Source: Lygos Inc. submitted to NRP
MICROBIAL PRODUCTION OF A HIGH-VALUE CHEMICAL FROM LIGNOCELLUSIC SUGARS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
SMALL BUSINESS GRANT
Reporting Frequency
Annual
Accession No.
1006241
Grant No.
2015-33610-23432
Cumulative Award Amt.
$100,000.00
Proposal No.
2015-00530
Multistate No.
(N/A)
Project Start Date
Jun 15, 2015
Project End Date
Sep 14, 2016
Grant Year
2015
Program Code
[8.8]- Biofuels and Biobased Products
Recipient Organization
Lygos Inc.
636 San Pablo Ave
Albany,CA 94706
Performing Department
(N/A)
Non Technical Summary
Lygos develops technologies for the production of chemicals from renewable raw materials, mainly sugar. Specifically, Lygos' scientists engineer microbes that grow in fermentation tanks to consume sugar and produce a target chemical, which is then purified from the fermenter for sale. Lygos' technology provides a method to produce the same chemicals currently made from petroleum but at both lower cost and from a renewable resource.Traditionally, sugars derived from cornstarch or sugarcane have been used to produce biochemicals (e.g., fuel ethanol). More recently, new technologies for the production of cellulosic sugars (those derived from non-food biomass) have been developed. One of the typical problems with cellulosic sugars is their lower purity, and there is typically a high concentration of various byproducts that inhibit the microbe from efficiently producing the target chemcial. In this Phase I SBIR, Lygos is developing new fermentation technologies designed to enable the microbes to more efficiently use cellulosic sugars and produce the target chemical efficiently.The expected outcomes of this project is a novel fermentation-based production method for an industrially useful organic acid from cellulosic sugars. If successful, over $1B in downstream derivative compounds may be produced from this chemical.
Animal Health Component
100%
Research Effort Categories
Basic
(N/A)
Applied
100%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
51140201060100%
Knowledge Area
511 - New and Improved Non-Food Products and Processes;

Subject Of Investigation
4020 - Fungi;

Field Of Science
1060 - Biology (whole systems);
Goals / Objectives
The goal of the Phase I SBIR proposal is to develop a fermentation process for the industrial production of a single, high-value organic acid using cellulosic sugars as the raw material. There are domestic agricultural and manufacturing benefits to our approach, which makes use of existing, low-value biomass resources as the feedstock for the fermentation. First, there is an abundance of waste agricultural and forestry biomass in the U.S. suitable for use with our technology. For example, corn stover is produced in an approximately 1:1 w/w ratio to corn grain, and over 350M MT of stover is estimated for the domestic 2014/2015 harvest. Second, with development of new microbial strains and fermentation technologies, this material could be cost-effectively converted into higher-value chemicals, providing an additional revenue source to relevant agricultural stakeholders.
Project Methods
One of the long-standing problems when using lignocellulosic sugars is that the presence of byproducts in the hydrolysate inhibits microbial growth and product formation during fermentation. In this Phase I work effort we will be developing fermentation processes for strains of microbes that exhibit a much higher tolerance to byproducts in the hydrolysate as compared to more commonly used microbes in industrial biotechnology, including S. cerevisiae and E. coli. The methods used are wet-lab experimentation using cellulosic hydrolysates from commercial providers. Performance of our microbial fermentations will be assayed with respect to yield (e.g., % molar conversion of sugar to product), titer (concentration of product formed), and productivity (rate of product formation).

Progress 06/15/15 to 09/01/16

Outputs
Target Audience:The target audience for this project includes the following market segments: bio- and petro-chemical producers and lignocellulosic sugar producers (e.g., biodiesel manufacturers). Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two employees further expanded and developed their expertise in yeast strain physiology,cultivation,and analytical chemistry through completion of thiswork plan. 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? The USDA and Lygos employees supported research into the proof-of-principle production of the target commodity chemical using a filamentous fungal production host. The target product was an organic acid derived from the tricarboxylic acid cycle. The ability to produce the target chemical was supported by information in the Literature indicating specific enzymes were present in these hosts to catabolize the desired catalysis. Lygos employees worked to identify the genes encoding the described reported enzymatic activities in these microbes. Specifically, strain cultivation assays were performed under different media and growth conditions and the resulting fermentation broths were analyzed by liquid chromatography with an initial goal ofidentifying specific strains and conditions resulting in the production of the target compound. The strains were cultured successfully; however, growth conditions could not be found that resulted in target product formation. Furthermore, results from published research papers were unable to be replicated in the lab.

Publications


    Progress 06/15/15 to 06/14/16

    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?Analysis of strain growth and product formation using different media formulations with product and pathway intermediate titers measured by liquid chromatography.

    Impacts
    What was accomplished under these goals? Lygos screened multiple strains of filamentous fungi for production of a tricarboxylic acid cycle organic acid. The strains were reported in the Literature to produce high concentrations of the target product when grown under specific medium conditions. Lygos scientists used multiple media formulations that contained different pathway intermediates for use as the sole carbon source in these strains. Of the strains screened, one was identified capable of catabolizing all pathway intermediates. This is an indirect indication the strain expresses the requisite pathway enzymes and may be capable of producing the target compound in fermentations.

    Publications


      Progress 06/15/15 to 02/14/16

      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?Analysis of strain growth and product formation using different media formulations with product and pathway intermediate titers measured by liquid chromatography.

      Impacts
      What was accomplished under these goals? Lygos screened multiple strains of filamentous fungi for production of a tricarboxylic acid cycle organic acid. The strains were reported in the Literature to produce high concentrations of the target product when grown under specific medium conditions. Lygos scientists used multiple media formulations that contained different pathway intermediates for use as the sole carbon source in these strains. Of the strains screened, one was identified capable of catabolizing all pathway intermediates. This is an indirect indication the strain expresses the requisite pathway enzymes and may be capable of producing the target compound in fermentations.

      Publications