Keywords: Synthetic biology; BioBrick™; BglBrick; Corynebrick; Methylobrick; Bacillus; Corynebacteria; Ralstonia; Streptococcus, Methylobacterium; Cyanobacteria
However, as is evident from searching the Registry of Standard Biological Parts (http://parts.igem.org), the largest and most accessible online repository of biological parts, the vast majority of bacterial synthetic biology parts were developed for Escherichia coli. Furthermore, a comparison of synthetic biology-related literature pertaining to E. coli, Bacillus subtilis, Corynebacterium glutamicum, Streptomyces, or Cyanobacteria published between 2004 and 2015 shows that publications for E.coli eclipse the others by approximately 10-fold (Figure 1).
Although E. coli has proven to be a useful tool for many decades, other bacteria may prove beneficial depending on the desired goal. For example, through native metabolic pathways, Ralstonia eutropha is able to produce polyhydroxyalkanoates [6]. These molecules can be extracted and processed to produce biodegradable plastics. Alternatively, the holy grail of synthetic biology may be to use cyanobacteria such as Synechococcus or Synechocystis to converts CO2 directly to biofuels or biochemical commodities via photosynthesis. For example, Synechococcus elongatus PCC7942 has been used to produce 2,3-butanediol [7].
Recently, a handful of synthetic biology-focused platforms
Notably, this work showed that the two inducible promoters, PliaI and PxylA, could be induced using inducer concentrations spanning three orders of magnitude with a resulting dynamic response of 300- and 100-fold activity. For applications of synthetic biology where multi-gene pathways are constructed,
Bacteria |
Reference |
Brief description |
Bacillus subtilis |
[11] |
BioBrick™compatible plasmids and toolbox geared toward easy assembly of genetic constructs |
Corynebacterium glutamicum |
[14][13] |
BglBrick and BioBrick™-like systems geared towards easy assembly of genetic constructs |
Ralstonia eutropha |
[15], [16] |
Plasmids and toolboxes(does not match an assembly standard), genetic part characterization |
Streptococcus pneumoniae |
[18] |
BglBrick platform and genetic part characterization |
Rhodobacter sphaeroides |
[24] |
BioBrick™ compatible platform, genetic part characterization |
Methylobacterium extorquens |
[17] |
Methylobricks, BioBrick™-compatible, toolbox of promoters, genetic part characterization |
Synechocystis sp. strain PCC6803.
|
[20]–[22] |
BioBrick™ compatible platform and genetic part characterization |
Synechococcus sp. strain PCC 7002
|
[23] |
Toobox of genetic parts with characterization |
One highlight of Ravasi, et al. [14] was the use of their platform to investigate combinatorial assemblies of promoters and RBS. Using various pairs of these genetic control elements, they were able to modulate protein expression of the fluorophores eGFP and mCherry [14]. On the other hand, Kang et al. demonstrate the utility of their system by introducing a functional xylose utilization pathway in C. glutamicum through heterologous expression of E. coli xylose isomerase and xylulose kinase [13].
In particular, transcriptional and translational control elements were characterized. Chemically inducible promoter systems driven by the common inducers xylose, Isopropyl Β-D- 1-Thiogalactopyranoside (IPTG), and anhydrotetracycline were shown to be active, and operate over a range of concentrations. Of note, the promoter PlacUV5, which is induced by IPTG, was only active when the transformed plasmid expressed galactose permease (lacY). Furthermore, of three RBS that were evaluated, the consensus sequence for the E. coli RBS was shown to provide the strongest protein translation capability. Finally, an operon was designed to produce the hydrocarbons pentadecane and heptadecene [15]. By altering the combinations of parts used for constructing the operon, the authors were able to modulate the titer and ratio of hydrocarbons produced.
As part of their toolbox, the authors evaluated the translational abilities of three constitutive promoters. Interestingly, these promoters are endogenous to M. extorquens AM1. Each promoter conferred a different degree of gene expression ability, thereby allowing researchers to design assemblies with modulated protein output. As a demonstration of the application of their system, the Methylobrick platform and toolbox was used to construct an operon for production of mesaconate using glyoxylate as the feedstock. Additionally, the authors demonstrate that their platform and toolbox is functional in other alpha proteobacteria: Agrobacterium tumefaciens, Caulobacter crescentus, and Paracoccus denitrificans.
Within the toolbox, the authors characterized several promoters. A set of synthetic constitutive promoters was developed and shown to modulate expression levels of the luciferase reporter gene over three orders of magnitude. Additionally, a Zn2+-inducible promoter system was developed and shown to modulate expression levels of reporter over three orders of magnitude in a Zn2+ concentration dependent manner. Further development is needed to expand the functionality of this platform and toolbox; however components have been used to study the dynamics of antibiotic resistance in S. pneumoniae [19].
However, the major difficulty regarding genetic manipulation of PCC6803 has been the development of reliable and controllable promoter systems. Extending the work mentioned above, the Anderson collection (http://parts.igem.org/Promoters/Catalog/ Anderson), a library of synthetic constitutive promoters was investigated for PCC6803 [21]. This widely used promoter collection was shown to operate over a wide range in PCC6803. Furthermore, a separate library of synthetic promoters, inducible by anhydrotetracycline, has also shown the ability to regulate translation over a broad range [22]. Taken together, the platform and toolbox of genetic parts is a promising starting point for advancing PCC6803 synthetic biology applications.
A toolbox has also been developed for another model cyanobacterium, Synechococcus sp. PCC 7002 [23]. The goal of this toolbox was to provide tools that can reliably control gene expression. In particular, through a combination of truncation and random mutagenesis, the authors built two sets of orthogonal constitutive promoter libraries that could regulate translation over 3 orders of magnitude. One set was based on the promoter PcpcB, which originated from PCC6803, the other set was derived from the E. coli σ70 consensus sequence. Of note is that while the original PcpcB promoter was sensitive to light, the resulting derivatives were not.
A set of promoters that could be induced by IPTG was also developed [23]. By introducing two lac operators into the PcpcB derivatives and further optimizing the promoter sequence, the authors were able to construct a system that could regulate expression over a range of 2 orders of magnitude when induced using different concentrations of IPTG. Use of this system required introducing and optimizing expression of the lacI repressor. Finally, the authors demonstrate further ability to modulate protein expression through the design of a set of optimized RBS sequences.
Another well-studied photosynthetic bacterium is the purple, non-sulfur photoheterotrophic Rhodobacter sphaeroides. The first attempt to develop a standardized system was produced by Tikh, et al. [24] through the introduction of a BioBrick™ compatible expression system [24]. Through this work, a truncated version of the native promoter, Ppuf, was developed and shown to have similar activity to the full-length version. To demonstrate the effectiveness of the system, the membrane protein proteorhodopsin was expressed at levels comparable to those previously reported for highly optimized strains of E. coli. Although this platform is still in its infancy, it presents a starting point for further development of synthetic biology tools for R. sphaeroides.
- Knight T. Idempotent Vector Design for Standard Assembly of Biobricks 2003.
- Vick JE, Johnson ET, Choudhary S, Bloch SE, Lopez-Gallego F, Srivastava P, et al. Optimized compatible set of BioBrickTM vectors for metabolic pathway engineering. Appl Microbiol Biotechnol. 2011;92(6):1275-86. Doi: 10.1007/s00253-011-3633-4.
- Scott SR, Hasty J. Quorum Sensing Communication Modules for Microbial Consortia. ACS Synth Biol. 2016 .
- Rubens JR, Selvaggio G, Lu TK. Synthetic mixed-signal computation in living cells. Nat Commun. 2016;7:11658. Doi: 10.1038/ncomms11658.
- Lian J, Zhao H. Reversal of the β-Oxidation Cycle in Saccharomyces cerevisiae for Production of Fuels and Chemicals. ACS Synth Biol. 2015;4(3):332-41. Doi: 10.1021/sb500243c.
- Peoples OP, Sinskey AJ. Poly-beta-hydroxybutyrate (PHB) biosynthesis in Alcaligenes eutrophus H16. Identification and characterization of the PHB polymerase gene (phbC). J Biol Chem. 1989;264(26):15298-303.
- Oliver JW, Machado IM, Yoneda H, Atsumi S. Cyanobacterial conversion of carbon dioxide to 2,3-butanediol. Proc Natl Acad Sci U S A. 2013 ;110(4):1249-54. Doi: 10.1073/pnas.1213024110.
- Anderson JC, Dueber JE, Leguia M, Wu GC, Goler JA, Arkin AP, et al. BglBricks: A flexible standard for biological part assembly. J Biol Eng. 2010;4(1):1. Doi: 10.1186/1754-1611-4-1.
- Kunst F, Ogasawara N, Moszer I, Albertini AM, Alloni G, Azevedo V, et al. The complete genome sequence of the Gram-positive bacterium Bacillus subtilis. Nature. 1997;390(6657):249-56.
- Westers L, Westers H, Quax WJ. Bacillus subtilis as cell factory for pharmaceutical proteins: a biotechnological approach to optimize the host organism. Biochim Biophys Acta. 2004;1694(1-3):299-310.
- Radeck J, Kraft K, Bartels J, Cikovic T, Dürr F, Emenegger J, et al. The Bacillus BioBrick Box: generation and evaluation of essential genetic building blocks for standardized work with Bacillus subtilis. J Biol Eng. 2013;7(1):29. Doi: 10.1186/1754-1611-7-29.
- Höfler C, Heckmann J, Fritsch A, Popp P, Gebhard S, Fritz G, et al. Cannibalism stress response in Bacillus subtilis. Microbiology. 2016;162(1):164-76. Doi: 10.1099/mic.0.000176.
- Kang MK, Lee J, Um Y, Lee TS, Bott M, Park SJ, et al. Synthetic biology platform of CoryneBrick vectors for gene expression in Corynebacterium glutamicum and its application to xylose utilization. Appl Microbiol Biotechnol. 2014 ;98(13):5991-6002. Doi: 10.1007/s00253-014-5714-7.
- Ravasi P, Peiru S, Gramajo H, Menzella HG. Design and testing of a synthetic biology framework for genetic engineering of Corynebacterium glutamicum. Microb Cell Fact. 2012;11:147. Doi: 10.1186/1475-2859-11-147.
- Bi C, Su P, Müller J, Yeh YC, Chhabra SR, Beller HR, et al. Development of a broad-host synthetic biology toolbox for ralstonia eutropha and its application to engineering hydrocarbon biofuel production. Microb Cell Fact. 2013;12:107. Doi: 10.1186/1475-2859-12-107.
- Li H, Liao JC. A Synthetic Anhydrotetracycline-Controllable Gene Expression System in Ralstonia eutropha H16. ACS Synth Biol. 2015;4(2):101-6. Doi: 10.1021/sb4001189.
- Schada von Borzyskowski L, Remus-Emsermann M, Weishaupt R, Vorholt JA, Erb TJ. A Set of Versatile Brick Vectors and Promoters for the Assembly, Expression, and Integration of Synthetic Operons in Methylobacterium extorquens AM1 and Other Alphaproteobacteria. ACS Synth Biol. 2015;4(4):430-43. Doi: 10.1021/sb500221v.
- Sorg RA, Kuipers OP, Veening JW. Gene Expression Platform for Synthetic Biology in the Human Pathogen Streptococcus pneumoniae. ACS Synth Biol. 2015;4(3):228-39. Doi: 10.1021/sb500229s.
- Sorg RA, Veening JW. Microscale insights into pneumococcal antibiotic mutant selection windows. Nat Commun. 2015;6:8773. Doi: 10.1038/ncomms9773.
- Huang HH, Camsund D, Lindblad P, Heidorn T. Design and characterization of molecular tools for a Synthetic Biology approach towards developing cyanobacterial biotechnology. Nucleic Acids Res. 2010;38(8):2577-93. Doi: 10.1093/nar/gkq164.
- Camsund D, Heidorn T, Lindblad P. Design and analysis of LacI-repressed promoters and DNA-looping in a cyanobacterium. J Biol Eng. 2014;8(1):4. Doi: 10.1186/1754-1611-8-4.
- Huang HH, Lindblad P. Wide-dynamic-range promoters engineered for cyanobacteria. J Biol Eng. 2013;7(1):10. Doi: 10.1186/1754-1611-7-10.
- Markley AL, Begemann MB, Clarke RE, Gordon GC, Pfleger BF. A synthetic biology toolbox for controlling gene expression in the cyanobacterium Synechococcus sp. ACS Synth Biol. 2015;4(5):595-603. Doi: 10.1021/sb500260k.
- Tikh IB, Held M, Schmidt-Dannert C. BioBrickTM compatible vector system for protein expression in Rhodobacter sphaeroides. Appl Microbiol Biotechnol. 2014;98(7):3111-9. Doi: 10.1007/s00253-014-5527-8.