolic system [19, 22]. Considering the potential association between the lipid metabolism and the soybean oil, series of feeding experiments were designed and carried out, including the main hydrolysates of soybean oil, such as oleic acid, linoleic acid and glycerol, as well as the soybean oil-like compounds, such as corn oil (0.1%, w/v), peanut oil (0.1%, w/v) and Tween 80 (polyoxyethylene sorbitan monooleate, 0.05%, w/v).…”

“…p6621 fermentation [19]. Additionally, soybean oil could induce the expression of lipase to produce CoA-esters, the precursors of FK506 by S. tsukubaensis [20, 22]. These three products have a similarity that they are produced by the de novo synthesis pathway.…”