Source: ZESTBIO, INC. submitted to
ENZYMATIC BIOPROCESSING OF PECTIN-RICH AGRICULTURAL SIDESTREAMS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
SMALL BUSINESS GRANT
Reporting Frequency
Annual
Accession No.
1028883
Grant No.
2022-33610-37831
Cumulative Award Amt.
$650,000.00
Proposal No.
2022-04467
Multistate No.
(N/A)
Project Start Date
Sep 1, 2022
Project End Date
Aug 31, 2024
Grant Year
2022
Program Code
[8.8]- Biofuels and Biobased Products
Recipient Organization
ZESTBIO, INC.
2715 HILLEGASS AVE
BERKELEY,CA 947051229
Performing Department
(N/A)
Non Technical Summary
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, there is a lack ofvalue-adding technologies for these abundantpeel and pulp byproducts.ZestBio, Inc. is developing biotechnologies for the production of specialty chemicals from these agricultural byproducts.The first chemical product from this bioprocessing technology can be formulated for water treatment applications. These formulations exhibit superior heavy metal binding compared to chemcials currently sold. Additionally, these formulations preventmild steel corrosion. Thus, this new ingredientsatisfiesgrowing demand for sustainable and high-performing chemicals.Implementation of our 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. New ingredients with improved performance in the water treatment industry can help owners of capital equipment that requirecorrosion treatment by extending thelifespan of their equipment and lowered operating costsfrom improved performance. Furthermore, this new ingredient will havean improved environmental footprint (protecting our US waterways) and provide a reliable chemicalsupply sourced from the USA instead of from China and elsewhere abroad.The overall project goal is to prepare the technology for scaling in pilot-scale bioreactors. First, we propose improving the stability of the enzymes we use in our chemical manufacturing process by tweaking their structure. Second, we propose improving the yieldof these enzymes by optimizing the production conditions. Third, we propose optimization and scaling of the chemical synthesis from citrus peel waste and sugar beet pulpinbioreactors. The anticipated results from this work plan are a technology ready for pilot-scale manufacturing.
Animal Health Component
0%
Research Effort Categories
Basic
0%
Applied
0%
Developmental
100%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
5110999100070%
5112010100030%
Knowledge Area
511 - New and Improved Non-Food Products and Processes;

Subject Of Investigation
2010 - Sugar beet; 0999 - Citrus, general/other;

Field Of Science
1000 - Biochemistry and biophysics;
Goals / Objectives
ZestBio, Inc. is developing technologies for the production of specialty chemicals from prevalent byproducts of the US fruit and vegetable processing industries.The main goal for thisprojectis to develop a benchscale process fora value-adding in vitro enzymatic conversion technology for the conversion of pectic monosaccharides into specialty chemicals. Achieving this goal will mark a key milestone in the technology development where the technology will be ready for scaling into larger pilot conversion reactions.There are three main objectives towards achieving this goal:Reductase screening and engineering with the target of identifying a reductase with a total turnover number of 2,000,000 in 24 hours.Optimization of protein expression for uronate dehydrogenase and the engineered reductase enzyme to improve enzyme units yield per liter of E. coli by 50%Scaling conversion reactions to 2 liter bioreactors via reaction validation and optimization in 200 mL bioreactors including a design of experiments analysis to build a model approximating the impact of different operating parameters on production.
Project Methods
Objective 1: Reductase screening and engineering with the target of identifying a reductase with a total turnover number of 2,000,000 in 24 hours.Methods:BLAST and literature search to identify reductase enzymes to screenCodon optimization of target reductase enzyme protein sequences for expression in E coli using published toolsOrdering of DNA sequencesPCR mutagenesis of select genes of interestCloning and transformation of DNA sequences for T7-inducible expression of proteins in E coliVerification of cloning success via DNA sequencingGrowth of E. coli for protein expression including induction of promotorChemical lysis of E coli cultureEnzymatic conversions setup using E coli lysate in either citrus peel waste or sugar beet pulpMeasurement of sugar and product titers in converted hydrolysate by sampling and running extracts on HPLCEvalulations:Restriction digests of plasmidsDNA sequencing of plasmidsMeasurement of protein activity units via NAD assaysMeasurement of substrate and product concentrations via HPLCObjective 2: Optimization of protein expression for uronate dehydrogenase and the engineered reductase enzyme to improve enzyme units yield per liter of E. coli by 50%Methods:Cloning of reductase and dehydrogenase genes into different expression plasmidsVerification of cloning success by restriction digest and DNA sequencingTransformation of verified plasmids into BL21 E coli cellsGrowth and protein expression for transformed E coli under an array of medias and growth conditionsMeasurement of cell density, chemical lysis, and measurement of activity units in lysateDesign of Experiments analysis of activity yield as a function of different growth conditionsGrowth and protein expression usingE coli in 2L bioreactorsEvaluations:Restriction digests of plasmidsDNA sequencing of plasmidsMeasurement of protein activity units via NAD assaysObjective 3: Scaling conversion reactions to 2 liter bioreactors via reaction validation and optimization in 200 mL bioreactors including a design of experiments analysis to build a model approximating the impact of different operating parameters on production.Methods:Processing of peels/pulp to low particle sizeEnzymatic hydrolysis of peels/pulp to release trapped sugarsFiltrations and washing to remove solids and recover released sugarsEvaporation to concentrate hydrolysatesSonication lysis ofE coli cell pellets and QC via NAD activity assaysSetup and operation of bioreactors with varying operating conditions and varying media compositionsSampling of bioreactors and feeding components such as additional e coli lysatesSample prep and HPLC analysis of sugar substrates and product concentrationsDesign of Experiments modeling of reaction outputs as measured by HPLCEvaluations:Measurement of protein activity units via NAD assaysMeasurement of bioreactor conditions: dissolved oxygen, pH, air flowMeasurement of substrate and product concentrations via HPLC

Progress 09/01/22 to 08/31/24

Outputs
Target Audience:• Crop growers, particularly those growing citrus and sugar beets as value of the crop and earnings from crops can be increased • Crop processors, businesses and employees that process citrus fruit into juice and sugar beets into sugar • Rural communities that will benefit from new jobs for chemical manufacturing from food wastes and the commercial output from growers and processors [including construction and taxes] • Chemical distribution businesses that will have new product offerings available • Water treatment businesses that use chemical products; being able to offer new products and products with improved environmental footprint and with reliable supply via chemical ingredients sourced from the USA instead of from China and abroad • Owners of capital equipment that requires corrosion treatment; these persons and businesses can benefit by extending capital lifespan and lowered operating costs from improved performance through new ingredients • Users of freshwater systems: reduction in harmful emissions into waterways by replacing chemicals such as phosphates which promote damaging algae blooms with biodegradable and less damaging chemicals Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Training was provided throughout the project for the technical staff performing the research through one-on-one mentoring with the program director and included mentoring on experimental design, data analysis, interpretation of results, scientific presentation of results, and report writing. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? All technical milestones originally proposed were achieved: Reductase screening and engineering identifieda reductase with a total turnover number of 4,000,000 in 24 hours. Optimization of protein expression yielded58% increase in dehydrogenase activity compared to starting baseline enzyme activity yield Optimization of protein expression yielded 300% increase in oxidase activity compared to starting baseline enzyme activity yield Optimized bioreactor conditions for key operating parameters to model impact of different operating parameters on production. Achieved high titer, high yield conversions within 24 hour reaction in bioreactors.

Publications


    Progress 09/01/22 to 08/31/23

    Outputs
    Target Audience: Crop processors, businesses and employees that process citrus fruit into juice and sugar beets into sugar Chemical distribution businesses that will have new product offerings available Water treatment businesses that use chemical products; being able to offer new products and products with improved environmental footprint and with reliable supply via chemical ingredients sourced from the USA instead of from China and abroad Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?As part of this work, the Project Director has been mentoring the scientific staff by assisting them in designing experiments and advising them on experiment analysis, providing feedback oninternal presentations of experimental findings and providing coaching for scientific report preparation and writing. How have the results been disseminated to communities of interest?A poster describing key findings was presented at the NIFA Bioeconomy Project Directors Meeting. What do you plan to do during the next reporting period to accomplish the goals?The plan in the next reporting period is to continue to pursue the work plan laid out in the proposal for achieving Objectives 2 and 3. This workplan includes: Objective 2: Cloning of reductase and dehydrogenase genes into different expression plasmids Verification of cloning success by restriction digest and DNA sequencing Transformation of verified plasmids into E coli cells Growth and protein expression for transformed E coli under an array of medias and growth conditions Measurement of cell density, chemical lysis, and measurement of activity units in lysate Design of Experiments analysis of activity yield as a function of different growth conditions Growth and protein expression using E coli in 2L bioreactors Objective 3: Processing of peels/pulp to low particle size Enzymatic hydrolysis of peels/pulp to release trapped sugars Filtrations to remove solids Evaporation to concentrate hydrolysates Sonication lysis of E coli cell pellets and QC via NAD activity assays Setup and operation of bioreactors with varying operating conditions and varying media compositions Sampling of bioreactors and feeding components such as additional e coli lysates Sample prep and HPLC analysis of sugar substrates and product concentrations

    Impacts
    What was accomplished under these goals? Goals that were accomplished during this project period include: Reductase screening and engineeringidentifiedreductases with a total turnover number exceeding 4,000,000 in 24 hours. An effective 58% increase in dehydrogenase activity compared to starting baseline enzyme activity yield. An effective 67% increase in reductase activity comopared to starting baseline enzyme activity yield.

    Publications