Source: ZYMOCHEM INC submitted to NRP
CARBON-CONSERVING MICROBIAL PRODUCTION OF 1-HEXANOL FROM BIO-BASED FEEDSTOCKS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
Reporting Frequency
Annual
Accession No.
1010133
Grant No.
2016-33610-25671
Cumulative Award Amt.
$600,000.00
Proposal No.
2016-03988
Multistate No.
(N/A)
Project Start Date
Sep 1, 2016
Project End Date
Feb 29, 2020
Grant Year
2016
Program Code
[8.8]- Biofuels and Biobased Products
Recipient Organization
ZYMOCHEM INC
4 ANCHOR DR STE 231
EMERYVILLE,CA 94608
Performing Department
(N/A)
Non Technical Summary
1-Hexanol has a global market size of ~200,000 MT/yr, wherein this alcohol is used as an endproduct or as an intermediate in producing other industrial chemicals with applications in the plastics, textile, perfume, and chemicals industries. 1-Hexanol is commercially produced from petrochemicalsvia processes the require substantial energy inputs, use toxic gases, and waste that is difficult to dispose of. Suffice to say, current approaches for making 1-hexanol are neither sustainable nor environmentally friendly.Microbial processes are a promising,sustainable alternative to petroleum-based approaches for making chemicals such as 1-hexanol. However, most of today's processes for the biological production of chemicals are simply not as carbon efficient. These inefficiencies are namely due to theto loss of ~33% of the sugar's carbon as carbon dioxide waste(CO2), which causes an increase in production costs and results in bioprocesses that are economically disadvantaged- arguably the most crucial factor for commercializing technology to produce chemicals. Thus, there is a clear need within the chemicals industry to develop bio-based renewable processes with improved yields, better economics, smaller environmental footprints, and greater sustainability than existing petroleum-based processes.We are developing a platform of enzyme-based pathways for converting renewable feedstocks such as corn-stover & woody biomass into chemicals, where essentially noneof the sugar's carbon is lost as CO2. Our"carbon-conserving" (C2) pathwaysenable microbes to make chemicals like 1-hexanol with improved theoretical yieldsand more economically compared to state-of-the-art approaches. During the previous Phase I project, we confirmedthe technical feasibility of our novel enzyme-based C2 pathway. In Phase II, we aim to integrate this C2 pathway into an engineered & optimizedindustrially-relevant microorganism with a robustability to produce1-hexanol from sugars, which will allow us to transition to larger scales for developing the entire bio-based processto make 1-hexanol.The commercialization of our C2 technology for producing1-hexanol will have several broad-reaching benefits such as alleviating dependence on foreign oil, decreasing adverse environmental consequences of current processes, and promoting sustainable manufacturing. Successful completion of our project will also provide a new foundation for sustainable industrial chemicals as well as diversify the use of agricultural and forest feedstocks. Doing so will create new markets for agricultural and rural businesses, thereby expanding economic opportunities and creating jobs in the renewable energy/chemicals sector.
Animal Health Component
80%
Research Effort Categories
Basic
(N/A)
Applied
80%
Developmental
20%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
51140992020100%
Goals / Objectives
Our primary technical objective for our priorPhase I project was to establish the feasibility of our carbon-conserving (C2) pathway for making 1-hexanolin vitrostarting from the initial substrates.During Phase I, we discovered and/or confirmed enzymes that catalyze all nine steps in the C2pathway for producing 1-hexanol and validated the pathway's functionality by demonstrating cell-free synthesis of 1-hexanol. We also carried out preliminary studies toward constructing the C2pathway for themicrobial production of 1-hexanol, confirming the in vivofunctionality of sections of the C2pathway.For this Phase II project, we have two high-level technical goals with multiple milestones and associated metrics to track progress toward completing the two objectives.Objective #1 -In vivovalidation of the C2 pathway by illustrating its functionality in an industrially relevant microorganism for converting the Step #1 substrates to 1-hexanol.Objective #2 -Engineering production strains and optimizing the C2 pathway toward improving 1-hexanol production metrics (increased titers, productivities, and yield) that will justify transitioning to pilot scale R&D.
Project Methods
Our work plan involvesof several tasks to achieve the milestones we have laid outto complete the two high-levels goalsfor this project. The overall work flow will consist of (but not be limited to) bioprospecting, high-throughputin vitroenzyme characterization & development, strain engineering via chromosomeediting, recombinant DNA synthesis,metabolic engineering & pathway construction, anaerobicin vivocell culture work, -omics studies,use of analytics,lab-scale bioreactor studies, and downstream process development toward product recovery.

Progress 09/01/16 to 02/29/20

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported 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?At present and following the Phase II, we have paused the development of our C2 technology for the production 1-hexanol and other related aliphatic small molecule products. One main reason for this are the technical challenges we faced during Phase II and the difficulty in resolving them. Our efforts have pivoted toward developing a similar C2 technology for the bio-based production of difunctional small molecule chemicals. The reason for this change is two-fold. [1] Through other efforts funded from other sources, we have successfully developed a different C2 technology that is more effectively able to produce such products. [2] Through our business development efforts, we have found that the markets and commercial attractiveness for the difunctional products offer a more promising route to commercialization.

Impacts
What was accomplished under these goals? We successfully completed all the tasks and milestones that we proposed for Year 1 of the project. Specifically, we successfully demonstrated the production of 1-hexanol within our industrial production microbe (the Go/No-go Milestone) via our C2 pathway, namely starting from the C2 pathway substrates. We also identified multiple bottlenecks in the C2 pathway, which were addressed & resolved to different extents during Year 2. Our efforts in Year 2 proved more challenging, where we were unable to achieve our target goals. The engineering of a highly effective biosynthetic pathway from glucose to the C2 pathway substrates proved to be the major obstacle. While we successfully engineered strains that could co-produce the different C2 substrates, the effectiveness (rates) were deemed inadequate to support a high productivity C2 pathway from glucose to 1-hexanol. Thus, we were unable to hit our target titer-rate-yield performance metrics. However, besides this objective, our other efforts in Year 2 proved more fruitful. We successfully improved the performance of the core C2 pathway by overcoming multiple pathway bottlenecks via enzyme development and strain engineering endeavors, in turn substantially improved 1-hexanol performance metrics (titers, rates, and yields) when starting from the C2 pathway substrates (not glucose).

Publications


    Progress 09/01/16 to 08/31/17

    Outputs
    Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported 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?During our year 1 studies we identified important bottlenecks of our technology and the underlying C2pathway.Specifically, we noted that some FACE/T pathway intermediates accumulated, which could be attributed to low enzyme activity towards the desired substrate and/or non-optimal enzyme expression levels.We have been anticipating this issue, and thus ongoing effortsare aimed at constructing and implementing engineered strains with genome integrated expression cassettes that use effective & validated promoters designs that are different than the plasmid-based expression system being used to finely control enzyme expression (Project Narrative - Task 5, Year 2). This will allow expression tuning of all the FACE/T pathway enzymes, which is important for enabling improved flux in a single strain. Additionally, once we confirm that low enzyme activity (due to poorsubstrate specificity) is an issue that resultsinFACE/T pathway intermediateaccumulation, we will be usingmultiple approaches (e.g. bioprospecting, protein engineering) to identify improved enzymes that enable higher flux through the pathway (Project Narrative - Task 5, Year 2). Additionally, these strain optimization efforts will be carried in conjunction withconstruction of a biosynthetic pathway to produce step 1 substrates from glucose (Project Narrative - Task 4, Year 2) toenable E. coli-based production of 1-hexanol solely from glucose.

    Impacts
    What was accomplished under these goals? Our work plan to develop the 1-hexanol FACE/T pathway in an industrially-relevant microbe involved two high-level objectives with six milestones. First (year 1), we will validate the FACE/T pathway within E. coli via the production 1-hexanol from step 1 substrates (Milestones 1-4). Second (year 2), we will improve production strains with the aim to reach target performance metrics (titers, rates, yields) that will enable pilot-scale testing within 12 months following this 2- year Phase II project (Milestones 5-6). During year 1, we successfully performed the following studies : 1) In vitro enzyme screening and characterization to validate≥3 suitably functional enzymes for each reactionofthe 1-hexanol pathway; 2) Developed a facile, high- throughout (HT) platform for genome editing to engineer chassis hosts (for screening better enzymes in a modular fashion)as well as create initial stable strains for 1-hexanol production; and 3) In vivo assembly and characterization of the FACE/T pathway in E. coli for making 1-hexanol from step 1 substrates. With these studies,we successfully completed all the tasks and milestones proposed during year 1 of the project. Specifically, we successfully demonstrated the production of 1-hexanol within E. coli (the Go/No Go Milestone) via our C2 pathway.

    Publications