Absence of the highly expressed small carbohydrate-binding protein Cgt improves the acarbose formation in Actinoplanes sp. SE50/110

Abstract

Actinoplanes sp. SE50/110 (ATCC 31044) is the wild type of industrial producer strains of acarbose. Acarbose has been used since the early 1990s as an inhibitor of intestinal human α-glucosidases in the medical treatment of type II diabetes mellitus. The small secreted protein Cgt, which consists of a single carbohydrate-binding module (CBM) 20-domain, was found to be highly expressed in Actinoplanes sp. SE50/110 in previous studies, but neither its function nor a possible role in the acarbose formation was explored, yet. Here, we demonstrated the starch-binding function of the Cgt protein in a binding assay. Transcription analysis showed that the cgt gene was strongly repressed in the presence of glucose or lactose. Due to this and its high abundance in the extracellular proteome of Actinoplanes, a functional role within the sugar metabolism or in the environmental stress protection was assumed. However, the gene deletion mutant Δcgt, constructed by CRISPR/Cas9 technology, displayed no apparent phenotype in screening experiments testing for pH and osmolality stress, limited carbon source starch, and the excess of seven different sugars in liquid culture and further 97 carbon sources in the Omnilog Phenotypic Microarray System of Biolog. Therefore, a protective function as a surface protein or a function within the retainment and the utilization of carbon sources could not be experimentally validated. Remarkably, enhanced production of acarbose was determined yielding into 8-16% higher product titers when grown in maltose-containing medium.

The expression of the acarbose biosynthesis gene cluster in Actinoplanes sp. SE50/110 is dependent on the growth phase

“…This effect is supposed to be caused by the reduced metabolic burden, since Cgt was identi ed to be one of the highest abundant proteins in Actinoplanes sp. SE50/110 [87][88][89]. The similar expression patterns shown in this study, support the suggestion, that by deletion of genes co-expressed with the acb gene cluster, the acarbose formation might be improved.…”

Application of Cas12j for Streptomyces Editing

“…Being almost half the size of the Cas9 family of proteins and less toxic as compared to SpCas9 or FnCas12a, we believe that the hypercompact AsCas12j-2 will be a valuable tool in the strategy to generate genomic perturbations in Streptomyces. Previous studies have also shown successful applications of various CRISPR-Cas strategies from Streptomyces to the rare Actinomycetes without modifications or the addition of helper proteins [27][28][29][30][31][32]. In the same manner, we predict that the usage of Cas12j-2 could also be applied across the Actinomycetes family.…”

The expression of the acarbose biosynthesis gene cluster in Actinoplanes sp. SE50/110 is dependent on the growth phase

“…The function of the gene product is unclear. Several functional analyses were carried out on the extracellular protein Cgt, but no enzymatic activity could be determined [82]. However, the deletion of cgt lead to an increase of acarbose formation in Actinoplanes sp.…”

“…However, the deletion of cgt lead to an increase of acarbose formation in Actinoplanes sp. SE50/110 [82]. This effect is supposed to be caused by the reduced metabolic burden, since Cgt was identi ed to be one of the highest abundant proteins in Actinoplanes sp.…”