Cell engineering

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Cells engineered to fluoresce under UV light. PGlo-UltraViolet.jpg
Cells engineered to fluoresce under UV light.

Cell engineering is the purposeful process of adding, deleting, or modifying genetic sequences in living cells to achieve biological engineering goals such as altering cell production, changing cell growth and proliferation requirements, adding or removing cell functions, and many more. Cell engineering often makes use of DNA technology to achieve these modifications as well as closely related tissue engineering methods. Cell engineering can be characterized as an intermediary level in the increasingly specific disciplines of biological engineering which includes organ engineering, tissue engineering, protein engineering, and genetic engineering.

Contents

The field of cellular engineering is gaining more traction as biomedical research advances in tissue engineering and becomes more specific. Publications in the field have gone from several thousand in the early 2000s to nearly 40,000 in 2020.

Overview

Improving production of natural cellular products

One general form of cell engineering involves altering natural cell production to achieve a more desirable yield or shorter production time. [1] A possible method for changing natural cell production includes boosting or repressing genes that are involved in the metabolism of the product. For example, researchers were able to overexpress transporter genes in hamster ovary cells to increase monoclonal antibody yield. [2] Another approach could involve incorporating biologically foreign genes into an existing cell line. For example, E.Coli , which synthesizes ethanol, can be modified using genes from Zymomonas mobilis to make ethanol fermentation the primary cell fermentation product. [3]

Altering cell requirements

Another beneficial cell modification is the adjustment of substrate and growth requirements of a cell. By changing cell needs, the raw material cost, equipment expenses, and skill required to grow and maintain cell cultures can be significantly reduced. For example, scientists have used foreign enzymes to engineer a common industrial yeast strain which allows the cells to grow on substrate cheaper than the traditional glucose. [4] Because of the biological engineering focus on improving scale-up costs, research in this area is largely focused on the ability of various enzymes to metabolize low-cost substrates. [5]

Augmenting cells to produce new products

Closely tied with the field of biotechnology, this subject of cell engineering employs recombinant DNA methods to induce cells to construct a desired product such as a protein, antibody, or enzyme. One of the most notable examples of this subset of cellular engineering is the transformation of E. Coli to transcript and translate a precursor to insulin which drastically reduced the cost of production. [6] Similar research was conducted shortly after in 1979 in which E. Coli was transformed to express human growth hormone for use in treatment of pituitary dwarfism. [7] Finally, much progress has been made in engineering cells to produce antigens for the purpose of creating vaccines. [8]

Adjustment of cell properties

Within the focus of bioengineering, various cell modification methods are utilized to alter inherent properties of cells such as growth density, growth rate, growth yield, temperature resistance, freezing tolerance, chemical sensitivity, and vulnerability to pathogens. [1] For example, in 1988 one group of researchers from the Illinois Institute of Technology successfully expressed a Vitreoscilla hemoglobin gene in E. Coli to create a strain that was more tolerant to low-oxygen conditions such as those found in high density industrial bioreactors. [9]

Stem cell engineering

One distinct section of cell engineering involves the alteration and tuning of stem cells. Much of the recent research on stem cell therapies and treatments falls under the aforementioned cell engineering methods. Stem cells are unique in that they may differentiate into various other types of cells which may then be altered to produce novel therapeutics or provide a foundation for further cell engineering efforts. [10] One example of directed stem cell engineering includes partially differentiating stem cells into myocytes to enable production of pro-myogenic factors for the treatment of sarcopenia or muscle disuse atrophy. [11]

History

The phrase "cell engineering" was first used in a published paper in 1968 to describe the process of improving fuel cells. [12] The term was then adopted by other papers until the more specific "fuel-cell engineering" was used.

The first use of the term in a biological context was in 1971 in a paper which describes methods to graft reproductive caps between algae cells. [13] Despite the rising popularity of the phrase, there remains unclear boundaries between cell engineering and other forms of biological engineering. [1]

Examples

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Escherichia coli ( ESH-ə-RIK-ee-ə KOH-ly) is a gram-negative, facultative anaerobic, rod-shaped, coliform bacterium of the genus Escherichia that is commonly found in the lower intestine of warm-blooded organisms. Most E. coli strains are harmless, but some serotypes such as EPEC, and ETEC are pathogenic and can cause serious food poisoning in their hosts, and are occasionally responsible for food contamination incidents that prompt product recalls. Most strains are part of the normal microbiota of the gut and are harmless or even beneficial to humans (although these strains tend to be less studied than the pathogenic ones). For example, some strains of E. coli benefit their hosts by producing vitamin K2 or by preventing the colonization of the intestine by pathogenic bacteria. These mutually beneficial relationships between E. coli and humans are a type of mutualistic biological relationship — where both the humans and the E. coli are benefitting each other. E. coli is expelled into the environment within fecal matter. The bacterium grows massively in fresh fecal matter under aerobic conditions for three days, but its numbers decline slowly afterwards.

β-Galactosidase Family of glycoside hydrolase enzymes

β-Galactosidase, is a glycoside hydrolase enzyme that catalyzes hydrolysis of terminal non-reducing β-D-galactose residues in β-D-galactosides.

<span class="mw-page-title-main">Synthetic biology</span> Interdisciplinary branch of biology and engineering

Synthetic biology (SynBio) is a multidisciplinary field of science that focuses on living systems and organisms, and it applies engineering principles to develop new biological parts, devices, and systems or to redesign existing systems found in nature.

In biology, a strain is a genetic variant, a subtype or a culture within a biological species. Strains are often seen as inherently artificial concepts, characterized by a specific intent for genetic isolation. This is most easily observed in microbiology where strains are derived from a single cell colony and are typically quarantined by the physical constraints of a Petri dish. Strains are also commonly referred to within virology, botany, and with rodents used in experimental studies.

<span class="mw-page-title-main">Cell culture</span> Process by which cells are grown under controlled conditions

Cell culture or tissue culture is the process by which cells are grown under controlled conditions, generally outside of their natural environment. After cells of interest have been isolated from living tissue, they can subsequently be maintained under carefully controlled conditions. They need to be kept at body temperature (37 °C) in an incubator. These conditions vary for each cell type, but generally consist of a suitable vessel with a substrate or rich medium that supplies the essential nutrients (amino acids, carbohydrates, vitamins, minerals), growth factors, hormones, and gases (CO2, O2), and regulates the physio-chemical environment (pH buffer, osmotic pressure, temperature). Most cells require a surface or an artificial substrate to form an adherent culture as a monolayer (one single-cell thick), whereas others can be grown free floating in a medium as a suspension culture. This is typically facilitated via use of a liquid, semi-solid, or solid growth medium, such as broth or agar. Tissue culture commonly refers to the culture of animal cells and tissues, with the more specific term plant tissue culture being used for plants. The lifespan of most cells is genetically determined, but some cell-culturing cells have been 'transformed' into immortal cells which will reproduce indefinitely if the optimal conditions are provided.

<span class="mw-page-title-main">Recombinant DNA</span> DNA molecules formed by human agency at a molecular level generating novel DNA sequences

Recombinant DNA (rDNA) molecules are DNA molecules formed by laboratory methods of genetic recombination that bring together genetic material from multiple sources, creating sequences that would not otherwise be found in the genome.

<i>Komagataella</i> Genus of fungus used industrially and as model organism

Komagataella is a methylotrophic yeast within the order Saccharomycetales. It was found in the 1960s as Pichia pastoris, with its feature of using methanol as a source of carbon and energy. In 1995, P. pastoris was reassigned into the sole representative of genus Komagataella, becoming Komagataella pastoris. Later studies have further distinguished new species in this genus, resulting in a total of 7 recognized species. It is not uncommon to see the old name still in use in the context of protein production, as of 2023; in less formal use, the yeast may confusingly be referred to as pichia.

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Bacterial display is a protein engineering technique used for in vitro protein evolution. Libraries of polypeptides displayed on the surface of bacteria can be screened using flow cytometry or iterative selection procedures (biopanning). This protein engineering technique allows us to link the function of a protein with the gene that encodes it. Bacterial display can be used to find target proteins with desired properties and can be used to make affinity ligands which are cell-specific. This system can be used in many applications including the creation of novel vaccines, the identification of enzyme substrates and finding the affinity of a ligand for its target protein.

Humanized antibodies are antibodies from non-human species whose protein sequences have been modified to increase their similarity to antibody variants produced naturally in humans. The process of "humanization" is usually applied to monoclonal antibodies developed for administration to humans. Humanization can be necessary when the process of developing a specific antibody involves generation in a non-human immune system. The protein sequences of antibodies produced in this way are partially distinct from homologous antibodies occurring naturally in humans, and are therefore potentially immunogenic when administered to human patients. The International Nonproprietary Names of humanized antibodies end in -zumab, as in omalizumab.

<span class="mw-page-title-main">Directed evolution</span> Protein engineering method

Directed evolution (DE) is a method used in protein engineering that mimics the process of natural selection to steer proteins or nucleic acids toward a user-defined goal. It consists of subjecting a gene to iterative rounds of mutagenesis, selection and amplification. It can be performed in vivo, or in vitro. Directed evolution is used both for protein engineering as an alternative to rationally designing modified proteins, as well as for experimental evolution studies of fundamental evolutionary principles in a controlled, laboratory environment.

<span class="mw-page-title-main">Mixed acid fermentation</span> Biochemical conversion of six-carbon sugars into acids in bacteria

In biochemistry, mixed acid fermentation is the metabolic process by which a six-carbon sugar is converted into a complex and variable mixture of acids. It is an anaerobic (non-oxygen-requiring) fermentation reaction that is common in bacteria. It is characteristic for members of the Enterobacteriaceae, a large family of Gram-negative bacteria that includes E. coli.

The non-mevalonate pathway—also appearing as the mevalonate-independent pathway and the 2-C-methyl-D-erythritol 4-phosphate/1-deoxy-D-xylulose 5-phosphate (MEP/DOXP) pathway—is an alternative metabolic pathway for the biosynthesis of the isoprenoid precursors isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). The currently preferred name for this pathway is the MEP pathway, since MEP is the first committed metabolite on the route to IPP.

<span class="mw-page-title-main">Metabolic flux analysis</span> Experimental fluxomics technique

Metabolic flux analysis (MFA) is an experimental fluxomics technique used to examine production and consumption rates of metabolites in a biological system. At an intracellular level, it allows for the quantification of metabolic fluxes, thereby elucidating the central metabolism of the cell. Various methods of MFA, including isotopically stationary metabolic flux analysis, isotopically non-stationary metabolic flux analysis, and thermodynamics-based metabolic flux analysis, can be coupled with stoichiometric models of metabolism and mass spectrometry methods with isotopic mass resolution to elucidate the transfer of moieties containing isotopic tracers from one metabolite into another and derive information about the metabolic network. Metabolic flux analysis (MFA) has many applications such as determining the limits on the ability of a biological system to produce a biochemical such as ethanol, predicting the response to gene knockout, and guiding the identification of bottleneck enzymes in metabolic networks for metabolic engineering efforts.

Biomolecular engineering is the application of engineering principles and practices to the purposeful manipulation of molecules of biological origin. Biomolecular engineers integrate knowledge of biological processes with the core knowledge of chemical engineering in order to focus on molecular level solutions to issues and problems in the life sciences related to the environment, agriculture, energy, industry, food production, biotechnology and medicine.

<span class="mw-page-title-main">Genetically modified bacteria</span> First organisms to be modified in the laboratory

Genetically modified bacteria were the first organisms to be modified in the laboratory, due to their simple genetics. These organisms are now used for several purposes, and are particularly important in producing large amounts of pure human proteins for use in medicine.

No-SCAR genome editing is an editing method that is able to manipulate the Escherichia coli genome. The system relies on recombineering whereby DNA sequences are combined and manipulated through homologous recombination. No-SCAR is able to manipulate the E. coli genome without the use of the chromosomal markers detailed in previous recombineering methods. Instead, the λ-Red recombination system facilitates donor DNA integration while Cas9 cleaves double-stranded DNA to counter-select against wild-type cells. Although λ-Red and Cas9 genome editing are widely used technologies, the no-SCAR method is novel in combining the two functions; this technique is able to establish point mutations, gene deletions, and short sequence insertions in several genomic loci with increased efficiency and time sensitivity.

<span class="mw-page-title-main">Microbial cell factory</span>

Microbial cell factory is an approach to bioengineering which considers microbial cells as a production facility in which the optimization process largely depends on metabolic engineering. MCFs is a derivation of cell factories, which are engineered microbes and plant cells. In 1980s and 1990s, MCFs were originally conceived to improve productivity of cellular systems and metabolite yields through strain engineering. A MCF develops native and nonnative metabolites through targeted strain design. In addition, MCFs can shorten the synthesis cycle while reducing the difficulty of product separation.

Recombinant antibodies are antibody fragments produced by using recombinant antibody coding genes. They mostly consist of a heavy and light chain of the variable region of immunoglobulin. Recombinant antibodies have many advantages in both medical and research applications, which make them a popular subject of exploration and new production against specific targets. The most commonly used form is the single chain variable fragment (scFv), which has shown the most promising traits exploitable in human medicine and research. In contrast to monoclonal antibodies produced by hybridoma technology, which may lose the capacity to produce the desired antibody over time or the antibody may undergo unwanted changes, which affect its functionality, recombinant antibodies produced in phage display maintain high standard of specificity and low immunogenicity.

Host cell proteins (HCPs) are process-related protein impurities that are produced by the host organism during biotherapeutic manufacturing and production. During the purification process, a majority of produced HCPs are removed from the final product. However, residual HCPs still remain in the final distributed pharmaceutical drug. Examples of HCPs that may remain in the desired pharmaceutical product include: monoclonal antibodies (mAbs), antibody-drug-conjugates (ADCs), therapeutic proteins, vaccines, and other protein-based biopharmaceuticals.

References

  1. 1 2 3 Cameron, Douglas C.; Tong, I-Teh (1993-01-01). "Cellular and metabolic engineering". Applied Biochemistry and Biotechnology. 38 (1): 105–140. doi:10.1007/BF02916416. ISSN   1559-0291. PMID   8346901. S2CID   28117582.
  2. ↑ Tabuchi, Hisahiro; Sugiyama, Tomoya; Tanaka, Saeko; Tainaka, Satoshi (2010). "Overexpression of taurine transporter in Chinese hamster ovary cells can enhance cell viability and product yield, while promoting glutamine consumption". Biotechnology and Bioengineering. 107 (6): 998–1003. doi:10.1002/bit.22880. ISSN   1097-0290. PMID   20661907. S2CID   9824980.
  3. ↑ Ingram, L O; Conway, T; Clark, D P; Sewell, G W; Preston, J F (1987-10-01). "Genetic engineering of ethanol production in Escherichia coli". Applied and Environmental Microbiology. 53 (10): 2420–2425. Bibcode:1987ApEnM..53.2420I. doi:10.1128/aem.53.10.2420-2425.1987. PMC   204123 . PMID   3322191.
  4. ↑ Ledesma-Amaro, Rodrigo; Nicaud, Jean-Marc (October 2016). "Metabolic Engineering for Expanding the Substrate Range of Yarrowia lipolytica". Trends in Biotechnology. 34 (10): 798–809. doi:10.1016/j.tibtech.2016.04.010. ISSN   0167-7799. PMID   27207225.
  5. ↑ Prieto, M A; Perez-Aranda, A; Garcia, J L (1993-04-01). "Characterization of an Escherichia coli aromatic hydroxylase with a broad substrate range". Journal of Bacteriology. 175 (7): 2162–2167. doi:10.1128/jb.175.7.2162-2167.1993. PMC   204336 . PMID   8458860.
  6. ↑ Goeddel, D. V.; Kleid, D. G.; Bolivar, F.; Heyneker, H. L.; Yansura, D. G.; Crea, R.; Hirose, T.; Kraszewski, A.; Itakura, K.; Riggs, A. D. (1979-01-01). "Expression in Escherichia coli of chemically synthesized genes for human insulin". Proceedings of the National Academy of Sciences. 76 (1): 106–110. Bibcode:1979PNAS...76..106G. doi: 10.1073/pnas.76.1.106 . ISSN   0027-8424. PMC   382885 . PMID   85300.
  7. ↑ Goeddel, David V.; Heyneker, Herbert L.; Hozumi, Toyohara; Arentzen, Rene; Itakura, Keiichi; Yansura, Daniel G.; Ross, Michael J.; Miozzari, Giuseppe; Crea, Roberto; Seeburg, Peter H. (October 1979). "Direct expression in Escherichia coli of a DNA sequence coding for human growth hormone". Nature. 281 (5732): 544–548. Bibcode:1979Natur.281..544G. doi:10.1038/281544a0. ISSN   1476-4687. PMID   386136. S2CID   4237998.
  8. ↑ Nascimento, I. P.; Leite, L. C. C. (December 2012). "Recombinant vaccines and the development of new vaccine strategies". Brazilian Journal of Medical and Biological Research. 45 (12): 1102–1111. doi:10.1590/S0100-879X2012007500142. ISSN   0100-879X. PMC   3854212 . PMID   22948379.
  9. ↑ Dikshit, Kanak L.; Webster, Dale A. (1988-10-30). "Cloning, characterization and expression of the bacterial globin gene from Vitreoscilla in Escherichia coli". Gene. 70 (2): 377–386. doi:10.1016/0378-1119(88)90209-0. ISSN   0378-1119. PMID   2850971.
  10. ↑ Li, Shengwen Calvin; Wang, Lang; Jiang, Hong; Acevedo, Julyana; Chang, Anthony Christopher; Loudon, William Gunter (2009-03-01). "Stem cell engineering for treatment of heart diseases: Potentials and challenges". Cell Biology International. 33 (3): 255–267. doi: 10.1016/j.cellbi.2008.11.009 . ISSN   1065-6995. PMID   19084605. S2CID   20486657.
  11. ↑ Fix, Dennis K.; Mahmassani, Ziad S.; Petrocelli, Jonathan J.; de Hart, Naomi M.M.P.; Ferrara, Patrick J.; Painter, Jessie S.; Nistor, Gabriel; Lane, Thomas E.; Keirstead, Hans S.; Drummond, Micah J. (2021-12-01). "Reversal of deficits in aged skeletal muscle during disuse and recovery in response to treatment with a secrotome product derived from partially differentiated human pluripotent stem cells". GeroScience. 43 (6): 2635–2652. doi:10.1007/s11357-021-00423-0. ISSN   2509-2723. PMC   8602548 . PMID   34427856.
  12. ↑ Thurber, W. C. (1968-05-01). "Closure to "Discussions of 'A Fuel Cell Power Plant for a Deep Diving Submarine'" (1968, ASME J. Eng. Ind., 90, pp. 266–267)". Journal of Engineering for Industry. 90 (2): 267. doi: 10.1115/1.3604626 . ISSN   0022-0817.
  13. ↑ Bonotto, S.; Kirchmann, R.; Manil, P. (1971-01-01). "Cell Engineering in Acetabularia: A Graft Method for Obtaining Large Cells with Two or More Reproductive Caps". Giornale Botanico Italiano. 105 (1): 1–9. doi:10.1080/11263507109431460. ISSN   0017-0070.
  14. ↑ Sadelain, Michel; Rivière, Isabelle; Riddell, Stanley (May 2017). "Therapeutic T cell engineering". Nature. 545 (7655): 423–431. Bibcode:2017Natur.545..423S. doi:10.1038/nature22395. ISSN   1476-4687. PMC   5632949 . PMID   28541315.
  15. ↑ Rita Costa, A.; Elisa Rodrigues, M.; Henriques, Mariana; Azeredo, Joana; Oliveira, Rosário (2010-02-01). "Guidelines to cell engineering for monoclonal antibody production". European Journal of Pharmaceutics and Biopharmaceutics. 74 (2): 127–138. doi:10.1016/j.ejpb.2009.10.002. hdl: 1822/11473 . ISSN   0939-6411. PMID   19853660.
  16. ↑ Mansouri, Maysam; Fussenegger, Martin (2021-09-29). "Therapeutic cell engineering: designing programmable synthetic genetic circuits in mammalian cells". Protein & Cell. 13 (7): 476–489. doi: 10.1007/s13238-021-00876-1 . ISSN   1674-8018. PMC   9226217 . PMID   34586617. S2CID   238217661.
  17. ↑ Clause, Kelly C.; Liu, Li J.; Tobita, Kimimasa (April 2010). "Directed Stem Cell Differentiation: The Role of Physical Forces". Cell Communication & Adhesion. 17 (2): 48–54. doi:10.3109/15419061.2010.492535. ISSN   1541-9061. PMC   3285265 . PMID   20560867.
  18. ↑ Pettinato, Mark C. (2021-10-27). "Introduction to Antibody-Drug Conjugates". Antibodies. 10 (4): 42. doi: 10.3390/antib10040042 . ISSN   2073-4468. PMC   8628511 . PMID   34842621.
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