Applications of Metabolic Engineering Summary
The key points from this module are as follows:
Genome editing technology – It employs generations of nucleases for genome editing and the DNA repair pathways used to modify target DNA. It is a technology for modifying target genomic DNA in vivo. It is reliably efficient and does not necessarily require the integration of selectable marker genes.
Promoters – They play an essential role in controlling the expression of genes in biosynthetic pathway. The native promoter can be replaced with artificial ones, either inducible or constitutive, or by expressing the gene with appropriate strength so that the encoded enzyme is present at optimal concentration.
Ribosome binding sites (RBS) – Changing the RBS region can control, gene expression at the translation level, consequently affecting the efficiency of protein production.
RNA-mediated gene modulation techniques – The RNA interference (RNAi), and small RNA sRNA can also decrease gene expression by translational repression of target genes.
Gene copy number – Gene expression levels are normally positively related with the gene copy number.
The following are strategies developed to control the chromosomal gene copy number:
Chemical inducible chromosomal evolution (CIChE)
Replicon-free markerless method (RMM) and Cre-LoxP-based methods
Chromosomal integration of genes with multiple copies (CIGMC)
Recombinase-assisted genome engineering (RAGE)
Strategies and methods for modulating the expression of pathway genes:
Multiplex automated genome engineering (MAGE)
Co-Selection MAGE (CoS-MAGE)
Tunable intergenic regions (TIGRs)
Customized optimization of metabolic pathways by combinational transcriptional engineering (COMPACTER)
A number of genome editing techniques, e.g. zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENS) are developed to facilitate chromosomal modification. The major shortcomings of these techniques are: High cost, Time-consuming process, and low editing efficiency.
Basic Ideas about CRSIPR (Clustered Regularly Interspaced Short Palindromic CRISPR Repeats)-Cas (CRISPR associated) system:
CRISPR-Cas is a gene editing tool that can manipulate genes (gene knockout or gene knock-in) and the genetic expression in plants, animals, and human. CRISPR contains short sections of bacterial DNA containing repetitive base sequences. CRISPR emerged as a robust technology that allowed:
(1) Effective application in various microorganisms, including both prokaryotes and eukaryotes
(2) Higher efficiency
(3) Lower cists
(4) Easily customizable target sequences
CRISPRS-array: a defining feature of the immune system. This genomic locus is composed of alternating identical repeats and unique spacers.
The CRISPR-Cas system has been assigned to two classes (6 types and 33 subtypes)
Class I CRISPR-Cas system (types I, III and IV): It employs multi-Cas protein complexes for interferences.
Class 2 CRISPR-Cas (types II, V and VI): Interference is accomplished with a single effector protein.
The Structure of the CRISPR-Cas locus
The CRISPR regions in bacterial or archaeal genome help to defend the microorganisms against invading viruses. The regions are composed of alternating identical repeats and unique spacers.
The properties of the CRISPR loci structure
- Repeat sequences of about 32bp are interleaved by variable spacers of about equal size.
- The number of repeat-spacer units varies greatly.
- CRISPR-associated (CaS) genes surround the CRISPR locus.
CRISPR-associated genes – CRISPR loci are surrounded by a cohort of conserved protein-coding genes, which appear in varying orientation and order. Homologous comparisons delineated four core CRISPR-associated gene families from Cas1-4, and later expanded to Cas5 and Cas6.
The steps of CRISPR-mediated immunity: The CRISPR immune system works to protect bacteria from repeated viral attack via three basic steps, which are:
Adaptation, Production of CRISPR RNA and Interference/Targeting
Functions of the Cas Proteins (the CRISPR effector) – Cas proteins drive the three phases of immunity: (1) adaptation (2) CRISPR RNA (crRNA) biogenesis and (3) Interference
Protospacer – Is basically the region which is cut, and cleaved, and converted to spacers to be integrated into the CRIPR array.
The advantages of the CRISPR-Cas system
CRISPR-Cas system has been applied in single cell microbes to maintain gnomic integrity by mitigating the effects of foreign or mobile genomic elements.
It enables simultaneous targeting of multiple loci (multiplexing)
It shows potential as scalable platforms for comprehensive genome-wide modifications of microorganism.
The three stages of CRISPR-Cas adaptive immunity:
The basis of Cas9 engineering tools – The simple way in which Cas9 nucleus can be guided to the desired DNA target denoted as protospacer, by CRISPR-RNA:trans-activating CRISPR RNA (crRNA:tracrRNA) hybrid complex:
The 5’-end of crRNA module (spcer) has to be complementary to the selected protospacer, and a specific short DNA motif –the protospacer adjacent motif (PAM) has to be present at the 3’-end of the selected protospacer. Based on this concept, the genome manipulation models were developed.
The advantages of Cas9 over other conventional tools:
The introduction of the double stranded breaks (DSBs) greatly increases the rates of recombination when used in conjunction with an appropriate DNA donor molecule. The use of the CRISPR-Cas9 and multiple sgRNAs together with multiple donor cassettes enabled the introduction of biosynthetic production pathways in both S. cerevisiae and Kluyveromyces lactis in a single transformation event.
Targeted DSBs mediated by Cas9 endonuclease activity facilitate the integration of linearized donor plasmids by homologous recombination.
Transcription control models
CRISPR interference (CRISPRi) – This transcriptional control allows for precise pathway flux control and avoids the negative effect of overexpression. It is an effective tool for modulating the expression level of any gene.
Advantages over genome manipulation modules
- Permanent genetic modifications (e.g. gene knockout) are not often the ideal means of exerting metabolic control in microorganisms to ensure high yields of the desired metabolite or chemical compounds.
- Constitutive expression of an exogenous or endogenous gene risks triggering feedback inhibition, slowing cellular growth and leading to cytotoxic effects.
Deactivated Cas9 – The mutation of key residues in the two nuclease domains of Cas9 protein results in a catalytically deactivated Cas9 (dCas9), which retains sequence-specific binding of a nucleic acid target but ablates endonuclease activity.
CRISPR activation (CRISPRa) – Is accomplished through the protein fusion of dcas9 to a transcriptional activation domain, and has been used for the transcriptional upregulation of select genes. In E. coli, the fusion of the dCas9 to a subunit of the RNAp complex effectively recruited other RNAp subunit components, improving the production of a protein target by about 250%.
CRISPR interference/activation (CRISPRi/a) – utilizes a dCas9:sgRNA complex and a transcriptional regulatory domain, which is species and application specific. The regulating domain may be recruited to the Cas nuclease by fusing an RNA-binding domain to the regulatory domain and adding an RNA hairpin to the 3’-end of the sgRNA resulting in the upregulation of transcription.
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