Jack Wang

This project will be a collaboration between the Forest Biotechnology Group in the Department of Forestry and Environmental Resources and the Forest Restoration Alliance in the Department of Entomology and Plant Pathology at North Carolina State University. We propose a integrative approach to understanding the genetic response to hemlock woolly adelgid (HWA) infestation in susceptible and resistant hemlock species, and how these genetic regulations are transduced to alterations in phenotypic traits associated with HWA susceptibility. The proposed project builds upon ongoing research in developing a CRISPR genome editing system for hemlocks funded by the SCBGP in 2020-21. Comparative transcriptomics and phenomics of hemlock variants with varying extent of HWA susceptibility will produce genetic insights that facilitate identification of candidate gene targets for editing using CRISPR-Cas to enhance HWA resistance. This project will focus on four key objectives: (1) controlled HWA infestation in putatively susceptible and resistant genotypes of hemlock species, (2) assessment of phenotypic response to infestation in hemlocks, (3) full transcriptomic analysis of hemlock response to HWA infestation, and (4) integration of transcriptomic and phenotypic responses to identify putative gene targets associated with HWA resistance. The putative genes identified in this project will be targeted for hemlock genome editing in a subsequent research that is beyond the scope of this project period.

The Equipment Grants Program (EGP) serves to increase access to shared special purpose equipment for scientific research for use in the food and agricultural sciences programs in our Nation’s institutions of higher education, including State Cooperative Extension System

This project will be a collaboration between the Forest Biotechnology Group and the Christmas Tree Genetics Program in the Department of Forestry and Environmental Resources at North Carolina State University. Our goal is to develop novel CRISPR-based genome editing technology that would accelerate the genetic improvement of Fraser fir. The proposed technology would enable the rapid production of new variants of Christmas trees edited for traits of ecological and economic values such as disease tolerance and post-harvest quality. Fraser fir is one of North Carolina’s most important specialty crops. Developing novel genomic tools and genome editing technology for Fraser fir will have a transformative impact on the North Carolina Christmas tree industry. This project builds on our recently established somatic embryogenic system and cell transfection method for Fraser fir (funded by SCBGP in 2018), which lay the foundation for optimizing efficient and robust CRISPR-Cas9 delivery and regeneration of enhanced Fraser fir from genome-edited somatic embryos. We propose three major objectives in this proposal: (1) Optimize the delivery of CRISPR-SpCas9 in Fraser fir somatic embryogenic protoplasts: we will test several experimental parameters to maximize transfection efficiency; (2) Regenerate and maturate CRISPR-SpCas9 edited protoplasts into Fraser fir plantlets: we will optimize an integrated protocol for regeneration and maturation of CRISPR-edited protoplasts originated from Fraser fir SE; (3) Validation of target gene editing in regenerated Fraser fir plantlets: we will genotype CRISPR-driven editing events in the regenerated fir plantlets. Subsequent to the funding period, the transgene-free CRISPR-based SE system will be used to edit superior clonal seedlings for Christmas tree field trials in the North Carolina Mountains.

We propose an innovative bioprocess that will produce high value cellulose nanocrystals (CNC) and butanol fuel from sustainable biomass feedstocks. Specifically, we will assess two biomass feedstocks: 1) poplar-derived market pulp and 2) CRISPR edited whole poplar biomass, as shown in Figure 1. Tailored hemicellulase and cellulase enzymes will be provided by Novozymes to selectively hydrolyze the hemicellulose and amorphous cellulose to generate free sugars and cellulose nanocrystals. The free sugars, both 5- and 6-carbon, will be fermented to butanol fuel via Clostridium saccharoperbutylacetonicum. After fermentation, butanol will serve two beneficial purposes for downstream separation operations: 1) butanol will act as a dispersant inhibiting hydrogen bonding and reducing nanocellulose agglomeration1 and 2) butanol will partially solubilize lignin thereby enhancing liquid/solid separation.2,3

The purpose of the Consortium on Sustainable and Alternative Fibers Initiative (SAFI) is to develop fundamental and applied research on the use of alternative and sustainable fibers for the manufacturing of market pulp, hygiene products and nonwovens. The idea for SAFI has grown out of societal needs for alternative yet sustainable materials. SAFI will study the potential of alternative fibers based on technical (performance), sustainable and economic principles. This enhancement project aligns with the goals and mission of SAFI and aims to harbor novel genome editing technologies to advance the development of new fiber feedstocks with unique properties to improve pulp production.

To realize the potential for producing fuels and chemicals from renewable resources in sustainable ways, bioprocess engineering strategies are needed that optimally integrate genetically modified feedstocks and metabolically engineered microorganisms. Extremely thermophilic bacteria from the genus Caldicellulosiruptor not only natively deconstruct lignocellulose to fermentable sugars but also can be engineered to subsequently produce industrial chemicals. By operating at high temperatures characteristic of extreme thermophiles, contamination risk can be virtually eliminated, thereby allowing bioreactor operation to align with well-established chemical processesing technology rather than require approaches akin to pharmaceutical manufacturing. Furthermore, by strategically choosing target chemicals, separation of product from fermentation broths can exploit product volatility at elevated temperatures (‘bioreactive distillation’), thereby significantly reducing energy costs associated with recovering dilute products from large aqueous streams. This project has the following objectives: 1) Using a novel, high temperature bioreactor configuration, characterize deconstruction and conversion of wild-type and transgenic poplar lines to fermentation products at high feedstock loadings by wild-type Caldicellulosiruptor bescii; 2) Improve existing transgenic poplar lines to minimize recalcitrance and maximize deconstruction and conversion of lignocellulose to fermentation products by wild-type C. bescii; 3) Metabolically engineer C. bescii to produce acetone from cellobiose, and then optimize conversion efficiencies and rates at bioreactor scale using a novel process intensification schemes (‘bioreactive distillation’); 4) Using optimized transgenic poplar and metabolically engineered C. bescii, produce acetone from lignocellulose at bioreactor scale, and determine the impact of feedstock loading and processing conditions on conversion efficiency, yields, volumetric productivity, and product recovery.

• We will continue our current efforts searching for surviving hemlocks using TreeSnap, collecting seeds and cuttings from the survivors, providing a portion of the seed collected or seedlings germinated to private nurseries and the NC Forest Service Linville nursery, and evaluating short-term growth and survival of hemlock seedlings and rooted cuttings by exposure to HWA in the screening facility as a measure of short-term tolerance or resistance to the insect. • We will continue to make hybrid crosses between native hemlocks and resistant and tolerant exotic hemlocks. Previously completed putative hybrid crosses will continue to be verified as hybrids using SSR markers in chloroplast DNA. New novel crosses will be attempted as part of this effort. • In addition, we will begin a long-term silvicultural study to determine appropriate techniques for best survival in ornamental and field plantings; and begin a long-term evaluation of all out-planted hemlocks to determine their growth, survival and infestation rates as measures of HWA resistance. • Transfer cultures of SE hemlocks from the Merkle Lab at UGA to NCSU, and add to the cultures from open-pollinated and control-pollinated seed as we collect more cones from sources to include putatively resistant native trees and new hybrid crosses. Where feasible, we will involve horticulture students and faculty at Haywood Community College in the induction of SE cultures and production of SE plants at their tissue culture lab in Clyde, NC. • Collect phenotypic data related to HWA resistance from potentially resistant and susceptible trees that we have identified to begin the process of developing molecular markers for resistance. Trees assessed for phenotype will include open-pollinated families, as well as some full-sib families in a single-pair mating design. We will collect and store tissue samples from the assessed trees suitable for future DNA extraction to be used in marker development. • Test CRISPR-based in vivo genome editing efficiency of Hemlock SE lines using CRISPR-SpCas9, LbCpf1, and LgaCas9 in Hemlock protoplasts and embryogenic masses.

The project aims to develop CRISPR-based genome editing using somatic embryogenesis (SE) to enable the strategic engineering of superior clonal Fraser fir Christmas trees. Fraser fir is one of North Carolina’s most important specialty crops. Developing novel genomic tools and genome editing technologies for Fraser fir will have a transformative impact on the North Carolina Christmas tree industry. We propose four major objectives to develop the CRISPR-based SE system: (1) Streamline SE platform: we will improve the final steps of the Fraser fir SE process to establish a complete platform from cryostorage to whole tree regeneration; (2) Identify superior SE clonal lines: we will produce 30 independent Fraser fir clonal lines and select the top three lines for genome editing; (3) Assemble CRISPR-ribonucleoprotein (RNP) complexes: we will assemble and test the mutagenic function of up to ten CRISPR-RNP complexes in vitro for Fraser fir genome editing; (4) In vivo validation of CRISPR-based genome editing: We will deliver the CRISPR-RNP complexes into Fraser fir protoplasts and embryogenic cell masses for transgene-free genome editing. Subsequent to the funding period, the CRISPR-based SE system will be used for engineering superior clonal seedlings for field trials in the North Carolina Mountains.

Angiosperm species, such as Eucalyptus globulus, have lignin composed of both syringyl and guaiacyl subunits. In contrast, gymnosperm species such as Pinus taeda have lignin which is devoid of syringyl monomer units. Favorable pulping efficiency with high pulp yields has long been associated to wood with lignin containing high syringyl component. Inducing the biosynthesis of syringyl subunits in gymnosperm lignin would be a direct approach to engineer gymnosperm wood for improved pulping efficiency and yields. Here, we propose to establish a strategy to demonstrate the induction of syringyl monolignol biosynthesis in gymnosperms through an in vitro protein activity assay system. In this system, we plan to spike the total protein mixtures isolated from the secondary differentiating xylem (SDX) of a gymnosperm with angiosperm syringyl-lignin specific recombinant proteins. We will assay these protein mixtures using coniferaldehyde, a common precursor to both guaiacyl and syringyl monolignols, and monitor the synthesis of sinapyl and coniferyl alcohol, precursors of syringyl and guaiacyl monolignol respectively. These assays will provide insights to the extent of syringyl monolignol biosynthesis we can induce into gymnosperms by the addition of angiosperm proteins.