andrew.tsao

Introduction

Genetically engineered strains are typically developed using specific microorganisms as chassis through methods such as mutation breeding and genetic engineering. Enhancing these engineered strains focuses on three primary research areas:

• Development of Enabling Technologies for Strain Creation: This involves advanced gene editing, gene expression regulation, and high-throughput screening to improve the genetic modification and metabolic regulation capabilities of microbial chassis.
• Systems Biology and Microbial Genetics Research: This research deepens the understanding of the genetic characteristics and physiological metabolic mechanisms of microbial chassis.
• Metabolic Pathway Design and Reconstruction: Building on existing knowledge, this involves redesigning metabolic pathways, screening key components for evolution, and reshaping the morphology and performance of microbial chassis to create a new generation of engineered strains.

Taking industrial fermentation as an example, high-quality engineered strains are the essential "chips" of the fermentation industry. For instance, Corynebacterium glutamicum has significant value in the amino acid fermentation industry. Over nearly half a century, extensive research on its genetic metabolic mechanisms and breeding technology development has been conducted. Gene editing is crucial for modifying microbial genetic information and reconstructing or regulating metabolic pathways. The complex cell wall membrane structure and high GC content of C. glutamicum's chromosome result in high structural stability, leading to low efficiency in exogenous DNA transformation and genetic modification, which is a major limiting factor for the iteration of industrial strains.

Researchers have addressed these challenges by preparing competent cells, optimizing the electroporation process, and transforming the cell wall synthesis pathway to improve the exogenous DNA transformation efficiency of C. glutamicum. Systematic optimization of the culture medium, cultivation process, and electroporation conditions for preparing competent cells has led to a transformation efficiency of 10^7 CFU/μg DNA under optimal conditions. Additionally, through comparative genomic analysis of industrial strains, researchers identified a ponA mutation site that increases DNA electroporation efficiency by about 20 times without affecting the strain's growth and metabolism. Alongside improving DNA transformation efficiency, breakthroughs in developing CRISPR-based gene editing technology have enabled efficient knockout and knock-in of large DNA fragments, minor chromosome modifications, and single nucleotide editing at multiple sites. Both CRISPR/Cas12a and CRISPR/Cas9 systems have been successfully applied to gene editing in C. glutamicum (Figure 1).

Genetically Engineered Bacterial Strains in Molecular Biology Applications

In molecular biology, genetically engineered strains, particularly competent cells, are crucial for efficiently absorbing naked DNA fragments like plasmids. These cells are commonly used in experiments such as DNA cloning, plasmid construction, and protein expression. Enhancing the performance of these bacterial strains involves several strategies:

• Enhancing Transformation Efficiency: The permeability of competent cell walls is increased to facilitate DNA entry. This can be achieved by optimizing the preparation of competent cells, including improving electroporation or chemical transformation conditions. Research often focuses on genes related to regulating cell wall synthesis or components.
• Reducing DNA Degradation: Genetic engineering techniques can delete unnecessary endogenous restriction enzyme systems or add genes related to plasmid stability, reducing the degradation of exogenous DNA. For instance, mutations in the endA gene, which expresses a non-specific nuclease, make exogenous DNA more stable and increase plasmid purity.
• Rapid Proliferation and High Replication Capacity: Strains are selected or modified to proliferate quickly and have high replication capacity, producing enough cells for transformation experiments more efficiently.
• Stress Response Mechanisms: Enhancing the strain's response mechanisms to stress encountered during the transformation process, such as temperature shock or electric shock, increases cell survival rates.
• Specificity Enhancement: For certain types of DNA, such as large molecular weight plasmids or DNA with special sequences, transformation efficiency can be improved by selecting or modifying specific genes. For example, mutations in the deoR gene of E. coli, which regulates the deo operon, can enhance the transformation efficiency of high molecular weight DNA.
• Plasmid Maintenance and Replication: Modifying host cells to improve plasmid stability and replication capacity involves studying genes related to maintaining plasmid stability.
• Metabolic Engineering: Optimizing bacterial metabolic pathways through metabolic engineering can reduce the production of harmful metabolic by-products, improving DNA transformation and expression.

E. coli is a widely used host for cloning, expression, and DNA manipulation in molecular biology. The development of competent E. coli strains, capable of taking up exogenous DNA, is fundamental to these procedures. Most common commercial laboratory E. coli strains are descendants of two separate isolates, the K-12 strain and the B strain. The K-12 strain, isolated in 1920, led to common laboratory strains like MG1655 and its derivatives DH5alpha and DH10B (also known as TOP10). The B strain, isolated in 1918, was designated the "B strain" in 1942, with the BL21 strain and its derivatives being the most common examples.

MDS42 is a streamlined version of E. coli created through genome reduction, derived from the well-studied, non-pathogenic E. coli K-12 strain. Engineered to remove non-essential genes and genetic elements unnecessary for laboratory growth, MDS42 has a reduced genome size, leading to more efficient cellular metabolism and easier genetic manipulation.

Key Features and Benefits of the MDS42 Strain

• Reduced Genome Size: Approximately 15% of the parental E. coli K-12 genome has been deleted in MDS42, removing around 1 million base pairs. This deletion includes insertion sequences, transposons, prophages, and other non-essential genes.
• Genetic Stability: By eliminating insertion sequences and transposons, which are frequently involved in genomic rearrangements, MDS42 exhibits a lower rate of spontaneous mutations, making it a more stable host for genetic engineering and synthetic biology applications.
• Improved Transformation Efficiency: The streamlined genome of MDS42 enables more efficient uptake and incorporation of foreign DNA, which is advantageous for cloning and other molecular biology techniques.
• Bio-production: MDS42 may demonstrate enhanced metabolic efficiency and faster growth rates, making it an excellent candidate for industrial biotechnology applications, including the production of proteins, biofuels, and other valuable metabolites.
• Research Tool: Serving as a simplified model organism, MDS42 aids in studying bacterial physiology and genetics, offering insights into the essential functions necessary for E. coli survival.

As a strain with a reduced genome, MDS42 was developed to minimize plasmid recombination events, potentially due to the absence of insertion sequence (IS) elements that could cause higher levels of recombination. MDS42 strains retain advantages common to other frequently used strains, such as rapid doubling time, T1 resistance, and high chemical competence, while improving DNA stability aspects. Significantly, MDS42 and its derivatives reduce the number of DNA constructs required for a given clone, which can substantially enhance the efficiency of even moderately high-throughput cloning.

Examples of Specialized E. coli Strains

• NEB Stable: Designed by New England Biolabs to improve cloning efficiency of direct repeats and other unstable DNA sequences. This strain has mutations that decrease recombination events (recA1), stabilizing cloned DNA. Additionally, it includes the endA1 mutation for cleaner DNA preparations and the hsdR17 mutation for efficient transformation of methylated DNA. NEB Stable is suitable for maintaining large plasmids and minichromosomes, which are often problematic in other strains.
• Thermo Scientific™ STL3: Tailored for cloning direct repeats and DNA with secondary structures that can cause instability. This strain contains mutations that reduce the activity of the RecBCD nuclease complex, which degrades linear DNA and facilitates homologous recombination. By limiting this activity, STL3 helps maintain repetitive sequences that would otherwise be challenging to clone.

Other Notable E. coli Strains

• DH5α: A highly competent strain commonly used for routine cloning, with endA1 and recA1 mutations that improve the quality of plasmid DNA and reduce recombination.
• BL21(DE3): Used for protein expression, containing the T7 RNA polymerase gene under the control of the lacUV5 promoter, allowing high-level expression of proteins from T7 promoter-containing plasmids.
• XL1-Blue: A versatile strain used for blue/white screening due to its lacZΔM15 mutation, also recA1 for stability and endA1 for cleaner DNA preps.
• Rosetta Strains: Contain tRNAs for rare codons, enhancing the expression of eukaryotic proteins with codon usage bias.

Each of these strains may carry additional mutations that confer antibiotic resistance, improve transformation efficiency, or provide other desirable traits for various applications. When choosing a host strain, considerations include the type of DNA being cloned, the purpose of the cloning (such as protein expression or library construction), and the need for specific features like blue/white screening or suppression of restriction systems.

GenScript Poly(A) Strain V2/V3 Provides Superior Poly(A) Stability

Choosing the right bacterial strain for replicating plasmids with a poly(A) tail is crucial to maintain the integrity of the poly(A) sequence. Bacteria strains exhibit varied levels of exonuclease activity, which can significantly affect the stability of the poly(A) tail in the plasmid DNA. endA1 mutation that minimizes plasmid DNA degradation, is a popular choice. However, even with NEB® Stable's design to handle repetitive sequences, it may not fully resolve the instability issues associated with extended poly(A) sequences. GenScript has introduced a unique strain tailored to enhance the stability of poly(A) tails. The GenScript Poly(A) Strain is composed of genetically edited genes and sequences, including RecA, which have a direct or indirect impact on the recombination of cloned DNA.

In conclusion, the development of various E. coli host strains has been driven by the need to overcome specific challenges associated with different types of genetic material and applications. By understanding the genetic characteristics of each strain, researchers can select the most appropriate host for their experiments, increasing the chances of successful cloning and expression of target genes. The ongoing development of new strains continues to expand the toolkit available to molecular biologists, enabling the advancement of research in genetics, biotechnology, and other life sciences.

References:

[1] Ruan YL, Zhu LJ, Li Q. Improving the electro- transformation efficiency of Corynebacterium glutamicum by weakening its cell wall and increasing the cytoplasmic membrane fluidity. Biotechnol Lett, 2015, 37(12): 2445-2452. DOI:10.1007/s10529-015-1934-x

[2] Liu J, Wang Y, Lu YJ, et al. Mutations in peptidoglycan synthesis gene ponA improve electrotransformation efficiency of Corynebacterium glutamicum ATCC 13869. Appl Environ Microbiol, 2018, 84(24): e02225-18

[3] Midon, M., Schäfer, P., Pingoud, et al. Mutational and biochemical analysis of the DNA-entry nuclease EndA from Streptococcus pneumoniae. Nucleic acids research, 2011,39(2), 623–634. https://doi.org/10.1093/nar/gkq802

[4] Mortensen, L., Dandanell, G., Hammer, K. Purification and characterization of the deoR repressor of Escherichia coli. The EMBO journal, 1989, 8(1), 325–331. https://doi.org/10.1002/j.1460-2075.1989.tb03380.x

[5] Chakiath CS, Esposito D. Improved recombinational stability of lentiviral expression vectors using reduced-genome Escherichia coli. Biotechniques. 2007, 43(4):466, 468, 470. doi: 10.2144/000112585. PMID: 18019337.