Establishing Chlamydomonas reinhardtii as an industrial biotechnology host

Abstract

Microalgae constitute a diverse group of eukaryotic unicellular organisms that are of interest for pure and applied research. Owing to their natural synthesis of value-added natural products microalgae are emerging as a source of sustainable chemical compounds, proteins and metabolites, including but not limited to those that could replace compounds currently made from fossil fuels. For the model microalga, Chlamydomonas reinhardtii, this has prompted a period of rapid development so that this organism is poised for exploitation as an industrial biotechnology platform. The question now is how best to achieve this? Highly advanced industrial biotechnology systems using bacteria and yeasts were established in a classical metabolic engineering manner over several decades. However, the advent of advanced molecular tools and the rise of synthetic biology provide an opportunity to expedite the development of C. reinhardtii as an industrial biotechnology platform, avoiding the process of incremental improvement. In this review we describe the current status of genetic manipulation of C. reinhardtii for metabolic engineering. We then introduce several concepts that underpin synthetic biology, and show how generic parts are identified and used in a standard manner to achieve predictable outputs. Based on this we suggest that the development of C. reinhardtii as an industrial biotechnology platform can be achieved more efficiently through adoption of a synthetic biology approach.

Keywords: Chlamydomonas reinhardtii; industrial biotechnology; metabolic engineering; rational design; synthetic biology; transgene expression.

Figure 1

The rise of C. reinhardtii as a model system for molecular biology.Major breakthroughs include first nuclear and chloroplast transformations (Boynton et al., ; Blowers et al., ; Kindle et al., 1989), mitochondrial transformation (Randolph-Anderson et al., 1993), systems analysis by proteomics (Hippler et al., 2001), the proposal of C. reinhardtii as a model organism (Harris, 2001), the production of the first therapeutic recombinant protein in the chloroplast (Mayfield et al., 2003), the sequencing of the nuclear genome (Merchant et al., 2007) and the publication of genome editing techniques (Sizova et al., ; Gao et al., ; Jiang et al., 2014) and the creation of a knock-out collection (Zhang et al., 2014).

Figure 2

Schematic of host optimisation via metabolic engineering and synthetic biology.The chronologic development of a biological host via metabolic engineering (blue), including the recent advent of systems biology and genome-scale metabolic models. Synthetic biology (red) has the potential to expedite this process in new biological systems, including microalgae, providing an opportunity to proceed directly from basic knowledge and capacity to the generation of highly optimised productive hosts.

Figure 3

Schematic representation of a synthetic biology workflow.An example investigation may aim to increase metabolite x through heterologous expression of gene y. Prior to this knowledge must be generated to describe the function of individual parts, promoters, 5′UTRs, transit peptides (TP), (trans)genes of interest (GOI) and 3′UTRs, and/or introns to enhance mRNA stability. Achieved through progress from design through construction application and analysis (including in silico models), when completed in a standardised manner this represents a single iteration of the cyclic process (a). The completion of several rounds of characterisation generates the knowledge required assign functional parameters to individual (generic) parts. These parts can be considered in a discrete manner (b) providing the opportunity to deconstruction of complex modules and recombine the parts a rational manner to generate novel functional devices (c) that have a predictable output.

Figure 4

A schematic of synthetic biology applied to create a circuit for enhanced lipid production.(a) A representation of a synthetic gene circuit in a starchless mutant cell line. Starch biosynthesis is complemented by the STA gene, in a light-regulated manner (Light; input). This regulation is overruled under nitrogen stress (nitrogen; input) through an OR logic gate. Down-regulation of starch biosynthesis coincides with the induction of native TAG biosynthetic pathways, providing a larger substrate pool for increased TAG production.(b) A schematic model employed to correlate the relationship of starch, nitrogen and lipids with the inputs of light and cellular nitrogen concentration.