From Natural Microbe Screening to Sustained Chitinase Activity in Exogenous Hosts

Genetic parts and hosts can be sourced from nature to
realize new
functions for synthetic biology or to improve performance in a particular
application environment. Here, we proceed from the discovery and characterization
of new parts to stable expression in new hosts with a particular focus
on achieving sustained chitinase activity. Chitinase is a key enzyme
for various industrial applications that require the breakdown of
chitin, the second most abundant biopolymer on the earth. Diverse
microbes exhibit chitinase activity, but for applications, the environmental
conditions for optimal enzyme activity and microbe fitness must align
with the application context. Achieving sustained chitinase activity
under broad conditions in heterologous hosts has also proven difficult
due to toxic side effects. Toward addressing these challenges, we
first screen ocean water samples to identify microbes with chitinase
activity. Next, we perform whole genome sequencing and analysis and
select a chitinase gene for heterologous expression.
Then, we optimize transformation methods for target hosts and introduce
chitinase. Finally, to achieve robust function, we optimize ribosome
binding sites and discover a beneficial promoter that upregulates
chitinase expression in the presence of colloidal chitin in a sense-and-respond
fashion. We demonstrate chitinase activity for >21 days in standard
(Escherichia coli) and nonstandard
(Roseobacter denitrificans) hosts.
Besides enhancing chitinase applications, our pipeline is extendable
to other functions, identifies natural microbes that can be used directly
in non-GMO contexts, generates new parts for synthetic biology, and
achieves weeks of stable activity in heterologous hosts.