14CONFAP UNDERSTANDING CELL WALL STRUCTURE AND HYDROLYSIS OF TWO LEADING C4 BIOENERGY CROPS TO IMPROVE SECOND GENERATION BIOETHANOL PRODUCTION

Summary: The transition from first-generation (1G) bioethanol to a more productive and sustainable bioeconomy based on plant biomass is essential for Brazil to remain at the forefront in clean energy production and to strengthen security of food and energy supplies, create jobs, and mitigate climate change. A key challenge associated with the transition from 1G to second-generation (2G) biofuels is the difficulty to release sugars from cell walls to produce bioethanol with economic viability. Understanding the factors that govern recalcitrance and engineering cell wall architectures and hydrolytic enzymes for enhanced sugar release is crucial for the commercial exploitation of lignocellulosic feedstocks and realization of the bio-economy concept. Another important aspect is the development of biomass crops that maintain biomass productivity and quality under water-scarce environments unsuited for growing food crops. This aspect is even more important in a scenario of predicted climate change, where we need to quickly adapt crops to challenging environmental conditions.

Activities comprise a mix of networking, research and landscape-scoping activities, integrated between the partners, across three different work packages:

WP1: Tailored enzyme cocktails matching feedstock & pretreatment. The aim within the framework of this 1-year award will be to provide preliminary data on the potential of cocktail-feedstock-pretreatment matching. Current commercial enzymes are based on the one-size-fits-all approach. Since enzymes are a major contributor to biomass processing costs, creating customized enzyme cocktails that are tailored to specific pretreatments and feedstocks, will reduce enzyme loading and therefore processing costs. This WP will deliver proof of concept for the enormous potential of matching enzyme cocktails to feedstock and pretreatment.

WP2: Engineering the hydrolytic enzymes and feedstocks of the future. Synthetic biology offers exciting engineering opportunities to facilitate the release of sugars from plant cell wall biomass. One example is that of "biological pretreatment", a concept based on exploiting and redesigning some of the endogenous hydrolytic enzymes and processes already taking place in plants. Another attractive strategy to improve cell wall deconstruction is through the in planta expression of thermostable cell wall degrading enzymes. We will organize a workshop to effectively capture the potential of such synthetic biology approaches and produce a strategy document based around engineered ideotypes for both "in planta deconstruction" and "multifunctional hydrolytic enzymes". The outcomes of this work-package provides the foundation for further implementation of synthetic biology approaches in the area of plant cell walls and will position the partners at the forefront of this emerging research area.

WP3: Effect of environmental and genetic factors on cell wall biomass quality and conversion. The composition and architecture of cell wall biomass can differ significantly depending on tissue, species, cultivar, and environmental conditions. Mapping variations in biomass quality is particularly important in the context of climate change and developing sugarcane varieties suitable for cultivation on marginal land. The integration of cell wall phenotyping data with those from saccharification assays will provide essential information on how differences in biomass tissue, varieties/genetics, and environmental conditions impact on cell wall quality and biomass deconstruction into its components sugars. The outcomes of this controlled environment experiment represent a platform for the translation and design of future field trial studies focussing on utilizing marginal land available in Brazil.

WP1: Tailored enzyme cocktails matching feedstock & pretreatment. The aim will be to provide preliminary data on the potential of cocktail-feedstock-pretreatment matching. Commercial bagasse samples will undergo different pretreatments and hydrolysis with tailored enzyme cocktails and a commercial cocktail as reference. Pretreatments and tailored cocktail compositions will be selected based on expertise and scoping exercises.

WP2: Engineering the hydrolytic enzymes and feedstocks of the future. Synthetic biology offers exciting opportunities to facilitate the release of sugars from plant biomass. The Dr Buckeridge group has fully characterized a cell wall modification system active during gas space (aerenchyma) formation in sugarcane root development. We will seek to provide proof of concept that by increasing cell wall separation and porosity with enzymes specific for grass walls, a biological pretreatment could lead to an increase in accessibility to the other polymers of the cell wall. We will also organize a workshop to effectively capture the potential of synthetic biology approaches.

WP3: Effect of environmental and genetic factors on cell wall biomass quality and conversion. The composition and architecture of cell wall biomass can differ significantly depending on tissue, species, cultivar, and environmental conditions. We will utilize facilities at the National Plant Phenomics Centre (NPPC-Aberystwyth) to begin assessment of this variation. Selected sugarcane and Miscanthus varieties will be grown in different soil types to mimic fertile, semi-fertile and poor conditions. Plants will undergo detailed phenotyping in the NPCC during different water treatments (severe drought, moderate drought and well watered). Harvested samples will undergo cell wall phenotyping. The integration of data will provide essential information on how differences in biomass tissue, genetics, and environmental conditions impact on cell wall quality and biomass deconstruction.

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