(6it) Biosensor Mediated Evolution of Biosynthetic Pathways for Biomanufacturing

Author: Niju Narayanan, Postdoctoral Associate, Los Alamos National Laboratory, Los Alamos, New Mexico

Research Interests: Depleting petroleum reserves, energy security concerns and global climatic changes have driven our search for alternate technologies in order to shift to a more bio-based sustainable society. Biosynthetic pathways provide an inexpensive route from renewable materials for the production of molecules that are currently sourced from petroleum. the success of a biosynthetic pathway is highly context dependent - microbial host, pathway genes and their expression levels, promoters, enzyme activities, product and intermediate metabolite titers, cofactor availability, and often their delicate balance is offset owing to the complexities of biology. Advances in synthetic biology methods has enabled the construction and rapid generation of billions of cells with varying metabolic design. However, the evaluation of such designs by traditional metabolite measurement techniques currently available, lag by several orders of magnitude. Genetically encoded biosensors that act as screens for productive cells can solve these problems, identify optimal combination of genetic components, and enhance product titers and yields.

My research interest is in the development of biosensors and their application in the engineering of microbial metabolic pathways for human needs; including polymers, fuels and pharmaceuticals.

Research Experience: My research background has provided me with a wide range of experience in synthetic biology, high throughput technology, metabolic engineering, protein engineering, recombineering, molecular biology, biochemistry, fermentation technology, downstream processing, and analytical methods, some are described below:

Postdoctoral Study:

1. “Genetic biosensors for small molecules to engineer biosynthetic pathways in Pseudomonas putida” Advisors: Dr. Ramesh Jha and Dr. Taraka Dale, Los Alamos National Laboratory

This project focusses on using transcription factor based whole cell biosensors with fluorescence readouts for enhancing product titers and yields, evolving their promoters for transition to alternate production hosts, enzyme evolution, strain performance and selection of best producers, adaptive evolution for better growth of engineered host strains, utility of renewable non-conventional feedstocks etc.

2. Biosynthesis of Isopentenol in Escherichia coli via the Methyl Erythritol Phosphate Pathway

Advisor: Prof. Greg Stephanopoulos, Massachusetts Institute of Technology

The project was focused on improving isopentenol titers produced by the MEP pathway by soluble and functional expression of dephosphorylase and the pathway enzymes, maturation of the Fe-S cluster proteins, investigation of possible iron donors, NADP regeneration routes for cofactor supply, development of product and pathway metabolite analysis methods, culture media and isopentenol extraction procedures.

3. Pathways for synthesis of Advanced biofuels

Advisor: Prof. Greg Stephanopoulos, Massachusetts Institute of Technology

This project was focused on isovaleryl coA pathway for synthesis of C5 compounds, identification, cloning, characterization and variants of putative pivalyl coA mutases, investigation of prenol synthesis from isovaleryl coA pathway, development of analytical methods for pivalic acid and isovaleric acid determination.

PhD Dissertation: “Molecular and genetic strategies to enhance the functional expression of recombinant proteins in Escherichia coli”

Advisors: Prof. C. Perry Chou and Prof. Bill Anderson, University of Waterloo

The research work during graduate study was focused on the exploration of ‘strategies for functional expression of recombinant proteins in E. coli using model proteins (lipase, penicillin acylase, yellow fluorescent protein, β-galactosidase, hCD83ext). They included posttranslational modifications, cytoplasmic and periplasmic expression, surface display, extracellular secretion, molecular chaperones, site directed and error-prone mutagenesis, stress regulatory pathways, host strains, and optimization of culture conditions.

Industrial collaboration project with Argos Therapeutics at University of Waterloo

“Development of ‘bioprocess for production of recombinant human therapeutic protein hCD83ext”

Advisor: Prof. Murray Moo-Young and Prof. C. Perry Chou

The project included optimization of culture media and bioreactor conditions for high level expression of hCD83ext, Development and optimization of purification protocol for hCD83ext: GST affinity column, thrombin cleavage, ion-exchange chromatography, ultrafiltration, analytical methods for GST assay and endotoxin assay, construction of hCD83 mutants for improved protein stability

Funding/Awards:

NSERC Postdoctoral Fellowship, 2013

Murray Moo-Young Biotechnology Scholarship, 2008

NSERC Graduate Studentship, 2009

International Doctoral Student Award, 2006-2009

Teaching Interests: My background in Chemical Engineering, coupled with my research training in Biological Engineering has equipped me to teach a variety of courses in Chemical Engineering, Biotechnology, Bioengineering, Biochemistry, Protein Engineering, Metabolic Engineering and Synthetic Biology. However, I would be particularly interested in teaching 1. Biochemical Engineering and 2. Synthetic Biology. The coursework would be designed to encompass theoretical and computational approaches, practical laboratory classes, experimental model systems, and current research & classic literature.

Teaching Experience: I have had the opportunity to be a teaching assistant for various courses at the University of Waterloo and at Indian Institute of Technology Delhi. Some of them include Bioprocess Engineering, Advanced Biochemical Engineering, Engineering Biology, Introduction to Biotechnology, Chemical Engineering Concepts, and Physical Chemistry. This has given me teaching experience for 5-6 years at undergraduate level and around 8 months at the graduate level. Teaching helped me to hone my technical knowledge in the curriculum subjects and develop my interpersonal and communication skills and I was able to enjoy a good rapport with my students and colleagues.

Supervisory experience: I have had the chance to mentor and work with 7 graduate students, 4 visiting scholars, 7 undergraduate co-op students and 3 technical staff during the course of my research career of graduate studies at UWaterloo, the Industrial collaboration project for therapeutic protein production at UWaterloo, and postdoctoral study at MIT and LANL. Most students who started without any background in biotechnology and bioengineering, developed the essential skills within a short period of time through research training. This is also demonstrated by their co-authorship on our publications.

Teaching Certificate Program: I have completed a teaching certificate program at MIT which was based on seven workshops aimed at development of teaching skills, exposure to research on teaching and learning, and structure of future learning. The workshop was followed by two microteaching sessions that are videotaped, evaluated and feedback provided.

Manuscripts in preparation

1. Narayanan, N., Dale, T., and Jha, R. K. Adapted laboratory evolution and muconate-sensor based screening for robust growth of Pseudomonas putida and high product titers on corn stover hydrolysate.
2. Narayanan, N., Henely, S., Dale, T., and Jha, R. K. Engineering a b-ketoadipate biosensor in Pseudomonas putida and evolution of aromatic catabolism pathway for biomanufacturing.
3. Jha, R. K., Narayanan, N., and Dale, T. Genetically encoded sensor for alleviating product inhibition in an industrially relevant enzyme: Implications to biosynthesis of cis, cis muconic acid in Pseudomonas putida KT2440
4. Narayanan, N., Edgar, S., Woolston, B., and Stephanopoulos, G. Maturation of Fe/S clusters of Methyl erythritol phosphate pathway enzymes in Escherichia coli.
5. Narayanan, N., Woolston, B., and Stephanopoulos, G. NADPH regeneration in Escherichia coli improves the biosynthesis of isopentenol via Methyl erythritol phosphate pathway.
6. Narayanan, N., Duggar, D., Pereira, B., and Stephanopoulos, G. Identifying and engineering of putative pivalyl CoA mutase for the biosynthesis of C5 branched chain compounds.

Publications in refereed journals

1. Narayanan, N., Khan, M., and Chou, C. P. (2011) Enhancing functional expression of heterologous Burkholderia lipase in Escherichia coli. Molecular Biotechnology. 47: 130-143
2. Srivastava, P., Singh, P., Narayanan, N. and Deb, J.K. (2011) Physiological and biochemical consequences of host–plasmid interaction – A case study with Corynebacterium renale, a multiple cryptic plasmid containing strain. Plasmid. 65: 110-117
3. Zhang, L., Narayanan, N., Brand, S. R., Nicolette, C. A., Baroja, M., Arp, J., Wang, H., Moo- Young, M., and Chou, C. P. (2010) Structural identification of recombinant human CD83 mutant variant as a potent therapeutic protein. Protein Expression and Purification. 73: 140-146
4. Narayanan, N., Khan, M. and Chou, C. P. (2010) Enhancing functional expression of heterologous lipase B in Escherichia coli by extracellular secretion. Journal of Industrial Microbiology and Biotechnology. 37: 349-361
5. Narayanan, N., and Chou, C. P. (2009) Alleviation of proteolytic sensitivity to enhance recombinant lipase production in Escherichia coli. Applied and Environmental Microbiology.75: 5424-5427
6. Narayanan, N., and Chou, C. P. (2008) Physiological improvement to enhance Escherichia coli cell- surface display via reducing extracytoplasmic stress. Biotechnology Progress 24: 293-301
7. Narayanan, N., and Chou, C. P. (2008) Periplasmic chaperone FkpA reduces extracytoplasmic stress response and improves cell-surface display on Escherichia coli. Enzyme and Microbial Technology. 42:506-513
8. Narayanan, N., Follonier, S., and Chou, C. P. (2008) In vivo monitoring and alleviation of extracytoplasmic stress to recombinant protein overproduction in the periplasm of Escherichia coli. Biochemical Engineering Journal. 42: 13-19
9. Narayanan, N., Xu, Y., and Chou, C. P. (2006) High-level gene expression for recombinant penicillin acylase production using the araB promoter system in Escherichia coli. Biotechnology Progress. 22: 1518-1523
10. Narayanan, N., Hsieh, M. Y., Xu, Y., and Chou, C. P. (2006) Arabinose-induction of lac-derived promoter systems for penicillin acylase production in Escherichia coli. Biotechnology Progress. 22: 617-625
11. Xu, Y., Hsieha, M. Y., Narayanan, N., Anderson, W. A., Scharer, J. M., Moo-Young, M., and Chou, C. P. (2006) Cytoplasmic overexpression, folding, and processing of penicillin acylase precursor in Escherichia coli. Biotechnology Progress. 21: 1357-1365
12. Xu, Y., Weng, C. L., Hsieh, M. Y., Narayanan, N., Anderson, W. A., Scharer, J. M., Moo-Young, M., and Chou, C. P. (2005) Chaperone-mediated folding and maturation of the penicillin acylase precursor in the cytoplasm of Escherichia coli. Applied and Environmental Microbiology. 71: 6247- 6253
13. Narayanan, N., Roychoudhury, P. K., and Srivastava, A. (2004) Isolation of adh mutant of Lactobacillus rhamnosus.sp for production of L(+) lactic acid. Electronic Journal of Biotechnology. 7: 72-84
14. Narayanan, N., Roychoudhury, P. K. and Srivastava, A. (2004) L (+) lactic acid fermentation and its product polymerization. Electronic Journal of Biotechnology.7: 167-179