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Fast-Growing Engineered Microbes: New Concerns for Gain-of-Function Research? 2018

https://www.frontiersin.org/articles/10....00207/full

Here, we review recent research and development of engineered fast-growing strains in industrial biotechnology, with a special focus on vaccine production using (synthetic biology) engineered pathogenic strains. We will discuss whether this represents a security concern and whether the industrial biotech sector needs to pay more attention to issues of Gain-of-Function (GoF) while developing and harnessing these fast-growing microbes.

Mycoplasma pneumoniae



Mycoplasma pneumoniae contains a very small genome, with only 816,394 base pairs (Himmelreich et al., 1996). Due to the small genome size, the genus of mycoplasma has been studied extensively, particularly the genomes of M. genitalium, M. mycoides, and M. pneumoniae. The bacteria from this genus attracted attention, not only as model organisms for the chemical synthesis of a full genome (Gibson et al., 2008; Lartigue et al., 2009; Hutchison et al., 2016), but also as model organisms to study factors relevant to cell growth. Among them, M. pneunomiae is of particular interest for cell growth. Despite its very small genome, M. pneumoniae is not simple – the cellular functions are rather complex, e.g., many M. pneumoniae proteins are part of more than one cellular machine or protein complex (Juhas et al., 2011). It is predicted that the genome of M. pneunomiae contains 677 ORFs, more than 75% of which show similarity to genes/proteins of other organisms. The alignment of the genome of M. pneumoniae with other organisms revealed that the reduced genome size of M. pneumoniae might result from the evolution of ancestral bacteria losing certain anabolic and metabolic pathways. Thus, although it can self-replicate, M. pneumoniae requires exogenous essential metabolites to survive (e.g., a certain amino acids and lipids). Investigating the co-evolution of mycoplasma and its hosts that led to a reduction of genome size, might hold the key to understand its relative slow growth. Artificially reduced genomes, in contrast, have recently been shown to exhibit a faster growth and higher mutation rate, demonstrating that there is no simple relation between genome size and growth rate (Nishimura et al., 2017). To better understand the minimal cellular machinery required for life, tandem affinity purification-mass spectrometry (TAP-MS) was applied to study the proteome organization in a genome-reduced bacteria based on the genome of strain M 129 (Kuhner et al., 2009). 62 homo-multimeric and 116 hetero-multimeric soluble protein complexes were identified, of which more than half were novel. By combining the pattern recognition and classification algorithms, the protein complexes identified by TAP-MS could be underpinned within the whole cell, while matching with the existing electron tomography. This knowledge from proteomics would help to better pinpoint the genes for cellular function, which could be harnessed for complementing the lost or weakened cellular functions linked to slow growth. The cellular blueprint of M. pneumoniae could allow scientists to complement those elements needed to facilitate cell growth by full analysis in silico of essentiality, as well as metabolomics, transcriptomics, and proteomics data (Eisenstein, 2017). All findings on the essential genes and interactions on metabolic and transcriptional networks provide clues of counter strategies to compensate the lost cellular function resulting from evolution – adding supplement genes or elements to the already reduced genome in order to enhance the growth rate. These strategies include supplementing the organisms with growth related genes from the fast-growing strain from the genus of mycoplasma or other fast-growing microbes (e.g., the common genes PHX, found in other fast-growing bacteria, as mentioned above), and complementing those metabolic functions the slow growth strains lack, such as lipid synthesis.


The European Commission funded Horizon 2020 project “MycoSynVac”1 is developing animal vaccines based on highly engineered pathogenic mycoplasma strains. To reach this goal, a number of obstacles have to be overcome, such as reducing pathogenicity, developing fast-growing strains for efficient production, implementing biosafety circuits to better control the new strains and developing a set of genetic engineering techniques (Figure 1).


FIGURE 1. Overview on using synthetic biological approaches to engineer mycoplasma vaccine strains with fast-growing properties. Image created by Biofaction, and used with permission.

The third GoF issue of fast-growing microbes concerns the research on making (originally) pathogenic microbes grow faster for research and/or vaccine production purposes, as in the case of mycoplasma. Given established evolutionary principles, there must be a balanced interaction between the infected host and the pathogenic microbes with slow growth – the microbes grow at a slow rate in order to not kill their hosts rapidly, to be persistent and/or to co-exist with the hosts. Within the genus of mycoplasma, there are fast-growing species that are mostly less virulent than the slow growth species. And the cell growth related genetic elements from these fast-growing species would be the elements that would graft to the slow growth species, taking examples of the research on fast-growing mycobacterium and mycoplasma strains. The risk from this type of research is that, if such balance is altered by granting the pathogenic organisms a fast-growing feature, the pathogenesis of infection would also change, with unknown consequences. Thus, the engineered fast-growing variants of the pathogenic species would have to be assessed on how virulent the fast-growing variants are compared to their wild type species in the host or potential host. The assessments of the potential newly gained pathogenesis should include both in vitro and in vivo analysis, as well as environmental assessments (the ability for pathogens to survive outside human hosts). Given how complicated and expensive it is to conduct these assessments, it would be better to take the security issues into consideration beforehand and implement the built-in safety controls. Some of the fast-growing microbes in development were also tested with built-in genetic safety guards as mentioned above for S. cerevisiae and M. pneumoniae. The GoF concern on this type of research might come from the possible unintended outcome of novel vaccine development- when turning (relatively) slow growing pathogens into fast-growing non-pathogenic variants, one can not rule out the risk that somewhere in the development process (or afterwards) a fast-growing pathogen could be created unintentionally. Given that very few pathogens get their pathogenicity via fast growth, and that many pathogens use slow growth as their evolutionary strategy, we do not expect this to be a major issue. To be sure, however, the R&D process should be aware of the possibility of this risk and implement measures to detect and minimize these risk.