(7be) 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.