key: cord-0926270-3e7828cd
authors: Vashee, Sanjay; Arfi, Yonathan; Lartigue, Carole
title: Budding yeast as a factory to engineer partial and complete microbial genomes
date: 2020-09-21
journal: Curr Opin Syst Biol
DOI: 10.1016/j.coisb.2020.09.003
sha: d167d1f82f6d0c19b35b400480216d42a6bad63e
doc_id: 926270
cord_uid: 3e7828cd

Yeast cells have long been used as hosts to propagate exogenous DNA. Recent progress in genome editing opens new avenues in synthetic biology. These developments allow the efficient engineering of microbial genomes in Saccharomyces cerevisiae that can then be rescued to yield modified bacteria/viruses. Recent examples show that the ability to quickly synthesize, assemble and/or modify viral and bacterial genomes may be a critical factor to respond to emerging pathogens. However, this process has some limitations. DNA molecules much larger than two megabase pairs are complex to clone, bacterial genomes have proven difficult to rescue, and the dual-use potential of these technologies must be carefully considered. Regardless, the use of yeast as a factory has enormous appeal for biological applications.

Laboratory workhorses such as Escherichia coli, Bacillus subtilis, and Saccharomyces cerevisiae have proved invaluable since they have been used as hosts to propagate and edit genetic material of other organisms. Initially, relatively small DNA fragments were cloned but, over time, this size has gradually increased and now reach the megabase range, including complete microbial genomes of native or synthetic origin [1] [2] [3] [4] .

In this review, we will discuss the ever-expanding use of yeast as an efficient propagating and editing factory for the genomes of various microbial species. This process involves the cloning or assembly of a full or partial genome into yeast, its engineering and rescue into a suitable recipient cell to rescue the designed function or live cells (Figure 1 ). This approach can be a novel method to study intractable organisms, genetically edit intractable organisms or build new living systems for basic and applied biology. Each component of the yeast factory cycle is detailed below. Potential barriers are discussed as well as the risks/benefits of such an approach.

Yeast has long been used as a host to clone DNA molecules, either as Yeast Artificial Chromosomes (YACs) or Yeast Centromeric plasmids (YCps), from a wide range of donor organisms. Many of the early examples involved cloning genomic DNA fragments from a range of eukaryotic[5,6] and prokaryotic species[7] as well as viruses [8, 9] for genome analysis, including physical maps of complex genomes and gene function studies. However, several issues of chimeras and instability of some cloned heterogeneous DNA in yeast reduced its use, while vectors in bacterial systems such as cosmids and bacterial artificial chromosomes (BACs) gained favor for genome analysis and development of reverse genetics tools.

Over the past decade, yeast has re-emerged as an attractive genome engineering host, bolstered by a groundbreaking experiment to assemble and boot-up the first "synthetic cell" [1] , and subsequently, by the cloning of several partial and full bacterial or eukaryotic genomes as well as assembly of viral genomes ( Table 1) .

Multiple approaches can now be used to clone large DNA fragments in yeast, including complete megabase-sized genomes. Depending on the characteristics of the donor organism or downstream applications, some approaches enable the cloning of native genomes whereas others permit the simultaneous cloning, editing or assembly of entire genomes from PCR-amplified, fully synthetic or TAR-cloned fragments. All of these methods require the presence of certain J o u r n a l P r e -p r o o f yeast genetic elements, including an autonomously replicating sequence (ARS) a centromere and a selection marker in order to replicate and maintain the cloned DNA. An ARS is not necessarily required for genomes with low G+C% (<40%) as the AT-rich consensus motif may naturally occur in their sequence ( Figure 1A) . These elements can be added before cloning, as a plasmid integrated in a bacterial genome. Then, the newly marked genome is isolated and transferred intact into yeast spheroplasts using the conventional yeast transformation procedure [10, 11] or by fusing the bacterial cell to yeast[12] ( Figure 1B) . The advantage of this approach is the selection of vector insertion sites that do not interfere with bacterial viability, which is convenient for genomes that are meant to be transplanted into a recipient cell to produce live cells. Another approach, TAR-cloning, exploits yeast's ability to efficiently recombine DNA fragments if they contain ends (~60 bp) that are homologous to a target sequence. In this case, the genome is isolated, linearized in vitro by a restriction enzyme or using the CRISPR-Cas9 system and cotransformed into yeast together with a linear yeast vector containing homology sequences[13-16] (Figure 1B) . A variation of this approach is CReasPy-Cloning which enables the simultaneous cloning and engineering of megabase-sized genomes in yeast[17] ( Figure 1B) . The TAR-cloning approach can be extended so that the yeast transformation is carried out with multiple overlapping fragments, either PCR-amplified, synthetic or previously TAR-cloned ( Figure 1C) , allowing for genome-wide engineering of microbial genomes.

Using these methods, many bacterial and viral genomes, both native and synthetic, have been cloned or assembled in yeast. Key examples are shown in Table 1 . For future target genomes, certain considerations can be factored into the choice of the cloning method. These include whether the organism is cultivable, transformable and/or has genetic tools. If the organism has all of these characteristics, then any of the outlined approaches can be used. For other organisms lacking one or more characteristics or for large scale editing, the in vitro or assembly methods are more appropriate.

Over the last decade, the cost of DNA synthesis has drastically reduced, almost reaching the 0.01$/base bar. Such low costs have enabled the engineering of organisms with fully synthetic DNA, with recent examples of re-coded or re-organized genomes [3, 4] . As a result, genome editing can now be performed by assembly of synthetic fragments in yeast. This approach remains nonetheless costly at the megabase scale and may be excessive for small, localized editing tasks. Therefore, depending on the need, it may be more appropriate to use one of the 

Once a microbial genome has been modified in yeast, it can be "rescued" using various approaches. For this review, "rescue" is defined as the process by which the cloned genome isolated from yeast is converted into the biological entity it encodes.

Since viruses are generally simpler systems, they are relatively easy to rescue (Figure 1E, For modified bacterial genomes, the rescue is more difficult, due in part to larger genome size, more complicated pathways, and cellular structure. One possibility to rescue a whole genome is to isolate intact edited microbial chromosomes from yeast and transfer them into recipient cells ( Figure 1E, left panel) 

Although potentially extremely powerful, the in-yeast cloning and editing of microbial genomes comes with a few drawbacks and bottlenecks.

Based on previous experience, we expect that the cloning of genomes in yeast to be more readily achievable compared to the subsequent rescue of the genomes. In addition, viral genomes have also proven much easier to clone and rescue than their bacterial counterpart. In-yeast engineering. Current methods are effective to perform a few modifications at a time.

TAR assembly alleviates this issue to some extent but it is somewhat limited by the number of fragments that can be used as well as the efficiency of homologous recombination. A potential improvement may be the use of yeast mutants impaired in competing repair pathways, such as non-homologous end-joining, as engineering hosts for microbial genomes. Rescue of viral genomes. For the most part, rescue of viral genomes is not a major concern.

However, there are still a few viruses, such as African swine fever virus (ASFV), whose is that DNA uptake may be limited by transformation efficiency and cell surface structure. To bypass these obstacles, improvement of methods to make spheroplasts/protoplasts in target organisms may be used to remove cell walls to increase DNA uptake. In addition, other DNA transfer methods, such as conjugation, can be used to transfer a genome from the donor species to the recipient. In conclusion, while it is clear that budding yeast is a powerful engineering factory, there is still room for improvement to fulfil its use for synthetic biology applications.

Creation of a bacterial cell controlled by a chemically synthesized genome

Design and synthesis of a minimal bacterial genome

Total synthesis of Escherichia coli with a recoded genome

have used assembly of synthetic E. coli 100kb fragments in yeast as an intermediate to generate an E., coli strain that used only 61 codons for protein synthesis

Chemical synthesis rewriting of a bacterial genome to achieve design flexibility and biological functionality