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ABSTRACT Despite the significant presence of plant-derived tricarboxylic acids in some environments, few studies detail the bacterial metabolism oftrans-aconitic acid (Taa) and tricarballylic acid (Tcb). In a soil bacterium,Acinetobacter baylyiADP1, we discovered interrelated pathways for the consumption of Taa and Tcb. An intricate regulatory scheme tightly controls the transport and catabolism of both compounds and may reflect that they can be toxic inhibitors of the tricarboxylic acid cycle. The genes encoding two similar LysR-type transcriptional regulators, TcuR and TclR, were clustered on the chromosome withtcuAandtcuB, genes required for Tcb consumption. The genetic organization differed from that inSalmonella entericaserovar Typhimurium, in whichtcuAandtcuBform an operon with a transporter gene,tcuC. InA. baylyi,tcuCwas not cotranscribed withtcuAB. Rather,tcuCwas cotranscribed with a gene, designatedpacI, encoding an isomerase needed for Taa consumption. TcuC appears to transport Tcb andcis-aconitic acid (Caa), the presumed product of PacI-mediated periplasmic isomerization of Taa. Two operons,tcuC-pacIandtcuAB, were transcriptionally controlled by both TcuR and TclR, which have overlapping functions. We investigated the roles of the two regulators in activating transcription of both operons in response to multiple effector compounds, including Taa, Tcb, and Caa.IMPORTANCEIngestion of Taa and Tcb by grazing livestock can cause a serious metabolic disorder called grass tetany. The disorder, which results from Tcb absorption by ruminants, focuses attention on the metabolism of tricarboxylic acids. Additional interest stems from efforts to produce tricarboxylic acids as commodity chemicals. Improved understanding of bacterial enzymes and pathways for tricarboxylic acid metabolism may contribute to new biomanufacturing strategies. 

Abstract The ambr250 high-throughput bioreactor platform was adopted to provide a highly-controlled environment for a project investigating genome instability in Chinese hamster ovary (CHO) cells, where genome instability leads to lower protein productivity. Development of the baseline (control) and stressed process conditions highlighted the need to control critical process parameters, including the proportional, integral, and derivative (PID) control loops. Process parameters that are often considered scale-independent, include dissolved oxygen (DO) and pH; however, these parameters were observed to be sensitive to PID settings. For many bioreactors, control loops are cascaded such that the manipulated variables are adjusted concurrently. Conversely, for the ambr250 bioreactor system, the control levels are segmented and implemented sequentially. Consequently, each control level must be tuned independently, as the PID settings are independent by control level. For the CHO cell studies, it was observed that initial PID settings did not resulted in a robust process, which was observed as elevated lactate levels; which was caused by the pH being above the setpoint most of the experiment. After several PID tuning iterations, new PID settings were found that could respond appropriately to routine feed and antifoam additions. Furthermore, these new PID settings resulted in more robust cell growth and increased protein productivity. This work highlights the need to describe PID gains and manipulated variable ranges, as profoundly different outcomes can result from the same feeding protocol. Additionally, improved process models are needed to allow process simulations and tuning. Thus, these tuning experiments support the idea that PID settings should be fully described in bioreactor publications to allow for better reproducibility of results. 

Abstract Background As bioprocess intensification has increased over the last 30 years, yields from mammalian cell processes have increased from 10’s of milligrams to over 10’s of grams per liter. Most of these gains in productivity can be attributed to increasing cell densities within bioreactors. As such, strategies have been developed to minimize accumulation of metabolic wastes, such as lactate and ammonia. Unfortunately, neither cell growth nor biopharmaceutical production can occur without some waste metabolite accumulation. Inevitably, metabolic waste accumulation leads to decline and termination of the culture. While it is understood that the accumulation of these unwanted compounds imparts a suboptimal culture environment, little is known about the genotoxic properties of these compounds that may lead to global genome instability. In this study, we examined the effects of high and moderate extracellular ammonia on the physiology and genomic integrity of Chinese hamster ovary (CHO) cells. Results Through whole genome sequencing, we discovered 2394 variant sites within functional genes comprised of both single nucleotide polymorphisms and insertion/deletion mutations as a result of ammonia stress with high or moderate impact on functional genes. Furthermore, several of these de novo mutations were found in genes whose functions are to maintain genome stability, such as Tp53, Tnfsf11, Brca1, as well as Nfkb1. Furthermore, we characterized microsatellite content of the cultures using the CriGri-PICR Chinese hamster genome assembly and discovered an abundance of microsatellite loci that are not replicated faithfully in the ammonia-stressed cultures. Unfaithful replication of these loci is a signature of microsatellite instability. With rigorous filtering, we found 124 candidate microsatellite loci that may be suitable for further investigation to determine whether these loci may be reliable biomarkers to predict genome instability in CHO cultures. Conclusion This study advances our knowledge with regards to the effects of ammonia accumulation on CHO cell culture performance by identifying ammonia-sensitive genes linked to genome stability and lays the foundation for the development of a new diagnostic tool for assessing genome stability.