March 24, 2016

expert reaction to a study on the synthesis of a minimal bacterial genome

A study by the research group of Craig Venter has been published in the journal Science in which they report the removal of a number of non-essential genes from the genome of a simple species of bacteria, a step towards reaching the minimum number of genes essential for life.

Prof Douglas Kell, Professor of Bioanalytical Science, University of Manchester, said:

“This is a very interesting paper and the amount of work involved was very considerable; in that sense it really is a ‘tour de force’.

“One point to make is that this ‘minimal genome’ is not absolute; a minimal genome is not that of a robust organism, and this is reflected via so-called ‘pseudo-essential genes’ that are not absolutely required, but are required for robust growth. That is why I tend to object to the term “a” minimal bacterial genome, as whether a genome is minimal (in terms of base pairs of genes) was not explored, and the minimality depends on physiological conditions. Also where there is redundancy in the synthetic genome, the authors recognised that both genes might individually appear non-essential, but only one could be removed (and either could); again this is not ‘a’ minimal genome (for n pairs of redundant genes there are 2^n possible ‘minimal’ genomes”.

“That said, there are three particularly interesting aspects. Firstly, of the roughly 473 genes left in the syn-3.0 organism (after around 440 were removed as being “non-essential”), 140 of those were of unknown function. This provides a clear indication of our imperfect knowledge even of the ‘minimal’ cell.

“Secondly, it is clear that the original organisms were carrying quite a high load of passenger genes, i.e. genes that could be dispensed with under at least some conditions. It is not clear as to the physiological (environmental) circumstances in which these might have been essential, but certain evolution theories see such genes as reservoirs of building blocks for novel genes.

“Thirdly, and somewhat related, is that the ‘stripped down’ syn-3.0 organism grew three times more slowly than the starting (syn-1.0) strain, but five times more quickly than the closely related M. genitalium organism. The fact that each of these ‘non-essential’ genes still contributed to growth rate meant that they gave an evolutionary advantage, albeit small. This is consistent with broadly neutral theories of microbial evolution, but we clearly have much to learn about all the genes that contribute to the control of growth rate.”

Prof John Ward, Professor of Molecular Microbiology, UCL, said:

“This latest paper from the Venter group revisits the work that led up to their papers on the first synthetic genome. Prior to those papers Hutchinson et al had been working towards answering the question ‘what is the smallest number of genes needed for free living organism (bacterium). This paper in Science returns to that question and uses the methodology that had been developed by them during the ensuing 6 years.

“Using gene synthesis and the yeast assembly methods they developed, the latest paper shows the synthesis of the minimal genome of Mycoplasma mycoides syn3.0. The answer is 438 protein and 35 RNAs all in a genome of 531 kilobases. They went back to using transposon mutagenesis (started by them in 1999) to investigate which genes were essential and found a class of genes named ‘i-genes’ that while not essential, caused growth impairments. They added these back to the designed minimal genome and also used a stepwise, iterative design cycle learnt from the last few years of synthetic biology ways of thinking.

“The outcome is a logically designed minimal genome that causes the bacterium to grow slower than the original synthetic M. mycoses syn1.0. They established a set of rules in the removal of non-essential genes which enabled them to speed up the designed deletion process. These largely gave viable bacteria but using biology e.g. Random mutations allowing growth in culture, gave small mutations that restored growth, in one case re-establishing a promoter that had been inadvertently deleted in the design.

“This paper sets the scene for starting to build minimal genomes for other bacteria. However as the authors of this paper acknowledge bacterial genomes such as B. sublilis and E. coli are large just so they have the metabolic and physiological flexibility to grow robustly and respond to many varied growth substrates and challenges. A truly minimal genome is an object lesson in what are the really essential functions for a freely living cell albeit one which has a huge number of requirements for complex carbon, nitrogen and essential small molecules such as vitamins.

“What are the minimal number of genes for a bacterium such as B. subtilis or E. coli to live on truly minimal media e.g. Glucose, ammonium ions, phosphate and sulphate with trace elements? Those questions can now begin to be designed into the next minimal genome projects along with many others such as the small photosynthetic genome.”

Dr Vitor Pinheiro, Lecturer in Synthetic Biology, UCL, said:

“The work is a remarkable tour de force and delivers the simplest free-living life form we know. The research started with a simple organism (Mycoplasma mycoides) and removed DNA sequences that were identified as unnecessary for growth in the standardised culture conditions being used – a point the authors also highlight. It shows that despite our efforts, there are still aspects of biology we don’t understand, exemplified by the 65 genes (nearly 14%) that have no known function. These knowledge gaps also point to the possible limitations of this research. For instance, the effect of genome organisation (position of individual genes along the chromosome) is not known and could in theory impact on the minimal genome size identified.

“Another example pertains how the research is conducted. It is an optimisation exercise, much like climbing a hill – as long as you keep going up, the expectation is that at some point you will reach the top. They have identified a quasi-minimum genome for mycoplasma but it is unknown if that is the same hill for all organisms – would a more complex bacteria require the same gene set? The fact that homologues for some of the essential genes are not found in other bacteria suggest that the minimal genome presented is not necessarily a universal answer.

“Syn3.0 is of considerable academic importance because it brings us closer to understanding what it takes to establish a free-living bacterium but its industrial impact will (at present) be limited. Syn3.0 is not optimised for growth in bioprocessors and any potential advantages (such as fewer side products, contaminants) are unproven.”

Prof. Paul Freemont, Head of Division of Molecular Biosciences, Imperial College London, said:

“One of the main goals of synthetic biology is to develop technologies and protocols to allow the construction of new biological cells and systems at the genetic level. This paper contributes a next step in the evolution of such techniques in enabling genome construction from synthetic DNA.

“I see this paper and work as a natural evolution of the synthetic biology field in relation to redesigning and building genomes and it does represent another landmark study in our ability to redesign natural systems at the genetic level using synthetic genes and design constraints. As such, the broader implications for society are our increasing ability to design and build genomes as well as edit them. This level of designed and targeted genetic interventions is unprecedented and opens up broader debates on what genomes and chromosomes should we be making synthetically.

“The concept of building a minimal cell is not a new idea and has been around a long time. There are many studies that have screened microbial cells for essential genes. However Venter uses a more systematic approach in line with synthetic biology, namely a Design Build Test (DBT) procedure. Interestingly, a completely designed minimal genome bottom up process actually did not work, suggesting that our knowledge of genomes, genes and cellular function are lacking. They instead used standard molecular biology techniques to identify which genes they could remove (nonessential) from their chosen genome and then reassembled those genes that are essential or are need for proper cell growth. This was done using the systematic DBT process described above.

“In terms of significance, it is a proof of concept but interestingly the minimal genome cell (based on Mycoplasma mycoses) they have produced is very similar to a natural version of mycoplasma named Mycoplasma genatalium (525 genes) which is the smallest known genome of a natural organism. The Venter minimal genome (473 genes) is around 52 genes less than a natural variant so in effect they have sort of done an accelerated reverse evolution experiment.

“The most significant aspect of the paper is the fact that it is possible to use systematic construction and protocols to build a synthetic genome from a set of genetic elements and then test these construction in cells and get them to work. However the future applications of such a technology are less clear given the new genome editing techniques that are now readily available and perhaps more accessible (CRISPr/CAS). In my opinion, while this work is fascinating, it likely will not provide the deep biological insights relevant to humans and mammals that some other significant genome projects, such as the synthetic yeast project, will, as the latter’s use of an accelerated evolution process will greatly increase our understanding of the relationships between gene organisation and living systems.”

‘Design and synthesis of a minimal bacterial genome’ by Hutchison III et al. will be published in Science on Thursday 24th March. 

Declared interests

Prof. Douglas Kell: No conflicts of interest to declare.

Prof. John Ward: I have no conflicts of interest with this work.

Dr Vitor Pinherio: I have no conflict of interest on the research being presented. I am employed by UCL and Birkbeck, who are not part of the research. My research funding comes from BBSRC and ERC, which are not funding that research. I am a member of the Royal Society of Biology but they are a learned society with no stakes on the research.

Prof. Paul Freemont: No conflicts of interest to declare