Seminar with Susan S. Golden and James W. Golden – University of Copenhagen

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Seminar with Susan S. Golden and James W. Golden

A Day in the Life of a Cyanobacterium:
integrating temporal and environmental information

Susan S. Golden, Division of Biological Sciences, University of California, San Diego, USA.

A circadian clock governs the transcription of essentially the entire genome in the cyanobacterium Synechococcus elongatus. This aspect of regulation has been studied vigorously, but much less is known about clock control of metabolism, or how the clock and environment work together to orchestrate cellular physiology in a diel cycle.

It has been demonstrated that a clock whose intrinsic period matches that of an external light-dark cycle provides a fitness advantage to the cyanobacterium, but the nature of that benefit is unknown. We are investigating the metabolic consequences for S. elongatus when it is grown with or without an intact clock, in continuous light or in a diel cycle, to determine how circadian timing contributes to fitness.

We found that wild-type cells are able to turn off nighttime metabolic pathways before dawn, enabling a switch from primary metabolism to the synthesis of more complex molecules early in the day. Mutations that lock the circadian oscillator in a specific temporal state that suppresses the master transcription factor RpaA, or that eliminate RpaA function, render cells unable to survive the dark in a diel cycle.

These mutants accumulate reactive oxygen species (ROS) during the day period, and cannot activate primary reductant-producing pathways at night. In an effort to identify all genes that are necessary to survive the night in this obligate photoautotroph, we created a bar-coded transposon library that enables the fitness contribution of every gene to be queried under particular growth conditions.

The initial outcome was a comprehensive picture of the set of genes that is essential for viability in S. elongatus, the first essential gene set for a photosynthetic organism. Mutants affected in growth under light-dark cycles emphasized the relationship between the clock and regulation of the night-time metabolic program.

Broad-host-range genetic tools for cyanobacteria and heterologous expression of natural products

James W. Golden, Division of Biological Sciences, University of California, San Diego, USA

Improved genetic tools for cyanobacteria provide new opportunities for scientific research and biotechnology. We have developed a set of broad host range standardized genetic parts and devices for genetic modification and engineering diverse cyanobacterial strains.

The parts and devices are carried in a library of donor vectors. These donor vectors contain origins of replication for E. coli, origins of transfer for conjugation, origins of replication and neutral sites for various cyanobacteria, antibiotic markers, expression cassettes with different promoters, and reporter cassettes.

This library of donor vectors allows the construction of modular shuttle vectors, designed for the component parts to be easily replaced or additional parts to be easily inserted. Different types of vectors can be assembled, including autonomously replicating vectors and integrating vectors for gene knockout and gene expression from the chromosome. Assembled modular vectors allowed a thorough characterization of different genetic parts and devices in several diverse cyanobacterial strains.

The system has been used for the characterization of mutant versions of the broad-host-range plasmid RSF1010, theophylline-inducible riboswitches, a set of constitutive promoters with different levels of activity, and vectors based on the S. elongatus plasmid pANS.

The genetic tool kit has been used for the heterologous expression of several genes, including a 20-kb gene cluster required for the biosynthesis of the polyunsaturated fatty acid EPA, a hs_bmp7 gene and hs_bmp7 to hs_bmp12 gene cluster involved in synthesis of polybrominated diphenyl ethers, and the dehydroascorbate reductase (DHAR) gene from Brassica juncea (BrDHAR).

The seminar is co-organized with DTU Biosustain.