Bi9540 Biotechnology and practical use
of algae and fungi
Lecture 13 – Engineering of algae and fungi
Metabolic engineering and synthetic biology
A) Synthetic biologists use a variety of parts to adjust the functioning of metabolic pathways. Transcription machinery, enzyme
promoters, ribosome binding sites (RBS), and translational machinery can be modified to adjust the concentration of an enzyme.
RNA devices can modulate mRNA degradation and translation efficiency. Pathway enzymes can be assembled on scaffolds to
optimize the spatial organization of a pathway. Genome editing approaches can be used to adjust host metabolism to improve flux
through the target pathway. (B) A “pipe” of key pathway enzymes can be tuned to increase product titers. In this conceptual
example, enzyme flux is represented by the size of the gray arrows. Metabolite concentrations are represented by the size of the
circles between enzymes. In this example, increasing the concentration of the second and third enzymes in the pathway increases
the titer of the product. Note that decreasing the concentration of intermediate metabolites can be beneficial; this is often the case
when intermediates are harmful to the host cell. Increasing enzymes does not always improve product titers and can in fact be
detrimental. In this review, we present synthetic biological parts that enable optimization of metabolic pipes.
Secondary metabolites
 Fungi employ great diversity of metabolic pathways for
production of secondary metabolites
 Engineering of the existing pathways is more efficient than
creation of novel pathways in other organisms
 Most prominent targets of fungal engineering are:
 Biofuels
 Pharmaceutical precursors
 Fine chemicals
CRISPR/Cas9 genome editing
 CRISPR is system of bacterial immunity
A. Wild-type Cas9 nuclease site specifically cleaves double-stranded DNA activating double-strand break repair machinery. In the
absence of a homologous repair template non-homologous end joining can result in indels disrupting the target sequence.
Alternatively, precise mutations and knock-ins can be made by providing a homologous repair template and exploiting the
homology directed repair pathway.
B. Mutated Cas9 makes a site specific single-strand nick. Two sgRNA can be used to introduce a staggered double-stranded break
which can then undergo homology directed repair.
C. Nuclease-deficient Cas9 can be fused with various effector domains allowing specific localization. For example, transcriptional
activators, repressors, and fluorescent proteins.
Agaricus bisporus genome editing
 First CRISPR/Cas edited organism to receive green light
from US Government
 targeting the family of genes that encodes polyphenol
oxidase (PPO) – enzyme activity reduced by 30 %
 Resistant to browning
 “APHIS does not consider CRISPR/Cas9-edited white
button mushrooms as described in your October 30, 2015
letter to be regulated”
Engineering of cyanobacteria and algae
 Engineering of photosynthetic organisms is promising area
of modern biotechnology
 Nuclear or chloroplast transformation can be selected
 Most prominent targets of the engineering projects are
biofuels and hydrogen production, followed by fine
chemicals
 Chlamydomonas and Synechocystis are the most
abundantly engineered organisms