Prokaryotic gene clusters: a rich toolbox for synthetic biology

Abstract

Bacteria construct elaborate nanostructures, obtain nutrients and energy from diverse sources, synthesize complex molecules, and implement signal processing to react to their environment. These complex phenotypes require the coordinated action of multiple genes, which are often encoded in a contiguous region of the genome, referred to as a gene cluster. Gene clusters sometimes contain all of the genes necessary and sufficient for a particular function. As an evolutionary mechanism, gene clusters facilitate the horizontal transfer of the complete function between species. Here, we review recent work on a number of clusters whose functions are relevant to biotechnology. Engineering these clusters has been hindered by their regulatory complexity, the need to balance the expression of many genes, and a lack of tools to design and manipulate DNA at this scale. Advances in synthetic biology will enable the large-scale bottom-up engineering of the clusters to optimize their functions, wake up cryptic clusters, or to transfer them between organisms. Understanding and manipulating gene clusters will move towards an era of genome engineering, where multiple functions can be "mixed-and-matched" to create a designer organism.

Figure 1. Gene clusters encode organelles and molecular machines

A schematic (left) and image (right) is shown for each system that appears in this review. (clockwise from top left) A reconstruction of the cryo-EM structure of the Salmonella type III secretion system is shown along with an image of multiple needle complexes spanning the inner and outer membranes [125, 126]. A schematic of the C. thermocellum cellulosome is shown with their surface localization [41]. The crystal structure of the R. sphaerodes Reaction center and LH1 (center) and LH2 (outer eight rings) are shown next to a high-resolution AFM of the photosynthetic membrane of Rsp. photometricum (inset) [127]. A cartoon of the type I pili from G. sulfurreducens (Fe³⁺ oxide is shown localized at the tip) [128] is compared to a SEM of the pilus-like appendages from S. oneidensis MR-1 [129]. The cryo-EM structure of the B. subtilis stressosome and their localization using RsbR-specific antibodies are shown [105]. An idealized {100}+{111} Fe₃O₄ crystal is shown with an image of a magnetosome chain [36]. An electron micrograph of a gas vesicle from Hfx. Mediterranei [29] and the packing of multiple vesicles in Microcystis sp. [30] are shown. Carboxysomes are shown from Synechocystis sp. PCC 6803 along with the pathway for carbon dioxide fixation [23]. All images reproduced with permission.

Figure 2. The gene clusters described in this review are compared

The colors of the genes loosely classify their functions. Many genes contain multiple functions. A classification of being “structural” includes genes genes that associate with a large complex and are necessary for function; for example ATPases in type I pili and type III secretion.

Figure 3. Utilization and breakdown pathways encoded in gene clusters are shown

The alkane degradation pathway from P. putida is adapted from Witholt and co-workers [51]. Nitrogenase is shown along with the pathway for the production of FeMoCo [57]. All images reproduced with permission.

Figure 4. Chemical production pathways are often encoded within gene clusters

The image is of an organelle containting 10-100 associated 2.5 megadalton NRPS-PKS megacomplexes from B. subtilis [130]. The erythromycin pathway is shown from Saccharopolyspora erythraea NRRL 2338 and echinomycin pathway from Streptomyces lasaliensis. For erythromycin, chemical groups added by post-assembly-line tailoring enzymes (two P450s and two glycosyltransferases) are shown in red. Abbreviations: A = adenylation, T = thiolation, C = condensation, E = epimerization, MT = methyltransferase, TE = thioesterase, AT = acyltransferase, KS = ketosynthase, KR = ketoreductase, DH = dehydratase, ER = enoylreductase. Bacteriochlorophyll (bottom left) is incorporated into light harvesting complexes. FeMoCo (bottom right) is produced by a metabolic pathway (Figure 4) and incorporated into nitrogenase (image from [131]). All images reproduced with permission.

Figure 5. Complex regulatory pathways can be encoded by gene clusters

The signaling network formed by the σ_B gene cluster is shown [106]. Environmental stress is received by the stressosome, whereas energy stress is sensed by a different branch of the pathway. Quorum sensing pathways from Pseudomonas aeruginosa [119] are shown with their synthetic use to build pattern-forming programs in E. coli [122]. All images reproduced with permission. The CRISPR image adapted from one drawn by James Atmos.