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

Abstract

Background Actinoplanes sp. SE50/110 is the natural producer of the diabetes mellitus drug acarbose, which is highly produced during the growth phase and ceases during the stationary phase. In previous works, the growth-dependency of acarbose formation was assumed to be caused by a decreasing transcription of the acarbose biosynthesis genes during transition and stationary growth phase. Results In this study, transcriptomic data using RNA-seq and state-of-the-art proteomic data from seven time points of controlled bioreactor cultivations were used to analyze expression dynamics during growth of Actinoplanes sp. SE50/110. A hierarchical cluster analysis revealed co-regulated genes, which display similar transcription dynamics over the cultivation time. Aside from an expected metabolic switch from primary to secondary metabolism during transition phase, we observed a continuously decreasing transcript abundance of all acarbose biosynthetic genes from the early growth phase until stationary phase, with the strongest decrease for the monocistronically transcribed genes acbA, acbB, acbD and acbE. Our data confirm a similar trend for acb gene transcription and acarbose formation rate. Surprisingly, the proteome dynamics does not follow the respective transcription for all acb genes. This suggests different protein stabilities or post-transcriptional regulation of the Acb proteins, which in turn could indicate bottlenecks in the acarbose biosynthesis. Furthermore, several genes are co-expressed with the acb gene cluster over the course of the cultivation, including eleven transcriptional regulators (e.g. ACSP50_0424), two sigma factors (ACSP50_0644, ACSP50_6006) and further genes, which have not previously been in focus of acarbose research in Actinoplanes sp. SE50/110. Conclusion In conclusion, we have demonstrated, that a genome wide transcriptome and proteome analysis in a high temporal resolution is well suited to study the acarbose biosynthesis and the transcriptional and post-transcriptional regulation thereof.

Enhancement of acarbose production by genetic engineering and fed-batch fermentation strategy in Actinoplanes sp. SIPI12-34

“…With the fed-batch strategy, the engineered strain SIPI2207 made a breakthrough in acarbose yield and reached the highest titer ever reported (Table 1 ). Julian Droste found that the transcription level of acarbose synthesis genes showed a continuously decreasing trend from the early growth stage to the stationary stage, and acarbose was highly produced during the early growth stage and its production ceased during the stationary stage [ 45 ]. This finding also gave us some guidance by extending the growth phase can be an effective strategy for enhancing the production of acarbose.…”

Sigma Factor Engineering in Actinoplanes sp. SE50/110: Expression of the Alternative Sigma Factor Gene ACSP50_0507 (σHAs) Enhances Acarbose Yield and Alters Cell Morphology

“…Actinoplanes sp. SE50/110 alternative sigma factor gene ACSP50_0507 is strongly transcribed during the transition and early stationary growth phase, with a maximal transcript amount at the early stationary growth phase and therefore possibly influencing the cells during growth phase transition [ 12 ]. This σ B/F/G family protein (IPRO014322) of 270 amino acids features RNA-polymerase binding and −10 promoter recognition region 2 (IPRO07027), RNA-polymerase and extended-10 binding region 3 (IPRO07624) and −35 promoter element recognition region 4 (cd06171).…”

“…Within the acb gene cluster, regulatory elements are scarcely known. Determination of transcription start sites (TSSs) and operon structures [ 11 ] as well as transcription dynamics and protein abundancies [ 12 ] were major steps for the broader understanding of acb gene regulation. In prior works, the MalR type regulator AcrC was identified to bind within the acb gene cluster in the intergenic region between the genes acbE and acbD , but gene deletion did not result in an increased acarbose yield [ 13 ].…”

The 4-α-Glucanotransferase AcbQ Is Involved in Acarbose Modification in Actinoplanes sp. SE50/110

“…AcbQ is one of the uncharacterized enzymes of Actinoplanes sp. SE50/110 and encoded within the acarbose biosynthesis gene cluster [22,30]. BLASTP analysis has shown similarities to MalQ proteins from other strains but also from its own Actinoplanes strain (ACSP50_7587, 42% identity, 56% similarity) [14].…”

“…Proteome analysis has shown that AcbQ features one of the highest protein stabilities during acarbose production compared to other Acb proteins. Its stability is post-translationally influenced, suggesting a high relevance in acarbose metabolism [22]. At a structural level, AcbQ has the highest similarity to MalQ from Haemophilus influenzae, which is a 4-α-glucanotransferase (EC 2.4.1.25) and belongs to the glycosyltransferase family [14,15].…”