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Host engineering

Generating single cell factories

Introduction

Synthetic biology doesn’t stop at the level of molecular systems. To expand the scope of this project we have chosen for a multi-level approach, in which we are working on biobricks, proteins, a pathway and also our host. In order to achieve the latter two strategies have been conceived of. In the first we have chosen to harness the power of directed evolution, a powerful tool that not often used in this competition. An explanation for this might lie in the fact that this approach is only semi-rational at best, however we like to argue that this does not make it any less of a powerful mechanism, and neither within the field of synthetic biology. Nonetheless, in order to explore new territories we have chosen for a second, fully rational approach in which we analyze the transcriptome of two distinct phenotypes; the mycelial and the single cell.

A scientific paper from 1971

Anderson, J. G. and J. E. Smith (1972). "Effects of Elevated-Temperatures on Spore Swelling and Germination in Aspergillus Niger." Canadian Journal of Microbiology 18 (3): 289-297.

Anderson and Smith found that at 44C germ-tube formation was completely inhibited in Aspergillus niger, although spherical growth could occur over a prolonged period to produce large spherical cells. More generally, there are more dimorphic fungi that display such a distinctive phenotypic transition at elevated temperatures. This made us ponder and let to the idea of generating a single cellular phenotype.

Rationale

The secretion capacity of Aspergillus niger is the feature mainly contributing to its status as excellent industrial workhorse. However, when we investigate this process in more detail, we find that only the hyphal tips of the mycelium are actively secreting. Since the vegetative mycelium poses a burden, generating single cells.

From a process-oriented perspective the potential of host engineering is also interesting. A single cellular phenotype will result in a higher surface to volume ratio, thus effectively increasing the exchange area, while at the same time it annihilates pore clogging. A unicellular strain could be cultured in a similar fashion as a yeast, such as Saccharomyces cerevisiae . The fact that this research intersects with a fundamental topic, the evolution of multicellularity, makes it even more interesting.

Figure 1) Schematic representation bioreactor

Strategy 1: Directed evolution

The power of experimental evolution with regard to complex adaptations has been demonstrated in recent research, allowing acquisition of multicellular Saccharomyces cerevisiae. The other way around one can envision evolution of a unicellular mutant of the filamentous fungus Aspergillus niger. Obtaining such a mutant could help greatly in identifying genes related to multicellularity in complex fungi, a field that is largely unexplored.

Aim

Obtain a single cell phenotypic Aspergillus niger strain by directed evolution

Approach

In this approach we select for cells with a reduced mycelial cohesiveness by using filters with different pore-sizes. The procedure is iterative; we grow the cells, vortex them, filter them and then grow the cells that were able to get trough the filter for the next round.


Figure 2) The iterative procedure in this directed evolution approach.

Research Methods

Mutagenised spores of A. niger N593 from which evolved strains were cultivated are stored at -80°C according to Appendix C for possible genomic comparison. Spores from A. niger N593 spores were mutagenized by exposure to a Philips TUV 30W lamp for 10 to 60 seconds. In order to apply selective pressure for the desired phenotypic trait, filters with different pore sizes are used. For filter steps in the evolution experiments BD Falcon® Cell Strainers of 40, 70 and 100 µm pore size and SEFAR NITEX® 3-150/50 nylon filter gauze of 150 µm pore size were used. Stainless steel mesh ‘cups’ with pore size 20 µm from Anping Yuansheng Mesh Cooperation were also used.


Figure 3) Materials used as filters

Strategy 2: Comparative transcriptomics

RNA sequencing is a next generation sequencing technology that is rapidly replacing the conventional DNA microarrays. Because, unlike microarrays, RNA sequencing does not rely on probes or primers, there is a smaller bias. It allows for more than analyzing differential gene expression, as it also allows for discovery of novel RNAs and analysis of isoforms of genes. Another advantage over DNA microarrays of RNA sequencing is that data can be reanalyzed once more information on the transcriptome becomes available. Combined with the existence of dimorphisms this allows for an interesting opportunity when it comes to the investigation of the genes involved in this phenotypic distinction.

Aim

Finding sets of candidate genes causative to the single cell phenotype in Aspergillus niger by transcriptome analysis.

Approach

Spores of A. niger N400 are grown at three distinct conditions. Growing A. niger in liquid medium at 45C will yield a single cell phenotype. When grown at the same temperature on a solid medium mycelium is formed. Mycelium is also formed when A. niger is grown at 30C. Thus, by varying temperature or phase-state of the medium one can obtain either a multi- or single cellular phenotype. Transcriptome analysis allows identification of the transcripts that are uniquely present or absent in the single cell phenotype. This knowledge can be used to genetically modify A. niger to obtain a single cell phenotype at a broad range of conditions, such as at room temperature.



Figure 4) Mapping reads onto the Aspergillus niger reference genome allows for discovery of patterns in gene expression that are unique to the single cell phenotype.

Research Methods

After cultivation of the Aspergilli at different conditions, RNA needs to be extracted according to Appendix H. This RNA will be sent out to a sequencing company and after some amount of time the data will be returned. For the analysis of the data a transcriptomics pipeline needs to be constructed. Since there is a reference genome available we don’t need to perform a de novo assembly.

Tophat
RNA-Seq generates millions of short sequence reads and therefore the mapping of RNA-Seq is an intensive computational task. This process is performed with the use of specific software. One of these software packages is Tophat. Most mapping algorithms depend on the known splice junctions. Tophat on the other hand is designed to map RNA-Seq reads without relying on these known splice junctions. Tophat detects splice sites ab initio by identifying reads that span exon junctions. The Tophat algorithm first maps the non-junction and short reads using Bowtie. Next the program creates a consensus of mapped reads. Then TopHat breaks up the initially unaligned reads into smaller pieces, Constructing a seed table and tracking of possible junctions, enabling them to be matched onto the reference genome.

Cufflinks
Cufflinks was developed to investigate differential gene expression. This program is designed to calculate the abundances of transcripts constructing an overlap graph. This graph is used to calculate the maximum likelihood of transcripts resulting in Fragment Per Kilobase per Milion (FPKM) values. This value, which represents the quantity of mapped fragments relative to the length of the transcript, normalizes for transcript length and machine yield such that expression of different transcripts (transcripts.gtf) can be compared. Expression levels of genes can simply be determined by summing the FPKM values of their respective isoforms.

Downstream analysis
The statistical program ‘R’ was used to visualise the final data output (http://www.r-project.org/). Extraction of gene ID’s from the fasta files into a text file allowed for manual input of this data into cytoscape (www.cytoscape.org). A hierarchical network can be inferred for all three Gene Ontology (GO) annotations for visualization of overrepresented terms in specific (sets of) nodes.