Design

Light Sensor

Another optogenetics system applied in our project design is a green/red photoreversible two-component signal transduction system (TCS) with transcriptional outputs in E. coli. The two-component system consists of the membrane-associated histidine kinase CcaS and its response regulator CcaR. The activating information stored in light is captured by phytochromes in situ. In phytochromes, a bilin-chromophore (in this case phycocyanobilin) binds at a conserved cysteine within an N-terminal GAF (cyclic GMP phosphodiesterase, adenylyl cyclase, FhlA) domain and imparts reversible photoactivation of signaling activity with maximal responses to 535-nm (green) and 672-nm (red) light. Absorption of green light increases the rate of CcaS autophosphorylation, phosphorylation of CcaR by phosphate transferring, and transcription from the promoter of the phycobilisome linker protein cpcG2, while absorption of red light reverses this process. The lower leakiness is reported to be acquired after removing the second putative promoter in cpcG2, which is thought to be constitutive and contributes to leakiness and low dynamic range.

We are inspired by work done by Professor Sode of making some improvements to the TCS, the leakiness is got lower while the dynamic range rises sharply by removing two PAS domains of unknown function within the CcaS sensor histidine kinase. In our project, we acquired #3、#4、#10 which were the variants of CcaS through repeating Professor Sode’s experiment

The domains Magnet derived from the homodimerizing photoreceptor VVD from the filamentous fungus Neurospora crassa consist of pMag and nMag, which bind to each other upon illumination. The T7 RNA polymerase from the T7 bacteriophage with high orthogonality is commonly used for overexpression heterologous proteins. In our design, split T7 RNA polymerases are fused with photoactivated dimerization fragments pMag and nMag, and through light-induced protein-protein interactions the split T7 RNA polymerases dimerize and reconstitute a complete RNA polymerase structure, then preforms its normal function. This light-inducible transcription system, with high spatiotemporal accuracy, low leakiness of gene expression in darkness and high expression strength when induced by blue light, can allow precise dynamic control of gene expression. We desire to apply this orthogonal blue light-responsive T7 RNAP in our project for precise dynamic control of gene expression.

Toggle Switch

Clustered regularly interspaced short palindromic repeats (CRISPR) is a family of DNA sequences found within the genomes of prokaryotic organisms such as bacteria and archaea, which have the function in binding and cutting DNA.^[1] There are three types of CRISPR system found in microorganisms to date: type I, type II and type III. In Type I and Type III systems the long precursor CRISPR RNA (pre-crRNA) is processed by CRISPR specific endoribonucleases into small CRISPR RNAs (crRNAs) that contain a repeat sequence flaked by portions of the adjacent CRISPR repeat sequence.^[2-7] In contrast, the pre-crRNA in Type II systems is processed by RNase III.^[8]

In Escherichia coli, the type I-E CRISPR system expressed endogenously shall be easy for internal regulation without causing metabolic burden in compared with the widely used type II system, which expressed dCas9 as an additional plasmid.

Similar to the other two CRISPR systems, crRNA in the Type I-E system sequences are recognized by ribonucleoprotein complex Cascade during target DNA binding.^[9,10] The ribonucleoprotein complex Cascade is composed of a 61 nt crRNA, and five different Cas proteins in an uneven stoichiometry: Cse1₁Cse2₂Cas7₆Cas5₁Cas6e₁, encoded by 8 genes separately (cas3, cse1, cse2, cas7, cas5, cas6e, cas1, cas2).^[11] The interference function of this system requires Cas3, a large protein with nuclease and helicase activities.^[12,13]

Therefore, by knocking out cas3 and activating the expression of CRISPR-associated complex for antiviral defence (Cascade), the CRISPR system is inactivated for its DNA-cutting function, with its DNA-binding function maintained, which can interference the expression of the target genes.^[14]

Thus we can use this CRISPRi system as a switch to regulate the gene of interest in Escherichia coli.

In our project, the target gene is gltA which encodes citrate synthase. The process catalysed by citrate synthase is irreversible and rate-limiting step in the TCA cycle. Furthermore, its substrate - acetyl-CoA is one of the most significant intermediates used to produce important chemicals such as 1,3-isopropanol^[15], 1-butanol^[16]. In principle, the type I-E CRISPRi system can redirect the metabolic flux from TCA cycle to target pathway via switching gltA off.^[17]

Fermentation

As we all know acetyl-CoA is a prominent precursor for TCA cycle, which is regarded as the most efficient pathway of energy producing for cell growth. However, acetyl-CoA is also required in many synthetic pathways, like biofuel 1-butanol^[18], solvent acetone ^[19] and bioplastic Polyhydroxybutyrate (PHB). PHB, a kind of biosynthesized polyester, is nowadays, one of the most promising bio-materials as the replacement of traditional petrochemical plastic for its outstanding biodegradable as well as other physical properties. Naturally, the synthesis of PHB requires three enzymes, phbA, phbB and phbC, by which acetyl-CoA can ultimately be converted into PHB. But the conflict between cell growth demands and the usage of acetyl-CoA for PHB accumulation is obviously the obstacle for PHB production. So here, we intend to introduce our light-induced switch and type I-E CRISPRi into PHB fermentation as an example of our growth-production switching system

[1] Barrangou R (2015). "The roles of CRISPR-Cas systems in adaptive immunity and beyond". Current Opinion in Immunology. 32: 36–41.
[2] Brouns SJJ, Jore MM, Lundgren M, Westra ER, Slijkhuis RJH, et al. (2008) Small CRISPR RNAs guide antiviral defense in prokaryotes. Science 321: 960–964.
[3] Haurwitz RE, Jinek M, Wiedenheft B, Zhou K, Doudna JA (2010) Sequence-and structure-specific RNA processing by a CRISPR endonuclease. Science 329:1355–1358.
[4] Carte J, Wang RY, Li H, Terns RM, Terns MP (2008) Cas6 is an endoribonuclease that generates guide RNAs for invader defense in prokaryotes. Gene Dev 22: 3489–3496
[5] rzybilski R, Richter C, Gristwood T, Clulow JS, Vercoe RB, et al. (2011) Csy4 is responsible for CRISPR RNA processing in Pectobacterium atrosepticum. RNA Biol 8: 517–528.
[6] am KH, Haitjema C, Liu X, Ding F, Wang H, et al. (2012) Cas5d protein processes pre-crRNA and assembles into a Cascade-like interference complex in subtype I-C/Dvulg CRISPR-Cas system. Structure 20: 1574–84.
[7] arside EL, Schellenberg MJ, Gesner EM, Bonanno JB, Sauder JM, et al. (2012) Cas5d processes pre-crRNA and is a member of a larger family of CRISPR RNA endonucleases. RNA 18: 2020–2028.
[8] Deltcheva E, Chylinski K, Sharma CM, Gonzales K, Chao Y, et al. (2011) CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature 471: 602–607.
[9] Westra ER, van Erp PB, Kunne T, Wong SP, Staals RH, et al. (2012) CRISPR immunity relies on the consecutive binding and degradation of negatively supercoiled invader DNA by Cascade and Cas3. Mol Cell 46: 595–605.
[10] Semenova E, Jore MM, Datsenko KA, Semenova A, Westra ER, et al. (2011) Interference by clustered regularly interspaced short palindromic repeat (CRISPR) RNA is governed by a seed sequence. Proc Natl Acad Sci U S A 108: 10098–10103.
[11] unin V, Sorek R, Hugenholtz P (2007) Evolutionary conservation of sequence and secondary structures in CRISPR repeats. Genome Biol 8: R61.
[12] Westra ER, van Erp PB, Künne T, Wong SP, Staals RH, Seegers CL, et al. CRISPR immunity relies on the consecutive binding and degradation of negatively super-coiled invader DNA by Cascade and Cas3. Mol Cell 2012; 46:595-605.
[13] Sinkunas T, Gasiunas G, Fremaux C, Barrangou R, Horvath P, Siksnys V. Cas3 is a single-stranded DNA nuclease and ATP-dependent helicase in the CRISPR/Cas immune system. EMBO J 2011; 30:1335-42.
[14] Easy regulation of metabolic flux in Escherichia coli using an endogenous type I‐E CRISPR‐Cas system.
[15] Nakamura, C.E., Whited, G.M., 2003. Metabolic engineering for the microbial production of 1,3-propanediol. Curr. Opin. Biotechnol. 14, 454–459.
[16] Dellomonaco, C., Clomburg, J.M., Miller, E.N., Gonzalez, R., 2011. Engineered reversal of the β -oxidation cycle for the synthesis of fuels and chemicals. Nature 10, 355–359.
[17] Metabolic flux redirection from a central metabolic pathway toward a synthetic pathway using a metabolic toggle switch.
[18] Dellomonaco C, Clomburg J M, Miller E N, et al. Engineered reversal of the β-oxidation cycle for the synthesis of fuels and chemicals[J]. Nature, 2011, 476(7360): 355.
[19] Bermejo L L, Welker N E, Papoutsakis E T. Expression of Clostridium acetobutylicumATCC 824 Genes in Escherichia coli for Acetone Production and Acetate Detoxification[J]. Applied and environmental microbiology, 1998, 64(3): 1079-1085.