Improvement of chloramphenicol production in Streptomyces venezuelae ATCC 10712 by overexpression of the aroB and aroK genes catalysing steps in the shikimate pathway

Abstract

Streptomyces venezuelae ATCC 10712 produces chloramphenicol in small amounts. To enhance chloramphenicol production, two genes, aroB and aroK, encoding rate-limiting enzymes of the shikimate pathway were overexpressed using the expression vector pIJ86 under the control of the strong constitutive ermE* promoter. The recombinant strains, S. venezuelae/pIJ86-aroB and S. venezuelae/pIJ86-aroK, produced 2.5- and 4.3-fold greater amounts respectively of chloramphenicol than wild type at early stationary phase of growth. High transcriptional levels of aroB and aroK genes were detected at the early exponential growth of both recombinant strains and consistent with the enhanced expression of pabB gene encoding an early enzyme in chloramphenicol biosynthesis. The results suggested that the increment of carbon flux was directed towards intermediates in the shikimate pathway required for the production of chorismic acid, and consequently resulted in the enhancement of chloramphenicol production. This work is the first report of a convenient genetic approach to manipulate primary metabolite genes in S. venezuelae in order to increase chloramphenicol production.

Modulation of salt tolerance in Thai jasmine rice (Oryza sativa L. cv. KDML105) by Streptomyces venezuelae ATCC 10712 expressing ACC deaminase

“…In addition, acdS expression under the ermE promoter of multi-copy plasmid pIJ86 in S . venezuelae without antibiotic selection was maintained up to 5 generations, consistent with the previous report 23 . Interestingly, S .…”

A comparative study at bioprocess and metabolite levels of superhost strain Streptomyces coelicolor M1152 and its derivative M1581 heterologously expressing chloramphenicol biosynthetic gene cluster

“…The residual L-glutamate in the fermentation broth was analyzed using LC-MS/MS following the method of intracellular amino acid analysis (described later), however using a shorter C18 column (1.7 µm, 2.1 × 50 mm) to reduce the analysis run time. 13 C L-glutamic acid (CIL, Inc.) was used as F I G U R E 1 Schematic representation of precursor and key intermediates involved in chloramphenicol, streptomycin, vancomycin, nystatin, and actinorhodin biosynthesis pathways (He et al, 2001;Vitayakritsirikul et al, 2016). Primary metabolic pathways are shown with blue arrows and gray text while secondary metabolic pathways are shown with green arrows and text.…”

“…Chlm biosynthesis utilizes the shikimate pathway by incorporating the primary metabolites phosphoenolpyruvate (PEP) from the glycolytic pathway and erythrose-4-phosphate (E4P) from the pentose phosphate pathway (PPP). Shikimate pathway produces chorismic acid as one of the intermediates, which acts as a precursor compound for the biosynthesis of aromatic amino acids (phenylalanine, tyrosine, and tryptophan) and the production of 4-amino-4deoxychorismate (ADC) (He et al, 2001;Vitayakritsirikul et al, 2016).…”

Genetic engineering approaches for the fermentative production of phenylglycines

“…This is a pivotal target for further studies as usually the aromatic amino acid biosynthesis pathway in bacteria is strictly regulated and limits the precursor supply (Huccetogullari et al 2019;Lee and Wendisch 2017;Rodriguez et al 2014;Sprenger 2007). It was shown for Streptomyces venezuelae that an improved flux through the shikimate pathway by overexpressing the genes of shikimate kinase (aroK) and dehydroquinate synthase (aroB) increased the production of the aromatic antibiotic chloramphenicol (Vitayakritsirikul et al 2016). Overexpression of the gene chorismate synthase (aroC) in Streptomyces tsukubaensis improved the production of the immunosuppressant tacrolimus (Wang et al 2017).…”