Recipient Organization
ZESTBIO, INC.
2715 HILLEGASS AVE
BERKELEY,CA 947051229
Performing Department
(N/A)
Non Technical Summary
ZestBio, Inc. is developing technologies for the production of specialty chemicals from prevalent byproducts of the US fruit and vegetable processing industries. The ZestBio mission is aligned with USDA NIFA priorities of developing new processes for manufacturing biobased chemicals from US agricultural resources. The United States is uniquely positioned as a world leader in fruit and vegetable processing, including sugar production from sugar beets (37 million tons/yr) and citrus juicing (3 million tons/yr). However, the peel and pulp byproducts from processing lack value-adding technologies. The proposed research aims to develop a value-adding in vitro enzymatic conversion technology for the conversion of pectic monosaccharides into specialty chemicals. This enzymatic strategy can enable rapid bioprocessing with reduced handling and capex requirements compared to conventional approaches such as chemical synthesis or fermentation. The first chemical product from this bioprocessing technology can be formulated as a chelant for water treatment applications. This formulation exhibits superior calcium and heavy metal binding compared to established biobased chelants. Thus, it satisfies the growing demand for sustainable and high-performing chemicals. The overall project goal is to engineer improved enzymatic activity for the in vitro manufacturing process. First, we propose screening the activity of a variety of natural enzymes and their homologs. Second, we propose mutagenesis of the enzyme sequence and identifying high activity mutants. The anticipated results from this work plan are improvements in enzyme activity sufficient for commercially viable enzymatic bioprocessing. Implementation of this biomanufacturing process can increase overall crop revenue. This offers reduced volatility in crop value, financial sustainability for farmers, and construction of rural-located fermentation facilities to create high paying technical jobs. Furthermore, by using existing crop byproducts, our products will have a lower carbon footprint compared to competing chemicals made from petroleum or purified sugar.
Animal Health Component
60%
Research Effort Categories
Basic
0%
Applied
60%
Developmental
40%
Goals / Objectives
The major goal for this project isto develop a value-adding in vitro enzymatic conversion technology for the conversion of pectic monosaccharides into specialty chemicals. This enzymatic strategy can enable rapid bioprocessing with reduced handling and capex requirements compared to conventional approaches such as chemical synthesis or fermentation. Implementation of this biomanufacturing process can increase overall crop revenue. This offers reduced volatility in crop value, financial sustainability for farmers, and construction of rural-located fermentation facilities to create high paying technical jobs.The overarching measurable objective for towards this goal is to produce and purify an enzyme for the conversion of the pectin sugar, D-galacturonic acid into a high value chemical intermediate. However, the current enzyme activity is limiting to production. Therefore the goal for this project are to improve enzyme activity.To achieve this overall objective, we have two main technical objectives:Screen enzyme homologs for high activty on pectin hydrolysate. We will rapidly synthesize and screen a variety of enzyme homologs using in vitro expression for high activity on D-galacturonic acid in pectin hydrolysate.High throughput enzyme engineering for improved activity. Using the best enzyme sequence from Objective 1, we will perform iterative PCR mutagenesis on this sequence and identify high activity mutants. Top hits from each round will be characterized for activity under production conditions and used as template for subsequent rounds of mutagensis.
Project Methods
This project is designed to develop a new chemical manufacturing method by taking D-galacturonic acid derived from pectin and converting it into value added chemicals. A key technical limitation for this is the activty of the enzyme. Therefore, this project aims toimprove enzyme activity. The methods applied will focus on utilizing strategies common to molecular biology, synthetic biology and metabolic engineering to express, purify, and assess enzymatic activity. No activity requires major departures from standard practices as the focus is on solving this importantenzyme activity problem with known methods.All work outlined below requires standard molecular biology equipment and analytical equipment, including HPLC for substrate and product quantification and a plate reader for absorbance and fluorescence measurements.The first part of the work plan is to design codon optimized versions of heterologous enzymes, synthesize this DNA via common third-party manufacturers, and then express the proteins off the resulting plasmids. These enzymes can then be assayed for activity by incubating with D-galacturonic acid. After the reaction has run, the reacted mixture can be run on HPLC to quantify production of ourproduct. By normalizing protein loading, the enzyme reactions that yield the highest product titers can be taken onto the next step for further engineering.The second part of the work plan is to perform PCR mutagenesis on the top enzymes from the initial screen. The PCR mutagenesiswill be done with standard published conditions and commercial kits that assist in inserting DNA mutations. To validate successful mutagenesis, individual plasmids from the reaction will be isolated and sequenced by Sanger Sequencing to measure the mutation rate.The resulting will be screened in high throughput with a growth selection. Hits from this screen will be isolated and sequenced and verified in an enzyme activity assay.This general approach of screening enzyme mutants with a growth screen has been used to great success in thepast by many other groups to engineering enzyme activities.