Metabolic Engineering for Robust Natural Product Biosynthesis in C. glutamicum

Jason Ronstadt, Chemical EngineeringMentor: Arul M. Varman, Assistant Professor

School for Engineering of Matter, Transport & Energy

Methodology

Current Results

pAJL01

• Resulted in many

background colonies

• Will assemble again once pAJL04

is successfully transformed

pAJL02

• Successfully assembled

and transformed

• Currently being sequenced

Future Work

Acknowledgements

• Finish assembling, transforming, and

sequencing all plasmids

• Determine baseline naringenin production

• Manipulate expression of ACC subunits and

measure change in naringenin production

Arren Liu- Graduate Mentor

Arul Varman- Principal Investigator

David Nielsen- Committee Member

Varman Lab

Objective• To engineer a pathway within C. glutamicum

that would allow the cell to produce naringenin.

• Significance: Naringenin is a natural product

that has pharmaceutical and agricultural

applications. This method will allow naringenin

to be produced more sustainably.

PCR to

amplify

GOIs

Purify

fragments

Gibson Assembly

Transform to cells

Colony PCR

Sequencing

Expected:> 5kb and > 3kb

Experimental Design

pAJL01

pAJL02

pAJL03

pAJL04

4CLpc CHSat CHI

4CLpc CHI

CHI

CHI

CHSph

4CL2at CHSat

CHSph4CL2at

pAJL03

• Had trouble cloning with 4CL2at

• Will assemble once pAJL04 is

confirmed

pAJL04

• In progress; successfully

assembled

• Currently running colony PCRs

to confirm transformation

0.5kb

1 kb

3 kb

Expected: 1.6 kb

CHSat Amplicons

pc_overhang at_overhang

pY-4CL2at gradient PCR

w/ old and new primers

Expected: 1.2 kb

3 kb

1 kb

0.5kb

4CL2at Amplicon

pAJL01

pAJL04

3 kb

1 kb

0.5kb

RD of pAJL02 plasmids w/ SacI

RD of pAJL01 & 02 plasmids w/ NdeI