Ph.D. Viva Voce of Mr. Girish H. Rajacharya (Entry number 2019SRZ8785) will defend his Ph.D.
Zoom link:
https://us02web.zoom.us/j/88233026519?pwd=fQib7SIeOmMyHBqF2okpB7Rchl9gd7.1
Abstract:
Recombinant protein production (RPP) in Escherichia coli (E. coli) is widely used in biotechnology and pharmaceuticals but imposes significant metabolic burden, leading to reduced growth, stress responses, and lower yields. This thesis presents a systems-level investigation of the metabolic, regulatory, and physiological factors governing RPP using an integrative multi-omics approach. In the first part of objective, our studies recombinant protein production (RPP) in Escherichia coli with the help of comprehensive multi-omics cellular reprogramming. Due to the significant metabolic burden RPP imposes on the host, the host triggers the stringent response, a global regulatory response that fine tunes the distribution of metabolic activities in the cell as a reaction to nutrient scarcity. To define the response, we used a defined chemical medium with an included amino acid upplementation strategy, and a control with no supplementation strategy. Also, we examined the recombinant and wild-type hosts with identical growth conditions to delineate the differences in gene expression, protein concentration, and metabolite composition because of the production stress. The results show that the availability of amino acids lessens the effect of the stringent response, alters the central metabolic system to a more active synthetic state, and increases the production of recombinant proteins. This study clearly demonstrates the importance of nutrient integrated strategy to control metabolic saturation.
Second objective explores the interplay between inducers and carbon source utilization in Escherichia coli to optimize recombinant protein production under proof of concept production strategies. The expression of genes involved in substrate metabolism is tightly regulated through mechanisms such as LacI repression and carbon catabolite repression, where the presence of preferred carbon sources influences the utilization of alternative substrates. To better understand these regulatory interactions, this study evaluates different recombinant proteins (simple and complex structure) and promoter systems under varying induction conditions, including lactose as a natural inducer, glycerol as a carbon source, and IPTG as a synthetic inducer. By analyzing substrate uptake and induction dynamics, the study provides insights into how co-utilization of carbon sources and inducer selection can modulate metabolic balance, improve expression efficiency, and reduce metabolic burden, thereby offering strategies to enhance recombinant protein production in E. coli.
The third objective of the study examines recombinant protein production's effects on the cell's biology and the metabolism of the cell itself, particularly Escherichia coli. The focus is on induction timing, host strain, and growth medium. The expression of recombinant proteins causes a diversion of the cell's energy and other building blocks needed for plasmids, transcription and translation, which causes a great deal of stress to the cell. In this study, induction during the early and middle log phases is compared and proteomics data is integrated for the growth and production phases for both host organisms (M15 and DH5α) that were grown in minimal and in complex media. From this study, a great deal is learned regarding the central metabolism and the production of the proteins regarding how the cellular resources are utilized, the overall stability of metabolism, and how the stress and adaptation of metabolism are adjusted. With these results, we start to understand the cellular mechanisms that need to be improved to increase the yield of the recombinant proteins and the overall efficiency of the processes involved.
The fourth objective analyzes the effects of genetic diversity and the fusion of dual recombinant enzymes on cellular metabolism and product incorporation using the AAR-ADO mediated pathway of alkane biosynthesis as an example. The single-enzyme AAR system showed high expression and high production of fatty alcohols in E. coli M15; however, the second enzyme, ADO, increased the pathway's metabolic and redox demands and negatively impacted the host. The integrated analysis of growth, metabolite, recombinant protein, and product formation indicates that E. coli DH5α is better than M15 in the complete AAR-ADO cascade. The proteome and pathway analysis indicates that the DH5α strain is adapted through a higher flux of NADPH, better electron flow, and better stability of the membrane, which is needed for more efficient flow through the double enzyme pathway. In M15, a metabolic instability is observed that includes a deficient central carbon metabolism, a redox imbalance, and increased stress responses, which limit the genetic pathway's efficiency even with high single enzyme expression. These results indicate that host adaptability, system-level metabolic coordination, and balance rather than only strong expression are important in the iterative operation of multi-enzyme biosynthetic pathways.