Research

Extreme makeover yeast edition: de novo synthesis of five chromosomes

Source: Rainis Venta, Creative Commons Attribution-Share Alike 3.0

Source: Rainis Venta, Creative Commons Attribution-Share Alike 3.0

– What I cannot create, I do not understand.

This sentence taken from Richard Feynman’s board at the time of his death essentially captures, if applied to a biological frame, the holy grail of synthetic biology; the understanding of life to such an extent that living systems can be rationally designed to gain and perform specified functions. Synthetic biology is perceived by many as an extension of metabolic engineering, where organisms are modified with end goal to produce a product. This is however part of the story, and the real strength of synthetic biology is the understanding via synthesis, the decomposition of a system to its building blocks and recombination of parts from the bottom up to elucidate the biological complexity.

The construction of a bacterial minimal genome, namely the reduction of the genetic content of Mycoplasma genitalium to 473 genes (1), was hailed as a landmark about a year ago. Last week, a series of seven research articles were published, describing the advancements in the construction of yeast synthetic chromosomes—and as the articles were published in Science, I use this journal’s citation format throughout this blog.

This work is a result of a large consortium, Synthetic Yeast 2.0 , which aims to design and implement a fully synthetic genome of Saccharomyces cerevisiae. The project has existed for several years, and started with the construction of a chromosome’s arms (2), and the first reported synthetic yeast chromosome (3). The recent publications report the synthesis of five more yeast chromosomes, totaling the synthetic sequences to more than one third of the full genome (410). The workflow started from the generation of 700-2000 bp DNA blocks, which are in vitro or in yeast assembled into 10 kb ‘chunks’. A number of chunks are chemically ligated to 30-60 kb ‘megachunks’, which are inserted sequentially into the genome.

Source:  Flickr  CC BY-NC 2.0

Source: Flickr CC BY-NC 2.0

The new chromosomes contain slight modifications from the native ones (4). The tRNA genes were removed from their original loci, and relocated to a specialized neochromosome. Repeating sequences were also removed, the TAG stop codon was replaced with TAA—leaving TAG open for repurposing, such as incorporation of a non-canonical amino acid. The design took place in BioStudio, an open-source computational framework that allows working in genome-scale (4).

Maybe the most important intervention is the inclusion of recombination sites every 10 kb and after every non-essential open reading frame. Consequently, one can implement SCRaMbLE (synthetic chromosome rearrangement and modification by loxP-mediated evolution) and produce numerous strains with rearranged genetic loci, thus acquiring a powerful tool for directed evolution and functional analysis experiments.

What comes next? According to Jeff Boeke, the consortium expects to have a fully synthesized genome by the end of the year (11). However, having obtained all synthetic chromosomes does not automatically mean that a fully ‘synthetic’ yeast strain will become immediately available;  although strains incorporating one heterologous chromosome do not display a particular phenotype, a strain with three of its chromosomes replaced had a growth defect (5). Issues arising from chromosome interactions and telomere function already show, while more unknown challenges are sure to appear.

The long-term goal of this colossal undertaking is to allow research labs and organizations to routinely use custom-made organisms. This dream seems to come closer and closer, but I do not think it will happen within the next few years. The cost for synthesizing the whole S. cerevisiae genome is estimated to 1-1.5 M dollars, without taking into account salaries and maintenance (4).  But I believe that the scientific insights obtained by this project will affect synthetic biology in multiple ways. And then, why not expand to more organisms? The recent proposal to synthesize a human genome gained a lot of publicity (and controversy).  A minimal or synthetic plant genome [see Marchantia polymorpha, a small liverwort (12), and Physcomitrella patens, a moss where homologous recombination happens with the same efficiency as in yeast (13)] will be leap forwards in understanding and harnessing photosynthesis…

Disclaimer: This post originally appeared at PLOS synbio (link) and is reproduced with permision

References

  1.    C. A. Hutchison et al., Design and synthesis of a minimal bacterial genome. Science. 351, 6253–6253 (2016).
  2.     J. S. Dymond et al., Synthetic chromosome arms function in yeast and generate phenotypic diversity by design. Nature. 477, 471–476 (2011).
  3.     N. Annaluru et al., Total Synthesis of a Functional Designer Eukaryotic Chromosome. Science. 344, 55–58 (2014).
  4.     S. M. Richardson et al., Design of a synthetic yeast genome. Science. 355, 1040–1044 (2017).
  5.     L. A. Mitchell et al., Synthesis, debugging, and effects of synthetic chromosome consolidation: synVI and beyond. Science. 355 (2017), doi:10.1126/science.aaf4831.
  6.     G. Mercy et al., 3D organization of synthetic and scrambled chromosomes. Science. 355 (2017), doi:10.1126/science.aaf4597.
  7.     Y. Wu et al., Bug mapping and fitness testing of chemically synthesized chromosome X. Science. 355 (2017), doi:10.1126/science.aaf4706.
  8.     W. Zhang et al., Engineering the ribosomal DNA in a megabase synthetic chromosome. Science. 355 (2017), doi:10.1126/science.aaf3981.
  9.     Y. Shen et al., Deep functional analysis of synII, a 770-kilobase synthetic yeast chromosome. Science. 355 (2017), doi:10.1126/science.aaf4791.
  10.   Z.-X. Xie et al., “Perfect” designer chromosome V and behavior of a ring derivative. Science. 355 (2017), doi:10.1126/science.aaf4704.
  11.   A. Maxmen, Synthetic yeast chromosomes help probe mysteries of evolution. Nature (2017), doi:10.1038/nature.2017.21615.
  12.   C. R. Boehm, B. Pollak, N. Purswani, N. Patron, J. Haseloff, Synthetic Botany. Cold Spring Harb. Perspect. Biol., a023887 (2017).
  13.   B. C. King et al., In vivo assembly of DNA-fragments in the moss, Physcomitrella patens. Sci. Rep. 6, 25030 (2016).

Synthetic metabolic route allows optimal use of xylose for bio-production of chemicals

Scientists from Toulouse, France, genetically modified Escherichia coli in order to assimilate (d)-xylose and direct it towards commercially interesting compounds in a novel manner.

 

Early, this July I attended the Synthetic and Systems Biology School in Taormina, Italy. Apart from the lectures and poster sessions, selected talks from the participants were also presented. Amongst others, I distinctly remember two postdoctoral researchers, Ceren and Débora, who enthusiastically described their work about producing interesting chemicals in E. coli. I was therefore very pleased when I saw their paper published in ACS Synthetic Biology a few weeks ago.

     
  
 
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      Native (blue) and synthetic (green) (D)-xylose assimilation pathway. The chemicals of interest (pink) and the first steps of glycolysis are also noted. The green numbers represent the heterologous enzymes that were used. Figure adapted with permission from Cam et al., 2015. Copyright American Chemical Society (2015).   

 

Native (blue) and synthetic (green) (D)-xylose assimilation pathway. The chemicals of interest (pink) and the first steps of glycolysis are also noted. The green numbers represent the heterologous enzymes that were used. Figure adapted with permission from Cam et al., 2015. Copyright American Chemical Society (2015).   

This article narrates a nice metabolic engineering approach to introduce a novel (D)-xylose assimilation metabolic route in E.coli. Naturally, E.coli phosphorylates (D)-xylose at C5 and incorporates it into the main metabolism. What the designed pathway does is assimilate the (D)-xylose through C1 phosphorylation, bypassing the pentose phosphate pathway and its native regulation. The sugars can now be introduced in the glycolysis pathway and thus be used for organism growth, while the desired chemical products are produced in a more targeted manner by expressing the respective biosynthetic enzymes. The theoretical product yields were computed and compared to the native metabolism and other engineering approaches, the novel enzymes that need to be expressed were identified and characterised, and microarray experiments and metabolite analysis were carried out to study the organism’s response to the new pathway. Finally, selected chemicals were produced by dedicated strains that performed impressively in terms of yields and product concentration.

So what's missing? It would be informative to see how the strains behave and what titres can be achieved in large batch-fermentation experiments. There is a small loss of growth rate of the modified strains. Moreover, it would be interesting to optimise the strains further and to test how generic this approach is by transferring the pathway to other organisms, such as baker’s yeast. Overall, however, this research paper is an easy-to follow biotechnology story that begins with the motivation and design and reaches the desired outcome of increased product formation.


There are two take-home messages that I would like to address after reading this article. The first comes from the use of (D)-xylose as a substrate. The rationale behind it is that xylose constitutes a large proportion of the unused cellulose and hemicellulose biomass that are byproducts of bio-refinery. This is a prime example of synthetic biology employed in sustainability efforts, where a waste product is converted into commercially interesting compounds. Those formed products are currently available in industrial scale as fossil fuel byproducts and their production using E.coli is an environmentally friendlier alternative. However, the bio-sustainability argument has some pitfalls and needs to be employed carefully. A fermentation production is itself an energy consuming process. Also, the use of sugars should not directly compete with or use up agriculture resources. Nevertheless, it is my opinion that the industry needs to disengage fast from oil as a feedstock, and synthetic biology is a powerful tool that can provide novel alternatives.

A second point is the use of systems biology together with synthetic biology. Taking into account that a system cannot be simply described by the addition of its components but also requires the interactions between those parts, likewise a bioengineering approach cannot rely on the simple expression of a heterologous pathway and the optimisation of the participating enzymes. This principle is illustrated in this article, where metabolic modelling and microarray analysis that provide invaluable insights during the pathway design and the strain optimisation steps respectively. Interdisciplinary research is also present in this work, as computational science, bioinformatics, and analytical chemistry are employed together with molecular biology and biochemistry. It becomes more and more obvious that single-focus and narrow approaches are getting obsolete. The new generation of scientists needs to speak the language of and understand collaborators coming from different backgrounds. Synthetic biology, which in principle combines parts to obtain new properties and novel functions, cannot fall behind in combining different disciplines in a way that facilitates research and strengthens innovation and creativity.

 

 

Research Paper: 

Yvan Cam, Ceren Alkim, Debora Trichez, Vincent Trebosc, Amélie Vax, François Bartolo, Philippe Besse, Jean Marie François, and Thomas Walther (2015) Engineering of a Synthetic Metabolic Pathway for the Assimilation of (d)-Xylose into Value-Added Chemicals. ACS Synthetic Biology DOI: 10.1021/acssynbio.5b00103

 

Written by:

Konstantinos Vavitsas

Konstantinos is a PhD student at the University of Copenhagen, working on the photosynthetic production of high-value compounds. 

Edited by: Devang Mehta

 

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