Selective Expansion of the Microbial Chemistry Repertoire for Metabolic and Protein Engineering

Post-Doctoral Research Fellow Since Sept. 2015

Research Interests:

Future Research

To better harness the potential of engineered biological systems, we must expand the repertoire of chemistry compatible with living processes. Access to additional functional groups and reaction chemistries within cells can enhance cell-based therapeutics, such as engineered probiotics, revolutionize high-throughput screens in drug development, and decrease reliance on petrochemical-based organic synthesis techniques used in manufacturing.

Two prominent examples of biochemical expansion are the introduction of non-standard amino acids (NSAAs) into the proteome and the biosynthesis of reactive aldehyde intermediates in the metabolome. NSAAs expand the structural and functional diversity of proteins, which span biocatalysts to therapeutic agents. Although systems have been engineered to enable NSAA incorporation into target proteins inside cells, these systems suffer from low selectivity for desired NSAAs. Selectivity also poses a problem for engineered biosynthesis of aldehyde intermediates inside cells, where native processes rapidly convert these molecules to alcohol byproducts and away from desired reactions.

In my research career, I have developed tools that overcome both selectivity problems, and I will use them as a faculty member to direct synthetic biology research in the following general areas:

Evolution of NSAA incorporation systems using proofreading proteins

(i)           Engineering high-throughput methods to selectively discriminate amino acids from structurally similar analogs

(ii)         Evolution of NSAA incorporation systems for greater selectivity

(iii)       Characterization of proofreading proteins and evolved NSAA incorporating systems to inform future engineering [collaborators: Bob Sauer (MIT) and Dieter Söll (Yale)]

Biosynthesis of metabolites containing novel functional groups obtained via aldehyde intermediates

(i)           Engineering of enzymes and pathways capable of converting aldehyde intermediates into further reactive bio-orthogonal chemistries, such as imines or enamines

(ii)         Rational introduction of NSAAs in enzyme active sites to alter specificity towards unnatural substrates, guided by sequence alignments, crystal structures, and computational protein design

Funding strategies associated with these aims target health, energy, and defense agencies. For more information, please visit my draft future lab website: www.kunjapurlab.org.

Graduate Research

“Microbial engineering for aldehyde synthesis” | Dr. Kristala L. J. Prather, MIT

I received training in metabolic engineering to enable microbial synthesis of aldehydes and aldehyde-derived non-alcohol products, such as fuels, flavors, and pharmaceutical intermediates. Through combinatorial deletion of redundant genes encoding aldehyde reductases, I engineered an E. coli strain that could enable production of model aromatic aldehydes such as vanillin for the first time. I also tested several aliphatic aldehydes and observed widespread partial stabilization based on the same deletions. Based on these insights, my colleagues and I harnessed this strain as a platform for obtaining products derived from aldehydes. We demonstrated diverse downstream enzymatic steps that led to production of a chiral pharmaceutical intermediate through carboligation or to production of gasoline constituents through oxidative deformylation. These reactions are merely the tip of an iceberg of new potential biochemistries enabled via aldehyde retention.

Skills Acquired: Design of heterologous metabolic pathways (1–3); genome engineering strategies to achieve aldehyde retention (1, 4); identification and resolution of kinetic bottlenecks and regulatory mechanisms (5); biochemical characterization of enzymes (6).

Post-Doctoral Research

“Engineering post-translational proofreading to discriminate non-standard amino acids” | Dr. George M. Church, Harvard

During my post-doctoral research, my main initiative has been to expand the repertoire of amino acids that can be incorporated into proteins. I discovered how the N-end rule pathway of protein degradation applies to commonly used NSAAs, and I then rationally engineered the N-end rule pathway for tunable control over how the N-end rule selectively degrades NSAA-containing proteins. I used these insights to engineer synthetic quality control, termed “Post-Translational Proofreading” (PTP). Using my engineered PTP system, my colleagues and I can engineer cells to incorporate specific NSAAs with a high level of accuracy.  This represents a dramatic change from earlier methods of NSAA incorporation, which were vulnerable to false positives that instead degrade rapidly using our system. We have used this method for evolution of more selective systems that incorporate NSAAs, and we have shown that the evolved systems improve the effectiveness of synthetic biocontainment. Thus far, this work has resulted in a first and co-corresponding author manuscript submission (7) and two provisional patent applications. I have also worked on adaptive evolution of a genomically recoded E. coli strain used for NSAA incorporation and am a co-first author on a submitted manuscript (8).

Skills Acquired: Site-specific in vivo non-standard amino acid incorporation; high-throughput screening of protein-encoding libraries; multiplexed genome engineering/editing; mass spectrometry; next-generation sequencing; PyRosetta computational protein design.

Past Proposals:

NIH NIBIB K99/R00 Resubmission (July 2017 cycle; original application score 34); DOE Genomic Sciences Center for Bioenergy 5-Year Renewal (co-author); NSF GRFP

Teaching Interests:

Teaching Experience

My interest in teaching began when I started a tutoring service that eventually served 14 clients before my high school graduation. At the University of Texas, I served as Tutor/Grader for the Transport Phenomena course for two semesters. In my final semester, I was given permission to serve as an undergraduate Teaching Assistant (TA) for the Material/Energy Balances course. At MIT, I served as a TA for two one-week courses tailored for biotechnology professionals. I also spent two summers co-teaching a seven-week “Microbial Chemical Factories” course for high-school juniors/seniors for whom a labmate and I co-designed the curriculum. Finally, I served as a TA for the graduate Kinetics and Reactor Design course at MIT, which is the most advanced course requirement for graduate chemical engineering students (my student evaluation: 6.7/7.0). My course preferences would be Kinetics, Material/Energy Balances, and Biochemical Engineering or Synthetic Biology electives.

Mentoring Experience

Parallels exist between teaching skills and research mentorship skills. In graduate school, I mentored five undergraduate research assistants, four of whom successfully co-authored publications with me (2, 5–6, 9), including one as a co-first author on a publication about the material properties of a non-ribosomally synthesized peptide (9). As a post-doc, I am mentoring two graduate students, one of whom has contributed as second author on the post-translational proofreading manuscript. Finally, having had many wonderful mentors in my own life, I support opportunities that give students access to additional mentors, and I started the SynBERC Industrial Mentorship and MIT Energy Club E-Mentors programs for this purpose.

Teaching Philosophy

My teaching philosophy for undergraduates focuses on four principles: broad relevance, participatory learning, peer-based reinforcement, and diversity.

Broad relevance: Drawing on experiences in both the petrochemical and pharmaceutical industries, and my professional network in other industries spanning consumer products and semiconductors, I will discuss scenarios and design problems that engage students with diverse career interests. Most students will not pursue academic careers, and my five undergraduate internship experiences helps me relate to those who are seeking employment after earning their bachelor’s degree.

Participatory learning: Pending class size, I will encourage various degrees of interactive learning, for example by posing a question or a blank space on every board/slide that I present. When students receive questions at regular intervals rather than a one-way deluge of information, students feel more knowledgeable about material and teachers receive faster feedback on what concepts are challenging students.

Peer-based reinforcement: I will advocate for peer-based reinforcement in the form of teamwork and group discussions in every curriculum. Learning from peers is particularly valuable when groups are diverse, and those who have different backgrounds and learning styles than myself (a visually-oriented learner) will benefit tremendously from alternative explanations by their peers.

Diversity: I am committed to promoting and supporting diversity in my classroom and in my lab. This includes diversity of experience and thought in addition to demographic diversity. I have worked with exceptionally talented people from all backgrounds, including underrepresented minorities such as my first undergraduate research assistant and my graduate advisor at MIT. Additionally, I will encourage open, respectful, and fact-based sharing of information among students, and I will seek out diverse role models to support students that I mentor.

Selected Publications:

1.        Kunjapur AM, Tarasova Y, Prather KLJ (2014) Synthesis and Accumulation of Aromatic Aldehydes in an Engineered Strain of Escherichia coli. J Am Chem Soc 136(33):11644–11654.

2.        Sheppard MJ, Kunjapur AM, Wenck SJ, Prather KLJ (2014) Retro-biosynthetic screening of a modular pathway design achieves selective route for microbial synthesis of 4-methyl-pentanol. Nat Commun 5:5031.

3.        Sheppard^† MJ, Kunjapur^† AM, Prather KLJ (2016) Modular and selective biosynthesis of gasoline-range alkanes. Metab Eng 33:28–40.

4.        Kunjapur AM, Prather KLJ (2015) Microbial engineering for aldehyde synthesis. Appl Environ Microbiol 81(6):1892–1901.

5.        Kunjapur AM, Hyun JC, Prather KLJ (2016) Deregulation of S-adenosylmethionine biosynthesis and regeneration improves methylation in the E. coli de novo vanillin biosynthesis pathway. Microb Cell Fact 15(1):1.

6.        Kunjapur AM, Cervantes B, Prather KLJ (2016) Coupling carboxylic acid reductase to inorganic pyrophosphatase enhances cell-free in vitro aldehyde biosynthesis. Biochem Eng J 109:19–27.

7.        Kunjapur AM, Stork DA, Kuru E, Vargas-Rodriguez O, Landon MM, Söll D, Church GM (2017) Engineering post-translational proofreading to discriminate non-standard amino acids. BioRxiv (preprint). DOI: 10.1101/158246.

8.        Wannier^† TM, Kunjapur^† AM, Rice DP, McDonald MJ, Desai MM, Church GM (2017) Long-term adaptive evolution of a genomically recoded organism. BioRxiv (preprint). DOI: 10.1101/162834.

9.        Khlystov^† NA, Chan^† WY, Kunjapur AM, Shi W, Prather KLJ, Olsen BD (2017) Material properties of the cyanobacterial reserve polymer multi-l-arginyl-poly-l-aspartate (cyanophycin). Polymer 109:238–245.