Source: ARCH INNOTEK, LLC submitted to NRP
DEVELOPMENT OF A LOW-COST NATURAL PIGMENT PRODUCTION IN A GRAS YEAST
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
Reporting Frequency
Annual
Accession No.
1015885
Grant No.
2018-33610-28262
Cumulative Award Amt.
$100,000.00
Proposal No.
2018-00445
Multistate No.
(N/A)
Project Start Date
Jul 1, 2018
Project End Date
Aug 31, 2019
Grant Year
2018
Program Code
[8.8]- Biofuels and Biobased Products
Recipient Organization
ARCH INNOTEK, LLC
4320 FOREST PARK AVE STE 303
SAINT LOUIS,MO 63108
Performing Department
(N/A)
Non Technical Summary
Carotenoids, the basic source of yellow, orange and red, are among the most common, naturally occurring pigments. Because of their antioxidant properties and colors, currently, carotenoids are used commercially as feed additives, natural food colorants, dietary supplements and cosmetics and pharmaceutical industries. Currently, most of carotenoids used industrially are chemically synthesized from petrochemical sources, a disadvantage as chemical synthesis yields food safety issue as well as not environmentally friendly. Carotenoids produced from their natural source are projected to be the fastest-growing market segment owing to increasing consumers' preferences toward natural products. A major challenge for manufacturers is the high production cost of natural products compared to the chemical synthetic versions, which limits the obtainable market. Without innovation, processes to isolate natural carotenoids cannot compete on price with the synthetic counterparts, particularly in aquaculture and poultry feed market. We aim to develop novel technologies which will allow us to reliably produce high-value carotenoids at low cost and sustainable commercial levels through yeast fermentation-based processes to satisfy the rapidly growing market demands of natural products for animal and human health. Our technology is based on bioengineering specialized General Regarded as Safe (GRAS) yeast which allows us the ability to make carotenoids more normally found elsewhere in nature. Our technologies have multiple benefits, including a clean "natural" label, better product quality, improved supply chain, reduced production cost, and improved sustainability. Compared with the current petroleum-based synthetic method, our approach will meet its high yield and lower-cost and will surpass it with a clean, natural label. Our approach will exceed the low yield and high cost of natural source extraction. After commercial-scale production, we believe that our product would be widely used by the aquaculture and poultry industries and would replace current chemically synthesized product in the animal feed market. The aquaculture and poultry industries, and thus consumers, will benefit from our wholesome product.
Animal Health Component
30%
Research Effort Categories
Basic
50%
Applied
30%
Developmental
20%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
51152301060100%
Goals / Objectives
The major of the project is to develop a clean and sustainable source of carotenoids through low-cost fermentation using metabolically engineered (GRAS) yeast. Since production of natural carotenoids from natural sources (plants or algae) is too expensive, most commercial carotenoids (e.g. astaxanthin) used in animal feed is synthetic and derived from petrochemical sources, which raises issues about food safety because the final product (salmon, trout, eggs, and chicken) will enter the human food supply chain. The major challenge for manufacturers is the high production cost from natural sources, which limits the obtainable market. Therefore, there is a strong desire and need in the aquaculture and poultry industries for a source of low-cost natural alternatives. The Phase I research is to demonstrate the feasibility of using a novel yeast fermentation process to produce carotenoids with the same stereoisomeric configuration as the natural-sourced product. Two objectives will be addressed in accomplishing the overall goal of the research. Objective #1: To identify novel key enzyme(s) by whole-genome sequencing method. The putative genes will be examined by heterologous expression in yeast strain. Comparison with the HPLC and LC-MS profiles of the control strain will reveal the enzymes' functions. Objective #2: To demonstrate the potential for a commercially-viable level of production. In order to demonstrate the potential for a commercially-viable level of production, the objective 2 will focus on increasing the supply of key precursor , acetyl-CoA, and fermentation optimization. We propose two new approaches to increase cytoplasmic acetyl-CoA supply. In parallel to strain engineering, we will perform fermentation process development. We will use shake flasks to screen for optimal conditions. Key cultivation factors including pH and carbon/nitrogen (C/N) ratio, will be optimized. Also substrate co-feeding strategy will be used to reduce metabolic burden for precursor synthesis. We will further validate these conditions ing bench-top bioreartor. After stepwise engineering and optimization of the fermentation process, we expect that over 1 g/L final product will be attainable after Phase I research.
Project Methods
The production of natural carotenoids from natural source is too expensive, most commercial carotenoids used in animal feed is synthetic and derived from petrochemical sources, which raises issues about food safety (potential toxicity and unknown by-products ). Although there are many disadvantages, the world market is still dominated by synthetic versions due to the lack of alternative sources. This Phase I project will apply synthetic biology and bioprocess optimization to develop a novel, safe, cost- effective fermentation production process via metabolically-engineered GRAS yeast. Via whole genome sequencing and synthetic biology, novel key enzymes will be identified and integrated, along with supporting pathways, into yeast chromosomes. Product synthesis will be maximized via genetic modifications and fermentation optimization.

Progress 07/01/18 to 02/28/19

Outputs
Target Audience:In the past year, we have developed a non-conventional yeast (Yarrowia lipolytica) platform to produce astaxanthin with the same structure as that naturally sourced from algae, the preferred source for animal feed and human dietary supplements. We successfully achieved the goal (1 g/L) and milestones for the yeast fermentation that we set up in our phase I project. After fermentation optimization, the platform strain can produce ~2 g/L astaxanthin with fed-batch fermentation in 5 L bioreactor. Furthermore, we have successfully cloned two diacylglycerol acyltransferase genes (DGAT1 and DGAT2) from the astaxanthin-producing Haematococcus pluvialis (UTEX 2505) strain by whole genome sequencing and RACE techniques. In addition, we have developed a HILIC-MS/MS in conjunction with dynamic 13C-tracking method to accurately quantify the concentration of intracellular metabolites. The results indicate limited TCA cycle activity and, combined with the shortage supply of GGPP may be limiting factors. The identifications of these bottlenecks/rate limiting steps inform rational further strain improvement. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Agraduate student, Jeffrey Czajka from Washington University in St. Louis,received industrial training through this grant by being involved inLC-MS methoddevelopment and analysis with Arch Innotek Scientists. Another graduate student, Lin Cheng from Washington University, performed independent research involved in bioprocess development and fermentation operationsat Arch Innotek under this grant support. A undergraduate, Justin Nathenson from Washington University in St. Louis, did research involved in product extraction/purification as summer intern at Arch Innotek. All three students received industrialexperience through this grant. How have the results been disseminated to communities of interest?Our work has attracted much attention from angel investors. We have received investment from BioGenerator and the Yield Lab. Several companies (Algae Health Sciences, Kemin, BASF etc) have also shown great interest in our technology/products and intend to establish a business partnership with Arch Innotek. We are planning on proceeding with pilot-scale bioreactor tests (1,000 L) at the Center for Crops Utilization Research at Iowa State University. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? In the past year, we have developed a non-conventional yeast (Yarrowia lipolytica) platform to produce astaxanthin with the same structure as that naturally sourced from algae, the preferred source for animal feed and human dietary supplements. We successfully achieved the goal (1 g/L) and milestones for the yeast fermentation that we set up in our phase I project. After fermentation optimization, the platform strain can produce ~2 g/L astaxanthin with fed-batch fermentation in 5 L bioreactor. Furthermore, we have successfully cloned two diacylglycerol acyltransferase genes (DGAT1 and DGAT2) from the astaxanthin-producing Haematococcus pluvialis (UTEX 2505) strain by whole genome sequencing and RACE techniques. In addition, we have developed a HILIC-MS/MS in conjunction with dynamic 13C-tracking method to accurately quantify the concentration of intracellular metabolites. The results indicate limited TCA cycle activity and, combined with the shortage supply of GGPP may be limiting factors. The identifications of these bottlenecks/rate limiting steps inform rational further strain improvement. Arch Innotek has successfully completed all the Phase I objectives. With the successful completion of the Phase I objectives, Arch Innotek has demonstrated that the feasibility of developing a safe, cost effective fermentation process using metabolically engineered yeast to produce at commercially-viable levels astaxanthin. The phase I success provides strong foundation for further improvements and development of a commercially-viable biomanufacturing process in phase II. The production of astaxanthin by fermentation will benefit the global society and environment by improving the renewability of the production process.

Publications

  • Type: Journal Articles Status: Under Review Year Published: 2019 Citation: Citation is not available, under review by iScience