Enhanced Biosynthesis of 2-Deoxy-scyllo-inosose in Metabolically Engineered Bacillus subtilis Recombinants.

Joo Hyun Lim,Hye Ji Kim,Hyun Ha Hwang,Yeo Joon Yoon,Hye Bin Son,Je Won Park,Jae Woo Lee,Eun Gyo Seo,Na Joon Lee

doi:10.3389/fmicb.2018.02333

Abstract

2-Deoxy-scyllo-inosose (DOI) has been a valuable starting natural product for the manufacture of pharmaceuticals or chemical engineering resources such as pyranose catechol. DOI synthase, which uses glucose-6-phosphate (Glc6P) as a substrate for DOI biosynthesis, is indispensably involved in the initial stage of the biosynthesis of 2-deoxystreptamine-containing aminoglycoside antibiotics including butirosin, gentamicin, kanamycin, and tobramycin. A number of metabolically engineered recombinant strains of Bacillus subtilis were constructed here; either one or both genes pgi and pgcA that encode Glc6p isomerase and phosphoglucomutase, respectively, was (or were) disrupted in the sugar metabolic pathway of the host. After that, three different DOI synthase–encoding genes, which were artificially synthesized according to the codon preference of the B. subtilis host, were separately introduced into the engineered recombinants. The expression of a natural btrC gene, encoding DOI synthase in butirosin-producing B. circulans, in the heterologous host B. subtilis (BSDOI-2) generated approximately 2.3 g/L DOI, whereas expression of an artificially codon-optimized tobC gene, derived from tobramycin-producing Streptomyces tenebrarius, into the recombinant of B. subtilis (BSDOI-15) in which both genes pgi and pgcA are disrupted significantly enhanced the DOI titer: up to 37.2 g/L. Fed-batch fermentation by the BSDOI-15 recombinant using glycerol and glucose as a dual carbon source yielded the highest DOI titer (38.0 g/L). The development of engineered microbial cell factories empowered through convergence of metabolic engineering and synthetic biology should enable mass production of DOI. Thus, strain BSDOI-15 will surely be a useful contributor to the industrial manufacturing of various kinds of DOI-based pharmaceuticals and fine chemicals.

Highlights

Pyranose compounds have been produced in the traditional petrochemical sector from petroleum as a raw material
When a btrC gene that encodes the DOI synthase from B. circulans was expressed in the B. subtilis host (BSDOI-02), an average of 2.3 g/L DOI was produced
When an artificial gene—genCopt synthesized on the basis of a previously described genC sequence template—was introduced into the host (BSDOI-03), DOI titer in the shake-flask fermentation broth remained at 0.8 g/L on average, showing a failure of the enhancement of DOI productivity through genCopt expression

Summary

Introduction

Pyranose compounds have been produced in the traditional petrochemical sector from petroleum as a raw material. 2-Deoxy-scyllo-inosose (DOI) synthase, which uses Glc6P as a substrate when catalyzing the synthesis of pyranose compound DOI was first discovered as BtrC in Bacillus circulans that produces 2-deoxystreptamine-containing aminoglycoside butirosins (Kudo et al, 1999). This enzyme participates in the biosynthetic steps necessary for the core 2-deoxystreptamine scaffold (Llewellyn and Spencer, 2006; Park et al, 2013), and its catalytic product DOI has been broadly utilized as a starting material or a precursor of agrochemicals and pharmaceuticals (Hansen and Frost, 2002). It was reported that by the concentration of the enzymatic reactants followed by a reaction with hydrogen iodide under mild acidic conditions, DOI can be converted even to catechol (Suzuki et al, 2013)

Methods

Results

Conclusion