Electron withdrawing group-dependent substrate inhibition of an α-ketoamide reductase from Saccharomyces cerevisiae.

Aldo-keto reductases remain enzymes of interest in biocatalysis due to their ability to reduce carbonyls to alcohols stereospecifically. Based on genomic sequence, we identified aldo-keto reductases of a S. cerevisiae strain extracted from an ancient amber sample. One of the putative enzymes, AKR 163, displays 99% identity with α-amide ketoreductases from the S288C and YJM248 S. cerevisiae strains, which have been investigated for biocatalytic applications. To further investigate AKR 163, we successfully cloned, expressed in E.coli as a glutathione-S-transferase fusion protein, and affinity purified AKR 163. Kinetic studies revealed that AKR 163 experiences strong substrate inhibition by substrates containing halogen atoms or other electron withdrawing groups adjacent to the reactive carbonyl, with Ki values ranging from 0.29 to 0.6 mM and KM values ranging from 0.38 to 0.9 mM at pH 8.0. Substrates without electron withdrawing groups do not display substrate inhibition kinetics and possess much larger KM values between 83 and 260 mM under the same conditions. The kcat values ranged from 0.5 to 2.5s-1 for substrates exhibiting substrate inhibition and 0.22 to 0.52s-1 for substrates that do not engage in substrate inhibition. Overall, the results are consistent with rate-limiting dissociation of the NADP+ cofactor after hydride transfer when electron withdrawing groups are present and activating the reduction step. This process leads to a buildup of enzyme-NADP+ complex that is susceptible to binding and inhibition by a second substrate molecule.

Improvement of glucaric acid production in E. coli via dynamic control of metabolic fluxes.

D-glucaric acid can be used as a building block for biopolymers as well as in the formulation of detergents and corrosion inhibitors. A biosynthetic route for production in E. coli has been developed (Moon et al., 2009), but previous work with the glucaric acid pathway has indicated that competition with endogenous metabolism may limit carbon flux into the pathway. Our group has recently developed an E. coli strain where phosphofructokinase (Pfk) activity can be dynamically controlled and demonstrated its use for improving yields and titers of the glucaric acid precursor myo-inositol on glucose minimal medium. In this work, we have explored the further applicability of this strain for glucaric acid production in a supplemented medium more relevant for scale-up studies, both under batch conditions and with glucose feeding via in situ enzymatic starch hydrolysis. It was found that glucaric acid titers could be improved by up to 42% with appropriately timed knockdown of Pfk activity during glucose feeding. The glucose feeding protocol could also be used for reduction of acetate production in the wild type and modified E. coli strains.

Actinomycetes scale-up for the production of the antibacterial, nocathiacin.

An Amycolatopsis fastidiosa culture, which produces the nocathiacin class of antibacterial compounds, was scaled up to the 15,000 L working volume. Lower volume pilot fermentations (600, 900, and 1,500 L scale) were conducted to determine process feasibility at the 15,000 L scale. The effects of inoculum volume, impeller tip speed, volumetric gas flow rate, superficial gas velocity, backpressure, and sterilization heat stress were examined to determine optimal scale-up operating conditions. Inoculum volume (6 vs. 2 vol %) and medium sterilization (R(o) of 68 vs. 92 min(-1)) had no effect on productivity or titer, and higher impeller tip speeds (2.1 vs. 2.9 m/s) had a slight effect (20% decrease). In contrast, higher backpressure, incorporating increased head pressure at the 15,000 L scale (1.2 vs. 0.7 kg/cm(2)) and low gas flow rates (0.25 vs. 0.8 vvm), appeared to be problematic (40-50% decrease). High off-gas CO2 levels were likely reasons for observed lower productivity. Consequently, air flow rate for this 25-fold scale-up (600-15,000 L) was controlled to match off-gas CO2 profiles of acceptable smaller scale batches to maintain levels below 0.5%. The 15,000 L-scale fermentation achieved an expected nocathiacin I titer of 310 mg/L after 7 days. Other on-line data (i.e., pH, oxygen uptake rate, and CO2 evolution rate) and off-line data (i.e., analog production, glucose utilization, ammonium production, and dry cell weight) at the 15,000 L scale also tracked similarly to the smaller scale, demonstrating successful fermentation scale-up.

Pilot-scale process development and scale up for antifungal production.

A pilot-scale fermentation was developed for an antifungal compound produced by a filamentous fungus. Replacement of galactose with lactose (20-fold cost savings) and a threefold phosphate reduction (15 to 5 g/L) improved productivity 2.5-fold. Addition of supplements--glycine, cobalt chloride, and trace elements--resulted in a further twofold productivity increase, greater process robustness, and less foaming which reduced antifoam addition tenfold (30 to <3 mL/L). Mid-cycle lactose limitations were addressed by raising initial lactose levels (40 to 120 g/L) resulting in another twofold productivity increase. Overall, peak titers increased tenfold from 45 +/- 9 to 448 +/- 39 mg/L, and productivities improved from 3 to 25 mg/L day. Despite its high productivity, process scale up was challenged by high broth viscosity (5,000-6,000 cP at 16.8 s(-1)). Gassed power requirements at the 600 L scale (4.7 kW/1,000 L) exceeded available power at the 15,000 L scale (3.0 kW/1,000 L), and broth transfer to the downstream isolation facility was hindered. Mid-cycle broth dilution with up to five 10 vol% additions of 12 wt% lactose solution or whole medium-reduced viscosity three- to fivefold (1,000-1,500 cP at 16.8 s(-1)), gassed power within scale-up limits (2.5 kW/1,000 L), and peak titer by up to 45%. The process was scaled up to the 15,000 L working volume based on constant aeration rate (vvm) and peak impeller tip speed, raising superficial velocities at similar shear. This strategy maximized mass transfer rates at target gassed power per unit volume levels, and along with controlled broth viscosity, precluded multiple dilution additions. A final titer of 333 mg/L with one dilution addition was achieved, somewhat lower than expected, likely owing to inhibition from some unmeasured volatile compound (not believed to be carbon dioxide) during an extended period of high back-pressure in the early production phase.

Isolation and structure elucidation of thiazomycin- a potent thiazolyl peptide antibiotic from Amycolatopsis fastidiosa.

Thiazolyl peptides are a class of rigid macrocyclic compounds richly populated with thiazole rings. They are highly potent antibiotics but none have been advanced to clinic due to poor aqueous solubility. Recent progress in this field prompted a reinvestigation leading to the isolation of a new thiazolyl peptide, thiazomycin, a congener of nocathiacins. Thiazomycin possesses an oxazolidine ring as part of the amino-sugar moiety in contrast to the dimethyl amino group present in nocathiacin I. The presence of the oxazolidine ring provides additional opportunities for chemical modifications that are not possible with other nocathiacins. Thiazomycin is extremely potent against Gram-positive bacteria both in vitro and in vivo. The titer of thiazomycin in the fermentation broth was very low compared to the nocathiacins I and III. The lower titer together with its sandwiched order of elution presented significant challenges in large scale purification of thiazomycin. This problem was resolved by the development of an innovative preferential protonation based one- and/or two-step chromatographic method, which was used for pilot plant scale purifications of thiazomycin. The isolation and structure elucidation of thiazomycin is herein described.

Sustainable reduction of bioreactor contamination in an industrial fermentation pilot plant.

Facility experience primarily in drug-oriented fermentation equipment (producing small molecules such as secondary metabolites, bioconversions, and enzymes) and, to a lesser extent, in biologics-oriented fermentation equipment (producing large molecules such as recombinant proteins and microbial vaccines) in an industrial fermentation pilot plant over the past 15 years is described. Potential approaches for equipment design and maintenance, operational procedures, validation/verification testing, medium selection, culture purity/sterility analysis, and contamination investigation are presented, and those approaches implemented are identified. Failure data collected for pilot plant operation for nearly 15 years are presented and best practices for documentation and tracking are outlined. This analysis does not exhaustively discuss available design, operational and procedural options; rather it selectively presents what has been determined to be beneficial in an industrial pilot plant setting. Literature references have been incorporated to provide background and context where appropriate.