Polyketide synthase (PKS) enzymes have a modular, deterministic logic that holds the potential to act as a flexible chemical factory for the biological production of a huge diversity of valuable small molecule compounds. However, engineering a custom PKS to produce a specific desired product currently requires years of trial and error, for reasons that remain poorly understood. In this project, we have developed a rapid, high throughput, Design-Build-Test-Learn (DBTL) cycle for polyketide synthases (PKSs) and demonstrate its utility for production of materials precursors. The objectives are 1) to develop a rapid, high-throughput (HT) DBTL cycle for PKSs that will enable production of a large number of unnatural, organic molecules on demand at high titer, rate, and yield (TRY); 2) to demonstrate the utility of the PKS DBTL cycle to produce three molecules: one commodity chemical (caprolactam or valerolactam) and two novel materials precursors (caprolactam or valerolactam derivatives); and 3) to demonstrate the utility of the PKS DBTL cycle to increase the TRY of one molecule (caprolactam or valerolactam). In this project, we have successfully demonstrated our high throughput PKS DBTL pipeline, and have biologically produced valerolactam and several other novel nylon monomers.

09 BIOMASS FUELS↗

DOE OSTI2022

Novel Approaches to the Isolation of Glucaric Acid from Fermentation Broth. Final report

Kalion has successfully demonstrated a fermentation route using glucose as the feedstock. This game-changing technology allows Kalion to economically produce large-scale quantities of high-purity glucaric acid products. Kalion has also developed a novel downstream processing strategy for isolating and purifying glucaric acid forms, including the free acid, monoammonium glucarate, and monopotassium glucarate. Despite the low solubility of these products, significant water removal is needed prior to crystallization, requiring energy intensive evaporation or reverse osmosis. Here, we work in collaboration with the Separations Consortium to develop novel, low-cost means of water removal in Kalion’s glucarate/glucaric acid purification process. This proposed CRADA project with national labs (ORNL, ANL, and NREL) evaluated various technologies developed by the Separations Consortium as applied to glucaric acid purification, intended to generate a scalable, cost-effective, energy-efficient process. Four approaches were evaluated with mock aqueous solutions of glucaric acid or glucarate, and the results analyzed with respect to separation effectiveness and potential cost savings. The leading technology will then be further developed and tested with actual clarified or non-clarified broth (depending on the approach) obtained from 20L fermentations provided by Kalion, that will contain inorganic ions, byproducts, and residual sugars in addition to the product.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2020

Outer membrane vesicles catabolize lignin-derived aromatic compounds in Pseudomonas putida KT2440

Lignin is an abundant and recalcitrant component of plant cell walls. While lignin degradation in nature is typically attributed to fungi, growing evidence suggests that bacteria also catabolize this complex biopolymer. However, the spatiotemporal mechanisms for lignin catabolism remain unclear. Improved understanding of this biological process would aid in our collective knowledge of both carbon cycling and microbial strategies to valorize lignin to value-added compounds. Here, we examine lignin modifications and the exoproteome of three aromatic–catabolic bacteria: Pseudomonas putida KT2440, Rhodoccocus jostii RHA1, and Amycolatopsis sp. ATCC 39116. P. putida cultivation in lignin-rich media is characterized by an abundant exoproteome that is dynamically and selectively packaged into outer membrane vesicles (OMVs). Interestingly, many enzymes known to exhibit activity toward lignin-derived aromatic compounds are enriched in OMVs from early to late stationary phase, corresponding to the shift from bioavailable carbon to oligomeric lignin as a carbon source. In vivo and in vitro experiments demonstrate that enzymes contained in the OMVs are active and catabolize aromatic compounds. Taken together, this work supports OMV-mediated catabolism of lignin-derived aromatic compounds as an extracellular strategy for nutrient acquisition by soil bacteria and suggests that OMVs could pote

biological funneling↗