SATPC0037617 Tab 04 4 SOW..pdf

Statement of Work

Background NASA will employ model microbial hosts extensively used in biomanufacturing—E.

coli, S. cerevisiae, and P. pastoris—none of which are recognized for high radiotolerance [1].

Our recent efforts as part of the B-SURE program have demonstrated the capacity (through targeted overexpression) to enable substantially higher tolerance to radiation sources. Thus, the combination of these effects in concert with strain engineering will be assessed here for lactic acid overproduction. Thus, our primary assessment metric will be the production of lactic acid (LA) as the prototype molecule for monomer formation. LA as described above was chosen as it is the key precursor to polylactic acid, which is used widespread as the primary filament in 3D printing [2, 3]. Since S. cerevisiae and P. pastoris do not naturally produce lactic acid, engineering efforts will be required [4, 5].

SOW

Task 1:

Initial efforts would aim to enhance the biosynthesis of LA in the two yeast organisms, P.

pastoris and S. cerevisiae, based upon established methodologies [4, 5]. Specifically, the Bos taurus lactate dehydrogenase gene (BtLDH) will be introduced to facilitate L-lactic acid production [4, 5]. Additionally, P. pastoris will be transformed with a putative lactate transporter, kpJEN1 recently reported to increase lactic acid production [4]. A similar strategy of LDH overexpression will be chosen for the E. coli host. After growth, production of lactic acid, substrate consumption, and by product formation in both planktonic and encapsulated modalities will be quantified on a daily fashion and compared using an established HPLC method [4]. Further strain engineering efforts can be used to redirect flux toward lactic acid accumulation and secretion.

Timeline: 0 – 3 months Deliverable: Establishment of at least 1 lactic acid producing strains capable of producing > 1 g/L production.

Task 2:

Once constructed, the above engineered strains will be evaluated using APMBR permeate as the aqueous feedstock for these cells. Production of LA as well as overall growth / tolerance information will be evaluated via HPLC and LogPhase600 systems. Target objective is to obtain >90% LA performance on wastewater as on pristine water.

Timeline: 3-10 months Deliverable: Establishment of at least 1 lactic acid producing strains capable of producing > 1 g/L production on APMBR permeate.

Task 3:

APMBR permeate fermentations of LA will be scaled up to the 2 L bioreactor setting at

UT-Austin and full FMBR at NASA and evaluated for overall production kinetics. Once established, this LA will be extracted from the bioreactor, purified using membrane-based purification, and polymerized using the dimer of lactic acid (lactide). As an alternative, it may be possible to perform a condensation polymerization of lactic acid directly, but the resultant polymer would be inferior in most properties. Both approaches will be evaluated. PLA levels will be characterized using Gas Chromatography (GC) techniques outlined in previous literature [6], as well as molecular weight analysis by GPC will be conducted.

Timeline: 6-12 months Deliverable: Delivery of PLA polymer (>500 mg) along with a established process for polymerization.

1. Zhang, Y.P., J. Sun, and Y. Ma, Biomanufacturing: history and perspective. J Ind Microbiol

Biotechnol, 2017. 44(4-5): p. 773-784.

2. Choi, S.Y., I.J. Cho, Y. Lee, S. Park, and S.Y. Lee, Biocatalytic synthesis of polylactate and its copolymers by engineered microorganisms. Methods Enzymol, 2019. 627: p. 125-162.

3. Huang, S., Y. Xue, B. Yu, L. Wang, C. Zhou, and Y. Ma, A Review of the Recent Developments in the Bioproduction of Polylactic Acid and Its Precursors Optically Pure Lactic Acids.

Molecules, 2021. 26(21).

4. de Lima, P.B., K.C. Mulder, N.T. Melo, L.S. Carvalho, G.S. Menino, E. Mulinari, V.H. de Castro, T.F. Dos Reis, G.H. Goldman, B.S. Magalhães, and N.S. Parachin, Novel homologous lactate transporter improves L-lactic acid production from glycerol in recombinant strains of Pichia pastoris. Microb Cell Fact, 2016. 15(1): p. 158.

5. Song, J.Y., J.S. Park, C.D. Kang, H.Y. Cho, D. Yang, S. Lee, and K.M. Cho, Introduction of a bacterial acetyl-CoA synthesis pathway improves lactic acid production in Saccharomyces cerevisiae. Metab Eng, 2016. 35: p. 38-45.

6. Jung, Y.K., T.Y. Kim, S.J. Park, and S.Y. Lee, Metabolic engineering of Escherichia coli for the production of polylactic acid and its copolymers. Biotechnol Bioeng, 2010. 105(1): p.

161-71.