Host and Consortia Engineering spans the development of cell-free systems, synthetic cells, single-cell organisms, multicellular tissues and whole organisms, and microbial consortia and biomes. Development of robust cell-free systems capable of diverse reactions, domestication and use of many single-cell hosts, targeted modification of multicellular organisms, and manipulation of microbial consortia.
Engineering biology has delivered new tools to engineer microorganisms, plants, and animal cell lines. There are now entirely new ways to construct hosts to perform tasks that nature cannot accomplish. While many of these efforts have focused on ‘traditional’ hosts represented by model microbes like E. coli and S. cerevisiae, there is a wealth of potential if the unique capabilities of a broader range of microbes can be harnessed for useful purposes. These might include microbes that are photosynthetic, such as cyanobacteria1Markley, A. L., Begemann, M. B., Clarke, R. E., Gordon, G. C., & Pfleger, B. F. (2015). Synthetic biology toolbox for controlling gene expression in the cyanobacterium Synechococcus sp. strain PCC 7002. ACS Synthetic Biology [Electronic Resource], 4(5), 595–603. View publication., that can utilize non-sugar feedstocks such as methane or lignocellulose2Sundstrom, E. R., & Criddle, C. S. (2015). Optimization of Methanotrophic Growth and Production of Poly(3-Hydroxybutyrate) in a High-Throughput Microbioreactor System. Applied and Environmental Microbiology, 81(14), 4767–4773. View publication.
Haitjema, C. H., Solomon, K. V., Henske, J. K., Theodorou, M. K., & O’Malley, M. A. (2014). Anaerobic gut fungi: Advances in isolation, culture, and cellulolytic enzyme discovery for biofuel production. Biotechnology and Bioengineering, 111(8), 1471–1482. View publication., or that can be engineered to produce and secrete complex macromolecules more efficiently than model hosts. The possibility that stable multi-organism consortia and biomes of defined compositions could be constructed is particularly tantalizing.
Cell-free biology has been a staple of life science research for more than 50 years. More recent technological achievements have created cell-free gene expression systems that can produce protein at titers reaching grams/liter, that can be constructed from non-model organisms, and that are greatly minimizing the time needed to prototype systems and circuits via the design-build-test cycle. The model-driven construction of complex cell-free systems as hosts could result in programmable hosts for advanced biosensing, for on-demand biomanufacturing, and even for the bottom-up construction of synthetic cells.
At present, engineering complex functions in non-model hosts remains difficult because many of the tools and approaches developed for model organisms cannot be applied with the same efficacy in non-model organisms. The profound impact that the development of similar tools and approaches for engineering non-model organisms would bring to two of the world’s most important industrial sectors, energy and chemical production, more than justify attention to these challenges. It will be crucial to ensure that sufficient attention is given to biosecurity and biodefense risks that will rise along with improved capabilities for engineering diverse microbes.3National Academies of Sciences, Engineering, and Medicine, Division on Earth and Life Studies, Board on Life Sciences, Board on Chemical Sciences and Technology, & Committee on Strategies for Identifying and Addressing Potential Biodefense Vulnerabilities Posed by Synthetic Biology. (2018). Biodefense in the age of synthetic biology. Washington (DC): National Academies Press (US). View publication. The pharmaceutical industry has long biomanufactured fermentation-based natural products in microbes and therapeutic proteins from mammalian cell culture, both of which could be dramatically improved through advancements in host engineering.
Compared to engineering in single cell hosts, the state-of-the-art for engineering multicellular systems and organisms is less well-developed. To date, these approaches have been primarily aligned with natural reproduction, where genetically identical cells and tissues are created by editing the gametes or embryos of plants or animals. Gene editing methodologies introducing biochemical and molecular changes have already resulted in plants and animals with desirable characteristics that may be difficult to obtain through traditional breeding techniques. Further developing the capacity to reliably, and selectively, edit and modify multicellular eukaryotes could be transformative for a broad range of environmental and agricultural applications.
Host and Consortia Engineering focuses on the advancement of tools and technologies required for the characterization and engineering of host cells and organisms, and the integration and interaction of these systems and the environment. This includes developing methods, tools, and models to: 1) generate synthetic cells and cell-free systems to accomplish tasks and processes that cannot be accommodated by existing natural hosts; 2) enable organismal transformation, modification, and reprogramming of cellular chemistry, biology, and transport; 3) predict and integrate inputs and outcomes from environmental signals; and 4) enable the control, definition, and determination of differentiation, three-dimensional architecture, and other aspects of complex multicellular systems and biomes.
Cell-free biology is the activation of complex biological processes without using intact living cells. While used for more than 50 years across the life sciences as a foundational research tool, a recent technical renaissance has made possible high-yielding cell-free gene expression systems (that produce protein in excess of grams/liter), the development of cell-free platforms from non-model organisms, and multiplexed strategies for rapidly assessing biological design-build-test cycles. These advances provide exciting opportunities to profoundly transform engineering biology through new approaches to model-driven design of genetic circuits, fast and portable sensing of compounds, on-demand biomanufacturing, building cells from the bottom up (i.e., synthetic cells), and next-generation educational kits. Key opportunities lie in understanding, harnessing, and expanding the capabilities of biological systems. For example, through the use of cell-free systems to inform cellular design, the efficiency of DNA synthesis can be amplified so that many different genes (encoding many different biomolecules) can be synthesized. The ability of cell-free systems to transcribe and translate a piece of DNA without the need to clone it into a specific vector and transforming into an organism (with all the limits associated with DNA transformation efficiencies) enables cell-free systems to shorten the time to testing, thus speeding up the overall design-build-test cycle, and enabling the scalable prototyping of gene function. However, to date, there are limited numbers of large datasets available that allow comparison of part performance between the cell-free environment and in cells (in vivo). One need is to make data and models available to the community so that others can build and test improved models leveraging already developed systems and data. In another direction of research, there is a need to investigate the use of cell-free systems in manufacturing. Imagine how rapid access to vaccines and therapeutics in remote settings could change lives, and how new biomanufacturing paradigms suitable for use in low resource settings might promote better access to costly drugs through decentralized production. “Just-add-water” freeze-dried, cell-free systems could offer a disruptive approach to emerging and re-emerging diseases threats. It is a paradigm shifting concept.
Today, we have a number of host organisms for which we have a satisfactory understanding of their metabolism and sufficient genetic tools that we can use for reliable engineering. However, there are a number of applications that beg for more suitable chassis. There is a need to develop new tools for existing organisms, as well as entirely new platform organisms, and capabilities compatible with high-throughput, data-driven workflows that are becoming increasingly favored in industrial biotechnology. Key capabilities needed across organisms include reliable transformation methods for plasmid delivery and genome integration, and well-characterized genetic parts (including promoters and terminators) to regulate gene expression.4Johns, N. I., Gomes, A. L. C., Yim, S. S., Yang, A., Blazejewski, T., Smillie, C. S., … Wang, H. H. (2018). Metagenomic mining of regulatory elements enables programmable species-selective gene expression. Nature Methods, 15(5), 323–329. View publication. Predictive models for gene and protein expression-timing and -levels are also needed. Through the successful engineering of a broader library of host cells and multicellular organisms, we can increase the number of reporters and tools to better understand biology, establish new living sensors and sentinels, and expand the production of polymers, metabolites, and numerous other products.
One area of host engineering where progress still needs to be achieved is the on-boarding of engineered genetic sequences, circuits, and pathways into host cells. Crucial to this is a detailed understanding of the central dogma machinery and a systems-level understanding of host physiology such that genes can be reliably expressed and (synthetic) pathways incorporated without negatively impacting fitness. This will include an understanding and prediction of the endogenous gene regulator elements, including transcription factors, important DNA cis elements, regulatory RNAs, and the role of chromatin and epigenetic markings. Furthermore, there is the need for fully sequenced and annotated genomes for the majority of organisms; this can be extended to fully annotated metabolic pathways and enzyme activities. Advancements in genetic transformations (or viral transductions) and the ability to manipulate the genome, and ultimately, the ability to transplant chromosomes, would enable more robust design, control, and/or domestication of host organisms and their functional cellular machinery.