Update on designing and building minimal cells

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Minimal cells comprise only the genes and biomolecular machinery necessary for basic life. Synthesizing minimal and minimized cells will improve understanding of core biology, enhance development of biotechnology strains of bacteria, and enable evolutionary optimization of natural and unnatural biopolymers. Design and construction of minimal cells is proceeding in two different directions: ‘top-down’ reduction of bacterial genomes in vivo and ‘bottom-up’ integration of DNA/RNA/protein/membrane syntheses in vitro. Major progress in the past 5 years has occurred in synthetic genomics, minimization of the Escherichia coli genome, sequencing of minimal bacterial endosymbionts, identification of essential genes, and integration of biochemical systems.

Introduction

Design-based engineering of biological systems (also known as synthetic biology) tests understanding of the living world and harnesses its diverse repertoire to solve society's problems [1, 2]. Ideally, an engineered system should be functionally robust and predictable. Yet these features are difficult to achieve when engineering biology [3] because of the poorly understood complexity of even the simplest single-celled organisms. An enticing way to simplify cellular complexity, test understanding, and potentially facilitate engineering is to synthesize minimal cells [4, 5, 6, 7]. Forster and Church reviewed plans of others to minimize small bacterial cells (in vivo ‘top-down’ approach) [5] and proposed detailed plans for synthesizing a minimal cell from biomolecular parts (in vitro ‘bottom-up’ approach) [4]. Here, we highlight progress, challenges, and prospects since these two reviews.

Section snippets

New tools

Minimal cells require minimal genomes, and minimal genomes require design, construction, and manipulation tools at an unprecedented scale. Great progress has been made in genome construction by the J. Craig Venter Institute (JCVI; Rockville, MD, USA). JCVI constructed the 582 kilobase pair (kbp) genome of Mycoplasma genitalium, the smallest known genome of a bacterium capable of independent growth [8]. This was done by commercial gene synthesis from oligodeoxyribonucleotides (oligos) and then

Top-down approach: in vivo reduction

Even the most highly reduced genome of M. genitalium contains 100 individually dispensable genes out of 528 annotated genes [20], so streamlining down to only essential genes is one route to minimal cells. So far, significant minimization has been carried out only in organisms with larger genomes such as E. coli (4640 kbp; 4434 genes) and Bacillus subtilis (4216 kbp; 4245 genes) aided by known sequences of closely related genomes. Genome reduction by up to 30% has proven surprisingly successful

Bottom-up approach: in vitro construction

The alternative direction to a minimal cell is bottom-up: synthesizing self-replication by pooling together essential purified biological macromolecules, their genes, and their small molecule substrates [4]. By this approach, cellular overhead including genes of unknown function can be removed, the system can be readily manipulated and tuned, and all of the components can be defined. One possibility is a DNA/RNA/protein system derived from the core replication machinery of today's simplest

Prospects for biotechnology

Minimal cell syntheses are still in their formative stages where the main rewards are new molecular tools and a better understanding of the core genetic and biochemical systems necessary for basic life. But applications in biotechnology are close at hand. Based on the improved stability, growth, and protein production of E. coli and other biotechnology workhorses upon reducing their genomes [21••, 22, 23], further minimized strains should replace most current commercial bacterial strains.

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

Acknowledgements

We are grateful to George Church for advice and comments on the manuscript and John Glass, John McCutcheon, and Michael Sismour for comments on the manuscript. This work was supported by the National Institutes of Health and National Academies Keck Futures Initiative (to MCJ and ACF), the National Science Foundation (to MCJ), and the American Cancer Society (to ACF).

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