The influence of sigma factors and ribosomal recognition elements on heterologous expression of cyanobacterial gene clusters in Escherichia coli

Abstract

Cyanobacterial natural products offer new possibilities for drugs and lead compounds but many factors can inhibit the production of sufficient yields for pharmaceutical processes. While Escherichia coli and Streptomyces sp. have been used as heterologous expression hosts to produce cyanobacterial natural products, they have not met with resounding success largely due to their inability to recognize cyanobacterial promoter regions. Recent work has shown that the filamentous freshwater cyanobacterium Anabaena sp. strain PCC 7120 recognizes various cyanobacterial promoter regions and can produce lyngbyatoxin A from the native promoter. Introduction of Anabaena sigma factors into E. coli might allow the native transcriptional machinery to recognize cyanobacterial promoters. Here, all 12 Anabaena sigma factors were expressed in E. coli and subsets were found to initiate transcription from several cyanobacterial promoters based on transcriptional fusions to the chloramphenicol acetyltransferase (CAT) reporter. Expression of individual Anabaena sigma factors in E. coli did not result in lyngbyatoxin A production from its native cyanobacterial gene cluster, possibly hindered by deficiencies in recognition of cyanobacterial ribosomal binding sites by native E. coli translational machinery. This represents an important step toward engineering E. coli into a general heterologous expression host for cyanobacterial biosynthetic gene cluster expression.

A Tripartite, Hierarchical Sigma Factor Cascade Promotes Hormogonium Development in the Filamentous Cyanobacterium Nostoc punctiforme

“…While the currently available data cannot differentiate between a posttranscriptional and posttranslational mechanism, posttranslational regulation of sigma factors, often by anti-sigma factors and/or regulated proteolysis, is common (33). A recent effort at heterologous expression of the full complement of Nostoc 7120 sigma factors in Escherichia coli was unsuccessful in the case of SigJ (34), possibly indicating inherent instability in the absence of some additional factor(s). This lends support to the theory that SigJ may be subject to regulated proteolysis.…”

Transcriptional factor engineering in microbes for industrial biotechnology

“…Fortunately, when 12 Anabaena sigma factors were expressed in E. coli, they were capable of initiating transcription under several cyanobacterial promoters. 32 Unlike the utilization of mutant endogenous RpoD that retains only mutants with the desired properties during the highthroughput screening (HTS), the application of compatible TF is slightly like a game of luck. However, some common considerations should be kept in mind to increase the possibility of success.…”

Emerging Species and Genome Editing Tools: Future Prospects in Cyanobacterial Synthetic Biology

“…Nostoc PCC 7120 contains twelve sigma factors that regulate gene expression according to environmental conditions [154]. In a recent study, Wells et al [155] tested the sigma factors from Nostoc PCC 7120 in E. coli and observed that several cyanobacterial promoters were able to drive transcription in E. coli only when sigma factors from Nostoc PCC 7120 were co-expressed. Similarly, sigma factors from Bacillus subtilis can be co-expressed in E. coli to construct an orthogonal expression system [156].…”