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

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.

SUBMITTER: Lim JH

PROVIDER: S-EPMC6170601 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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

Lim Joo Hyun JH Hwang Hyun Ha HH Lee Na Joon NJ Lee Jae Woo JW Seo Eun Gyo EG Son Hye Bin HB Kim Hye Ji HJ Yoon Yeo Joon YJ Park Je Won JW

Frontiers in microbiology 20180927

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 recom ...[more]

PMID: 30319595

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Project description:Saccharomyces cerevisiae is a versatile industrial host for chemical production and has been engineered to produce efficiently many valuable compounds. 2-Deoxy-scyllo-inosose (2-DOI) is an important precursor for the biosynthesis of 2-deoxystreptamine-containing aminoglycosides antibiotics and benzenoid metabolites. Bacterial and cyanobacterial strains have been metabolically engineered to generate 2-DOI; nevertheless, the production of 2-DOI using a yeast host has not been reported. Here, we have metabolically engineered a series of CEN.PK yeast strains to produce 2-DOI using a synthetically yeast codon-optimized btrC gene from Bacillus circulans. The expression of the 2-Deoxy-scyllo-inosose synthase (2-DOIS) gene was successfully achieved via an expression vector and through chromosomal integration at a high-expression locus. In addition, the production of 2-DOI was further investigated for the CEN.PK knockout strains of phosphoglucose isomerase (?pgi1), D-glucose-6-phosphate dehydrogenase (?zwf1) and a double mutant (?pgi1, ?zwf1) in a medium consisting of 2% fructose and 0.05% glucose as a carbon source. We have found that all the recombinant strains are capable of producing 2-DOI and reducing it into scyllo-quercitol and (-)-vibo-quercitol. Comparatively, the high production of 2-DOI and its analogs was observed for the recombinant CEN.PK-btrC carrying the multicopy btrC-expression vector. GC/MS analysis of culture filtrates of this strain showed 11 times higher response in EIC for the m/z 479 (methyloxime-tetra-TMS derivative of 2-DOI) than the YP-btrC recombinant that has only a single copy of btrC expression cassette integrated into the genomic DNA of the CEN.PK strain. The knockout strains namely ?pgi1-btrC and ?pgi1?zwf1-btrC, that are transformed with the btrC-expression plasmids, have inactive Pgi1 and produced only traces of the compounds. In contrast, ?zwf1-btrC recombinant which has intact pgi1 yielded relatively higher amount of the carbocyclic compounds. Additionally, 1H-NMR analysis of samples showed slow consumption of fructose and no accumulation of 2-DOI and the quercitols in the culture broth of the recombinant CEN.PK-btrC suggesting that S. cerevisiae is capable of assimilating 2-DOI.

Project description:BackgroundPhenylpropanoids are the precursors to a range of important plant metabolites such as the cell wall constituent lignin and the secondary metabolites belonging to the flavonoid/stilbene class of compounds. The latter class of plant natural products has been shown to function in a wide range of biological activities. During the last few years an increasing number of health benefits have been associated with these compounds. In particular, they demonstrate potent antioxidant activity and the ability to selectively inhibit certain tyrosine kinases. Biosynthesis of many medicinally important plant secondary metabolites, including stilbenes, is frequently not very well understood and under tight spatial and temporal control, limiting their availability from plant sources. As an alternative, we sought to develop an approach for the biosynthesis of diverse stilbenes by engineered recombinant microbial cells.ResultsA pathway for stilbene biosynthesis was constructed in Escherichia coli with 4-coumaroyl CoA ligase 1 4CL1) from Arabidopsis thaliana and stilbene synthase (STS) cloned from Arachis hypogaea. E. coli cultures expressing these enzymes together converted the phenylpropionic acid precursor 4-coumaric acid, added to the growth medium, to the stilbene resveratrol (>100 mg/L). Caffeic acid, added in the same way, resulted in the production of the expected dihydroxylated stilbene, piceatannol (>10 mg/L). Ferulic acid, however, was not converted to the expected stilbene product, isorhapontigenin. Substitution of 4CL1 with a homologous enzyme, 4CL4, with a preference for ferulic acid over 4-coumaric acid, had no effect on the conversion of ferulic acid. Accumulation of tri- and tetraketide lactones from ferulic acid, regardless of the CoA-ligase expressed in E. coli, suggests that STS cannot properly accommodate and fold the tetraketide intermediate to the corresponding stilbene structure.ConclusionPhenylpropionic acids, such as 4-coumaric acid and caffeic acid, can be efficiently converted to stilbene compounds by recombinant E. coli cells expressing plant biosynthetic genes. Optimization of precursor conversion and cyclization of the bulky ferulic acid precursor by host metabolic engineering and protein engineering may afford the synthesis of even more structurally diverse stilbene compounds.

Project description:UnlabelledThe 2-thiouridine (s(2)U) modification of the wobble position in glutamate, glutamine, and lysine tRNA molecules serves to stabilize the anticodon structure, improving ribosomal binding and overall efficiency of the translational process. Biosynthesis of s(2)U in Escherichia coli requires a cysteine desulfurase (IscS), a thiouridylase (MnmA), and five intermediate sulfur-relay enzymes (TusABCDE). The E. coli MnmA adenylates and subsequently thiolates tRNA to form the s(2)U modification. Bacillus subtilis lacks IscS and the intermediate sulfur relay proteins, yet its genome contains a cysteine desulfurase gene, yrvO, directly adjacent to mnmA. The genomic synteny of yrvO and mnmA combined with the absence of the Tus proteins indicated a potential functionality of these proteins in s(2)U formation. Here, we provide evidence that the B. subtilis YrvO and MnmA are sufficient for s(2)U biosynthesis. A conditional B. subtilis knockout strain showed that s(2)U abundance correlates with MnmA expression, and in vivo complementation studies in E. coli IscS- or MnmA-deficient strains revealed the competency of these proteins in s(2)U biosynthesis. In vitro experiments demonstrated s(2)U formation by YrvO and MnmA, and kinetic analysis established a partnership between the B. subtilis proteins that is contingent upon the presence of ATP. Furthermore, we observed that the slow-growth phenotype of E. coli ΔiscS and ΔmnmA strains associated with s(2)U depletion is recovered by B. subtilis yrvO and mnmA. These results support the proposal that the involvement of a devoted cysteine desulfurase, YrvO, in s(2)U synthesis bypasses the need for a complex biosynthetic pathway by direct sulfur transfer to MnmA.ImportanceThe 2-thiouridine (s(2)U) modification of the wobble position in glutamate, glutamine, and lysine tRNA is conserved in all three domains of life and stabilizes the anticodon structure, thus guaranteeing fidelity in translation. The biosynthesis of s(2)U in Escherichia coli requires seven proteins: the cysteine desulfurase IscS, the thiouridylase MnmA, and five intermediate sulfur-relay enzymes (TusABCDE). Bacillus subtilis and most Gram-positive bacteria lack a complete set of biosynthetic components. Interestingly, the mnmA coding sequence is located adjacent to yrvO, encoding a cysteine desulfurase. In this work, we provide evidence that the B. subtilis YrvO is able to transfer sulfur directly to MnmA. Both proteins are sufficient for s(2)U biosynthesis in a pathway independent of the one used in E. coli.

Dataset Information

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

Publications

Enhanced Biosynthesis of 2-Deoxy-<i>scyllo</i>-inosose in Metabolically Engineered <i>Bacillus subtilis</i> Recombinants.

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