Protein and Pathway Engineering for Biosynthesis or Aromatic Compounds

A type I fatty acid synthase, FAS-B from Brevibacterium ammoniagenes, and a type I polyketide synthase, 6-MSAS from Penicillium patulum, were successfully engineered via rational pathway design to synthesize TAL in vivo with a maximal yield of 1.7 g/L. The direct biosynthesis of phloroglucinol from D-glucose was achieved by the discovery of a novel type III polyketide synthase, PhlD, from Pseudomonas fluorescens. Expression of PhlD in E. coli led to the production of phloroglucinol in vivo with an estimated yield of 0.7 g/L under the shake flask condition and 20 g/L using a continuous fermentation for 5 days. In vitro assay revealed that PhlD catalyzed the synthesis of phloroglucinol from three molecules of malonyl-CoA, and exhibited broad substrate specificity, which was successfully altered via saturation mutagenesis guided by a homology structural model of PhlD. To improve phloroglucinol production for industrial manufacturing, a two-prone approach was undertaken. Firstly, directed evolution was carried out to improve the poor properties of PhlD. Family shuffling of 52 PhlD homologous genes using synthetic DNA shuffling technique gave two improved PhlD mutants, which showed 4 fold increased enzymatic catalytic efficiency and/or thermostability. Secondly, the availability of substrate for phloroglucinol synthesis, malonyl-CoA in the host E. coli, was improved via metabolic engineering. The combination of various metabolic engineering strategies led to a total of 15 fold elevated malonyl-CoA level in E. coli. Ultimately, when combined, these two manipulations would translate synergistically into improvement in phloroglucinol production.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Chemical Engineering

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2015

Author dc:creator

• Zha, Wenjuan

Contributors dc:contributor

• Zhao, Huimin

• Engineering, Chemical

Language dc:language

eng

Identifier

(MiAaPQ)AAI3290452

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/82402