Improved PKS Gene Expression With Strong Endogenous Promoter Resulted in Geldanamycin Yield Increase

Abstract

The type I polyketide geldanamycin is a potent anti-tumor reagent. Its biosynthesis includes three steps: the biosynthesis of precursors, such as 3-amino-5-hydroxybenzoic acid (AHBA), the polyketide synthase (PKS) chain extension, and the post-PKS modifications. According to the genomic and transcriptomic analysis, the PKS chain extension was deduced to be the rate-limiting step for geldanamycin production in Streptomyces hygroscopicus XM201. In order to improve the expression of PKS genes, a strong endogenous promoter 5063p was obtained based on the transcriptomic analysis and XylE enzymatic assay. By replacing the native PKS promoter gdmA1p with 5063p, the expression of the PKS genes during geldanamycin fermentation was increased by 4-141-folds, and the geldanamycin yield was increased by 39%. Interestingly, AHBA feeding experiment showed that the supply of AHBA in turn become a new rate-limiting factor for geldanamycin production. Further combined overexpression of the 6-gene AHBA biosynthetic cassette and PKS genes increased the yield of geldanamycin by 88%, from 773 mg L of the wild-type to 1450 mg L in the derived strain. Our results suggested that improved expression of all PKS genes in a particular biosynthetic gene cluster is important for the yield increase of the corresponding polyketide natural product.

A dynamic control strategy to produce riboflavin with lignocellulose hydrolysate in the thermophile Geobacillus thermoglucosidasius

“…Among them, the strain DSM2542-DCall-MSS exhibited highest ribo avin production. This has once again showed that tuning the expression levels of biosynthetic genes is critical to achieve the maximal biosynthetic capacity of a pathway [54][55][56] . The bunch of newly identi ed xylose-inducible and glucose-insensitive promoters with a broad range of strengths, provide expedient genetic tools for the further engineering of G. thermoglucosidasius DSM 2542.…”

A severe leakage of intermediates to shunt products in acarbose biosynthesis

“…The reverse transcription was performed using RevertAid TM H Minus First Strand cDNA Synthesis Kit (Thermo Fisher). The transcription of the related genes was monitored by quantitative real-time PCR using Maxima TM SYBR Green/ROX qPCR Maxter Mix (Thermo Fisher) 58 . The relative transcription values of the target genes were calculated by the 2 -ΔΔCt method 59 with the housekeeping gene hrdB (ACPL_1268) as an internal control.…”

Regulation of Geldanamycin Biosynthesis by Cluster-Situated Transcription Factors and the Master Regulator PhoP

“…Transcriptional studies of the geldanamycin polyketide synthase genes in S. hygroscopicus XM201 suggested that expression of the polyketide synthase genes was limiting for geldanamycin biosynthesis [21]. These authors replaced the native polyketide synthase promoter upstream of gdmA1 by a strong endogenous S. hygroscopicus XM201 promoter selected on the basis of its high transcriptional activity.…”

“…Then, in this overproducing strain, biosynthesis of the AHBA starter unit became limiting. Combined overexpression of the polyketide synthase gene and the amino shikimate gene cluster resulted in an increase of 88% in geldanamycin production [21].…”

“…The balance between utilization of nitrogen and carbon sources is very important in the biosynthesis of geldanamycin because of the involvement of the mC 7 N precursor unit that requires an amino group to form the AHBA starter unit. The availability of the genome sequence of both S. hygroscopicus XM-201 [21] and S. autolyticus CGMCC 0516 [23] provides new useful “omics” information to dissect the genome of these geldanamycin producing strains. Transcriptomic analysis of expression of different genes involved both in the biosynthesis of structural components of geldanamycin and in the control of the biosynthesis of this antitumor agent needs to be emphasized.…”