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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

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