Xyleco’s Redacted MCAN Submission T. reesei, strain 3CH-3 J-19-0001

January 24, 2020

145854099.4

i. Introduction

Xyleco is seeking to use a modified T. reesei during its cellulase enzyme-production process using, e.g., corn or other inductant to induce the production of -degrading cellulases. These cellulases are to be utilized for the manufacture of sugars such as xylose and glucose, or sugar-derived substances such as lactic acid. The EPA has previously determined that modified T. reesei is not likely to present an unreasonable risk, is not pathogenic to humans or animals, and has a long history of safe use for production of enzymes in a variety of industries, including sugar production, food and animal feed, pharmaceuticals, textiles, and paper processing.1

Xyleco’s enzyme preparations derived from strain 3CH-3, originating from Bio-Safety Level I RUT- C30 strain, is not likely to present an unreasonable risk. As required under 40 CFR § 725.155, Xyleco submits the following information regarding the organism, the enzyme’s safe attributes, as well as the Company’s benign production and disposal of the organism.

Section I provides Submitter identification.

Section II provides the microorganism information including the features and historical background of strain RUT-C30 from which the modified production strain 3CH-3 will be derived.

Section III summarizes the purpose of using strain 3CH-3 for its enzyme production.

Section IV details the genetic construction of the microorganism to enzyme production. Pursuant to § 14(c) of the Toxic Substances Control Act (TSCA), as amended by the Frank R. Lautenberg Chemical Safety for the 21st Century Act, the Company requests that the information be treated as Confidential Business Information (“CBI”) exempt from public disclosure. Xyleco believes this information does not require substantiation under 15 U.S.C. § 2613(c)(2).

Section V describes Xyleco's proposed manufacturing process, including the fermentation process, recovery of the enzyme product, an inactivation step prior to saccharification, saccharification, disposal procedures and production volumes. Pursuant to § 14(c) of TSCA, the Company requests that the information be treated as CBI exempt from public disclosure. Xyleco believes this information does not require substantiation under 15 U.S.C. § 2613(c)(2).

Section VI provides a description of the intended categories of use by function and application of the enzyme preparation and byproducts from the manufacturing of the organism.

Section VII provides information on worker exposure and procedures to minimizes releases to the environment.

1 See TSCA Section 5(a)(3)(C) Determination for Microbial Commercial Activity Notice (MCAN) J-17-0022.

1 145854099.4 Section VIII includes the certification required under 15 U.S.C. § 2613(c)(5).

Appendices A – I include information, details and data which might be helpful to reference about the topics discussed in this notice.

2 145854099.4 I. Submitter Identification

(1) The name and headquarters of the submitter.

Xyleco, Inc.2 360 Audubon Road Wakefield, MA 01880

(2) The name, address, and telephone number of the principal technical contacts representing the submitter.

Xyleco, Inc. Attn.: Craig Masterman 360 Audubon Road Wakefield, MA 01880 (t) 781.557.5578 [email protected]

Perkins Coie LLP Attn: Jeffrey Hunter 1120 NW Couch Street, 10th Floor Portland, OR 97209 (t) 503.727.2265 [email protected]

2 A Delaware Corporation.

3 145854099.4 II. Microorganism Information

A. Historical Overview and Phenotypic Traits

T. reesei is believed to be an anamorph (asexual stage) of the fungus Hypocrea Jecorina. The T. reesei strain QM6a (wild type) was originally isolated from the Solomon Islands during World War II due to its degradation of canvas and garments of the US Army. It is commonly present on decaying plant matter. Trichoderma species exist within soils of all climates. All strains of T. reesei currently used in biotechnology and basic research are derived from this one isolate.

In a 1970’s mutagenesis program at Rutgers University, a combination of UV light, electron beam radiation and chemical mutagenesis was used to generate QM6a mutant strains with higher cellulase production. One of the strains isolated was the hypersecreting RUT-C30, or T. reesei, modified.3

RUT-C30 is classified as Biosafety Level I (BSL1), meaning it is a well-characterized agent not known to present a risk to human health, while presenting minimal potential hazards to laboratory personnel and the environment. Many BSL1 organisms including T. reesei RUT-C30 have been utilized in food production processes.4

The EPA has previously determined that T. reesei RUT-C30 is not likely to present an unreasonable risk, is not pathogenic to humans or animals, and has a long history of safe use for production of enzymes.4

B. Taxonomic Designation

Kingdom: Fungi Phylum: Ascomycota Class: Sordariomycetes Order: Hypocreales Family: Hypocreaceae Genus: Trichoderma Species: reesei Production strain: 3CH-3 Name of the Strain modified to produce Xyleco T. reesei 3CH-3: NRRL11460 (equivalent to ATCC 56765 or RUT-C30)

C. Morphological and Physiological Features

T. reesei is a mesophilic filamentous fungus. The hypersecreting feature of RUT-C30 was found to be associated with changes in ultrastructure of the strain, such as higher endoplasmic reticulum (ER) content.5

3 Peterson R. & Nevalainen H. (2012) Trichoderma reesei RUT-C30--thirty years of strain improvement. Microbiology. 2012 Jan;158(Pt 1):58-68. doi: 10.1099/mic.0.054031-0 4 See TSCA Section 5(a)(3)(C) Determination for Microbial Commercial Activity Notice (MCAN) J-17-0022. 5 Increased endoplasmic reticulum content of a mutant of Trichoderma reesei (RUT-C30) in relation to cellulase synthesis, Ghosh, Enzyme Microb Technol 4, 110–113.

4 145854099.4 RUT-C30 was also reported to lack the typical stacked appearance of the Golgi body which instead resembled individual ER-associated saccules.6

6 Subcellular fractionation of a hypercellulolytic mutant, Trichoderma reesei Rut-C30: localization of endoglucanase in microsomal fraction, Glenn, Appl Environ Microbiol 50, 1137–1143.

5 145854099.4 III. Purpose of Enzyme

A. General

RUT-C30, the hyper-secreting, hyper-producing mutant strain of T. reesei is a workhorse industrial strain and serves as the recipient strain for genetic modification, by introduction of β- glucosidase (cel3a). T. reesei RUT-C30 has an extensive history of safe use in industrial enzyme production in a variety of industries, including sugar production, food and animal feed, pharmaceuticals, textiles, and paper processing. See TSCA Section 5(a)(3)(C) Determination for Microbial Commercial Activity Notice (MCAN) J-19-0001.

One well-known bottleneck in the RUT-C30 cellulolytic process is the relatively low expression of endogenous β-glucosidase (cel3a), resulting in low cellobiose hydrolysis and therefore lower monosaccharide yields. β-glucosidase has also been attributed to several other applications such as in food technology, the cosmetic industry, and various other commercial applications.7

The term cellulases broadly describes enzymes that catalyze the hydrolysis of the β-1,4- glycosidic bonds between individual glucose units within . The mechanism of cellulosic breakdown involves the synergistic action of endoglucanases, cellobiohydrolases, and β-glucosidases. Β-glucosidase (cel3a) is an extra-cellular enzyme which hydrolyzes cellobiose and cellooligosaccharides to release fermentable D-glucose.

B. Common or Usual Name of Substance

The enzyme preparation is a biological material containing secreted cellulases (the “Cocktail”), along with any organic and inorganic material derived from cultivation and fermentation processes. The over-expressed enzyme is β-glucosidase. Β-glucosidase is also commonly known as cellobiase or Bgl2, EC no. 3.2.1.21.

Other designations include β-D-glucosidase and β-glucoside glucohydrolase, gentiobiase, emulsin, elaterase, aryl-β-glucosidase, β-glucoside glucohydrolase, arbutinase, amygdalinase, p- nitrophenyl β-glucosidase, primeverosidase, linamarase, salicilinase, and β-1,6-glucosidase.

7 Vaithanomsat, P., Songpim, M., Malapant, T., Kosugi, A., Thanapase, W., & Mori, Y. (2011). Production of β- Glucosidase from a Newly Isolated Aspergillus Species Using Response Surface Methodology. International Journal of Microbiology, 2011, 949252. doi: 10.1155/2011/949252 Juhasz T, Kozma K, Szengyel Z, Reczey K. (2003) Production of β-glucosidase in mixed culture of Aspergillus Niger BKMF 1305 and Trichoderma reesei RUT C30. Food Technol Biotechnol. 2003; 41:49–53 Karray R., Hamza M., Sayadi S. (2015) Evaluation of ultrasonic, acid, thermo-alkaline and enzymatic pre-treatments on anaerobic digestion of Ulva rigida for production. Bioresour Technology 2015;187:205-213. doi: 10.1016/j.biortech.2015.03.108.

6 145854099.4 IV. Genetic Construction A. Recipient Organism The T. reesei RUT-C30 host strain was genetically engineered to overexpress modified endogenous β-glucosidase gene.

1. The name of the enzyme is β-glucosidase. See Section III B for other designations.

2. The name of the protein production strain is Trichoderma reesei 3CH-3. B. Donor Organism The homologous enzyme β-glucosidase gene described in this submission was selected in order to overcome bottleneck β-glucosidase activity of the parent strain, thus enhancing cellulosic degradation and bioconversion of lignocellulosic biomass to bioethanol and other biofuels. The native nucleotide sequence of the gene was modified to include an 8-His polyhistidine affinity tag at its C' terminus in order to distinguish from unlabeled endogenous cel3a. C. Production Organism The T. reesei RUT-C30 3CH-3 strain was constructed for overexpression of β-glucosidase (cel3a). An expression cassette carrying the cel3a gene was used to transform and integrate into the genome of RUT-C30. The production strain 3CH-3 differs from its recipient strain (RUT- C30) in its enhanced β-glucosidase activity. The resultant strain secretes an increased amount of β-glucosidase, including native and His-tagged protein, into the culture media, as compared to RUT-C30. In addition, the 3CH-3 strain also produces the other endogenous cellulases found in RUT-C30 such as cellobiohydrolases and endoglucanases.

The techniques used in transforming and culturing T. reesei have been previously described (Sambrook and Russell, 2001)8. The production organism also meets the criteria for safe production of microorganisms9. The T. reesei strain is non-pathogenic, non-toxigenic, and considered a safe host for other harmless gene products10. The production strain is capable of sporulating, indicating that the inserted cel3a does not interfere with major metabolic function. The spores constitute the master cell bank, from which the production seed culture is made.

8 Sambrook, J., and Russell, D.W. (2001) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press. 9 Pariza M., Johnson E. (2001). Evaluating the Safety of Microbial Enzyme Preparations Used in Food Processing: Update for a New Century. Regul Toxicol Pharmacol. 2001 Apr v33(2):173-86. doi.org/10.1006/rtph.2001.1466 10 Peterson R. & Nevalainen H. (2012) Trichoderma reesei RUT-C30--thirty years of strain improvement. Microbiology. 2012 Jan;158(Pt 1):58-68. doi: 10.1099/mic.0.054031-0

7 145854099.4 V. Process Description of the Manufacturing, Processing, and Use Operations

A. Fermentation Overview

3CH-3 production is submerged-batch fermented under controlled conditions. The production process includes: spore banks, seed cultures to obtain sufficient biomass for large scale fermentation, the main enzyme fermentation, inactivation prior to saccharification, saccharification and recovery, including further downstream processing. The production values and the production diagram are illustrated in Appendix C.

The Company’s fermentation steps include:

• Spore cultivation; • Seed train cultures; • An inactivation step prior to saccharification; and • Enzyme fermentation.

8 145854099.4 VI. Byproducts

The enzymes produced during the fermentation of 3CH-3 will be used to saccharify agricultural waste to simple sugars. No viable cells have been detected in the products after saccharification. See Appendix E "Viability testing during fermentation and saccharification."

The 3CH-3 enzyme Cocktail, including the over-produced β-glucosidase enzymes, are secreted during the main fermentation. The Cocktail is intended for use during the saccharification process to convert cellulose and hemicellulose to glucose and xylose, and to serve as a fermentable substrate for the production of lactic acid and other specialty food or chemical products.

During saccharification, the proposed dose of the Cocktail is 1.6 g/L in a solution containing approximately 26% w/v pre-treated corncob (to reduce its recalcitrance). One hundred percent of the 3CH-3 Cocktail is transferred to saccharification tanks for the completion of all relevant downstream processes. Adding the enzyme effectively reduces the concentration of pre-treated biomass in the tank from 30% w/v to 26% w/v.

Although 3CH-3 is not present in any product, byproduct or waste, it is again worth noting that the EPA has previously determined that T. reesei modified is not likely to present an unreasonable risk, is not pathogenic to humans or animals, and has a long history of safe use for production of enzymes in a variety of industries, including biofuel production, food and animal feed, pharmaceuticals, textiles, and paper processing.11

11 See TSCA Section 5(a)(3)(C) Determination for Microbial Commercial Activity Notice (MCAN) K-17-0022.

9 145854099.4 VII. Worker Exposure and Environmental Release Information

A. Site Identity

The production strain 3CH-3 will be produced at two sites. Site one, the Massachusetts pilot plant, is located in Wakefield, MA. Site two, the Washington state manufacturing facility, is in Moses Lake, WA. Spore banks will be produced, in certified mobile clean rooms, and maintained separately on site at each location. Only one operator is required to produce and maintain the spore bank. He/she follows all recommended industry practices and procedures for clean room operations.

B. Process Controls

Each of the first two seed culture stages is incubated in sterile flasks fitted with 0.45um filter tops. The transfers of cultures are performed aseptically inside a mobile clean room containing a Class II Type A2 biosafety cabinet. The final transfer of the seed culture to the fermentation tank is done using a closed, sterilized process hose ensuring no exposure to the environment. All seed tanks along with the main fermentation tank are equipped with several sterile 0.01 micron air filters to ensure that any exhaust is filtered and free of 3CH-3 cells and their secreted enzymes prior to expulsion from the building.

In order to test and document ambient air conditions, an SAS Super 100 air sampler was used at each step of the process. Points of transfer and all flask and bioreactor filters were sampled for possible aerosol escape of spores or mycelia. Air samples were plated on both selective and non- selective media. No viable cells appeared on the selective plates, verifying the efficacy of the tank vent filters and pilot plant air cycling and filtration.

See Appendix F for the air sampler plate results.

The whole broth fermentation product is transferred to the saccharification tank through a closed, sterilized process hose. The saccharification tank is equipped with the same filters in place. After saccharification no viable 3CH-3 cells are found. All equipment is cleaned in place (CIP) with an industry standard cleaning solution, sterilized with chlorhexidine gluconate, a medical grade disinfectant, and finally steamed in place (SIP).

See Appendix D for air filter information and Appendix G for more details on CIP procedures used.

C. Worker Exposure

There is minimal 3CH-3 exposure to workers. The production cell bank is made in a mobile clean room equipped with level II biosafety cabinets and filter/UV sterilized air. During seed culture and other steps of fermentation, the employees wear industry standard appropriate PPE, such as lab coats, hair nets, masks, eye protection, and gloves. If a worker is exposed to 3CH-3, the procedure consists of washing the affected area with water. If a spill occurs during any step in the fermentation process, the material is promptly cleaned up and the soiled materials placed

10 145854099.4 in bio-waste disposal bins for proper handling by a third party disposal company, followed by decontamination treatment of the affected area with chlorhexidine gluconate. A mock spill was performed to test cleanup procedures. The spill was soaked with 10% bleach then chlorhexidine for 30 minutes each, wiped clean, sprayed with 70% ethanol and the area was swabbed and plated on non-selective plates. No colonies were present on the plates after 72 hours.

All equipment used in manufacturing is CIP’d, cleaned with chlorhexidine gluconate, and steamed in place (SIP). Wastewater is sent to the wastewater treatment portion of the facility, where it is treated and then discharged in accordance with state and federal regulation and required permits. All clothing and PPE are disposed of using bio-waste containers and sent to a third party for decontamination/disposal in accordance with state and federal regulations. To test our cleaning procedures swabs were taken from each reactor port after each cleaning step. (See Appendix G)

D. Environmental Exposure

The manufacturing process is performed in a controlled environment. The possibility of a release of 3CH-3 or associated cellulases to the environment is remote. Testing has demonstrated that no cells is present post-saccharification. The organism is not present in any final products and there is no exposure to downstream users and consumers. All byproducts produced are common ingredients found in food

11 145854099.4

Appendix A.1 Redacted

13 145854099.4 Appendix A.2 Redacted

14 145854099.4 Appendix C Production Summary & Volumes Production Diagram

15 145854099.4

Appendix D Air Filtration Bioreactor Venting

All large scale bioreactors use vent filters in order to maintain approximately atmospheric pressure during the seed cultures and fermentation. The vent filters used are Donaldson P-SRF N sterile air pleated depth filter elements, which have a “retention rate of ≥99.999% for all particles 0.01 µm and larger” (See URL below for manufacturer’s spec sheet).

https://www.donaldson.com/content/dam/donaldson/compressed-air-and-process/literature/north- america/compressed-air-and-gas/filter-elements/sterile-elements/p-srf-n/f117010-eng/P-SRF-N- Sterile-Air-Pleated-Depth-Filter-Elements.pdf

Air samples were taken at various points throughout fermentation via a hand-held air sampler with a plate containing growth media mounted inside. Sample air volume was 60 L, taken at a rate of 100 L/min. Plates used were PDA +/- hygromycin b in order to discern the amount of process microbes present in pilot plant ambient air. Samples were also taken via plates left out and open for two hours in areas where fermentation processes are frequent; the pilot plant and the R&D lab where fourteen 20 L bioreactors are frequently operated for fermentation process development. Samples were also collected from the open air outside the facility. Plates were cultured for 7 days at 28 C. Refer to Appendix F for results.

Pilot Plant ambient air

The manufacturing area (high bay) air is cleaned using four wall-mounted HEPA filter / UV fan units. They are hospital/clean room grade APS-625 type units with HEPA Pleat-II filters which are sterilized using a built-in UV lamp assembly. These will each clean 625 cubic feet per minute (CFM). The high bay is roughly 90’x90’x20’ so the four filter units will cycle the air approximately once per hour. See the URLs below for relevant equipment specifications.

https://www.hepa.com/standard-filters/hepa-pleat-ii-%C2%AE

https://www.berriman- usa.com/air purifiers air cleaners/aps/model 625 hepa hospital air cleaner for isolation.htm

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MCAN Submission J-19-0001 Xyleco Revised Appendix E January 23, 2020

T.reesei 3CH-3 Cell Kill Study at 48C

4.0E+04

3.5E+04

3.0E+04

2.5E+04

2.0E+04

1.5E+04 Forming Units (CFU) per mL per (CFU) Units Forming - 1.0E+04 Colony 5.0E+03

0.0E+00 0h 0.5h 1h 2h 6h CFU per mL 37,000 10,000 6,000 4,300 0

Time in hours (h)

146995064.1 Appendix F Air Sampling/CFU Air sampling was conducted to collect viable aerosols onto culture plates at various possible release points for T. reesei 3CH-3. Aerosols were collected onto both non-selective and selective (for hygromycin-resistant) plates. Results were recorded in terms of CFU per L of air aspirated.

Hygromycin-resistant colonies indicate presence of production strain 3CH-3. These appeared only on positive control plates.

20 145854099.4 Appendix G CIP inactivation of T. reesei 3CH-3 CIP method Reactor ports were swabbed and samplings were streaked onto selective (hygromycin) and non-selective plates. Results were recorded in terms of CFU's from each plate.

CFU selective plate (+Hygro) CFU non-selective plate Before CIP too many to count too many to count After CIP no colonies observed 1

Clean-In-Place (CIP) procedures

After completion of fermentation (7-12 days), the tanks are drained and the whole enzyme broth stored in totes. For cleaning validation, samples were taken from the tank by swabbing the interior surfaces inside the top third and bottom third sight glass ports. The tanks are then given an initial rinse with approximately 30 L of water, sprayed through a rotating spray-ball assembly in order to rinse the bulk of the remaining solids from the walls, impeller, sparge tube and sensors. This rinse water and solids mixture is recirculated via the bottom valve, a pneumatic baffle type pump, and through the spray-ball back into the tank. After sufficiently rinsed, the 30 L of water and solids is then drained via the bottom valve and pumped to the in-house waste-water treatment tank for processing.

To thoroughly clean the tanks after rinsing, a solution containing 1 L of CIP-100 (STERIS) is prepared by filling the tank with 100 L of water, installing a bag filter canister in line with the recirculation loop immediately downstream of the pneumatic pump (filter pore size 100 µm) and adding the 1 L of CIP- 100. The resulting pH is 11-12 and the cleaning solution is then pumped through the spray-ball at a pressure sufficient to spray all surfaces and can be done through multiple ports if necessary. Any remaining solids are removed by the bag filter, and the solution is heated to 70 C by the tank’s built-in jacket heating system. The heated caustic is then cycled through the recirculation loop for a full hour after reaching temperature. This solution is then drained in the same way and sent to the in-house waste- water treatment tank for processing. After CIP completion, the tank is cooled and rinsed twice with 100 L of fresh water, or until pH becomes approximately neutral. The rinse water is sent to the same waste- water tank.

To validate the cleaning, swab samples were again taken from the same locations and cultured for 72 hours on dextrose agar (PDA) plates at room temperature, as were the initial pre-cleaning samples.

21 145854099.4 Appendix H

T. reesei forms spores under distress. RUT-C30 and 3CH-3 both form recognizably round to ovoid shaped spores of about 4um diameter. Spore formation, however, has not been observed in our fermentation process. Below are two microscope images comparing a typical 3CH-3 culture with the same culture spiked with approximately 105 spores. Cells were treated with Schaeffer and Fulton stain to better distinguish spore from vegetative cells.

before spiking after spiking

22 145854099.4 Appendix I Analytical Reports

Development Report, Determination of Paracelsin in Xyleco’s Trichoderma Reesei Strain 3CH-3 Enzyme Broth, Induced on Biomass Using Submerged Fermentation, December 5, 2019 (Redacted)

Development Report, Determination of Paracelsin in Broth from Biomass Saccharification with Enzymes Produced from Xyleco’s Trichoderma Reesei Strain 3CH-3, December 9, 2019 (Redacted)

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