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.

TML1 AND TML2 SYNERGISTICALLY REGULATE NODULATION BUT NOT ARBUSCULAR MYCORRHIZA IN MEDICAGO TRUNCATULA.

Two symbiotic processes, nodulation and arbuscular mycorrhiza, are primarily controlled by the plant's need for nitrogen (N) and phosphorus (P), respectively. Autoregulation of Nodulation (AON) and Autoregulation of Mycorrhization (AOM) share multiple components - plants that make too many nodules usually have higher arbuscule density. The protein TML (TOO MUCH LOVE) was shown to function in roots to maintain susceptibly to rhizobial infection under low N conditions and control nodule number through AON in Lotus japonicus. M. truncatula has two sequence homologs: MtTML1 and MtTML2. We report the generation of stable single and double mutants harboring multiple allelic variations in MtTML1 and MtTML2 using CRISPR-Cas9 targeted mutagenesis and screening of a transposon mutagenesis library. Plants containing single mutations in either gene produced twice the nodules of wild type plants whereas plants containing mutations in both genes displayed a synergistic effect, forming 20x more nodules and short roots compared to wild type plants. The synergistic effect on nodulation was maintained in the presence of 10mM nitrogen, but not observed in root length phenotypes. Examination of expression and heterozygote effects suggest genetic compensation may play a role in the observed synergy. However, plants with mutations in both TMLs had no detectable change in arbuscular mycorrhizal associations, suggesting that MtTMLs are specific to nodulation and nitrate signaling. The mutants created will be useful tools to dissect the mechanism of synergistic action of MtTML1 and MtTML2 in M. truncatula nodulation as well as the separation of AON from AOM.

The Regulation of Nodule Number in Legumes Is a Balance of Three Signal Transduction Pathways.

Nitrogen is a major determinant of plant growth and productivity and the ability of legumes to form a symbiotic relationship with nitrogen-fixing rhizobia bacteria allows legumes to exploit nitrogen-poor niches in the biosphere. But hosting nitrogen-fixing bacteria comes with a metabolic cost, and the process requires regulation. The symbiosis is regulated through three signal transduction pathways: in response to available nitrogen, at the initiation of contact between the organisms, and during the development of the nodules that will host the rhizobia. Here we provide an overview of our knowledge of how the three signaling pathways operate in space and time, and what we know about the cross-talk between symbiotic signaling for nodule initiation and organogenesis, nitrate dependent signaling, and autoregulation of nodulation. Identification of common components and points of intersection suggest directions for research on the fine-tuning of the plant's response to rhizobia.