AUTHOR=Choi Si-Sun , Seo Seung-Yeul , Park Sun-Ok , Lee Han-Na , Song Ji-soo , Kim Ji-yeon , Park Ji-Hoon , Kim Sangyong , Lee Sang Joung , Chun Gie-Taek , Kim Eung-Soo 

TITLE=Cell Factory Design and Culture Process Optimization for Dehydroshikimate Biosynthesis in Escherichia coli

JOURNAL=Frontiers in Bioengineering and Biotechnology

VOLUME=7

YEAR=2019

URL=https://www.frontiersin.org/journals/bioengineering-and-biotechnology/articles/10.3389/fbioe.2019.00241

DOI=10.3389/fbioe.2019.00241

ISSN=2296-4185

ABSTRACT=<p>3-Dehydroshikimate (DHS) is a useful starting metabolite for the biosynthesis of muconic acid (MA) and shikimic acid (SA), which are precursors of various valuable polymers and drugs. Although DHS biosynthesis has been previously reported in several bacteria, the engineered strains were far from satisfactory, due to their low DHS titers. Here, we created an engineered <italic>Escherichia coli</italic> cell factory to produce a high titer of DHS as well as an efficient system for the conversion DHS into MA. First, the genes showing negative effects on DHS accumulation in <italic>E. coli</italic>, such as <italic>tyrR</italic> (tyrosine dependent transcriptional regulator), <italic>ptsG</italic> (glucose specific sugar: phosphoenolpyruvate phosphotransferase), and <italic>pykA</italic> (pyruvate kinase 2), were disrupted. In addition, the genes involved in DHS biosynthesis, such as <italic>aroB</italic> (DHQ synthase), <italic>aroD</italic> (DHQ dehydratase), <italic>ppsA</italic> (phosphoenolpyruvate synthase), <italic>galP</italic> (D-galactose transporter), <italic>aroG</italic> (DAHP synthase), and <italic>aroF</italic> (DAHP synthase), were overexpressed to increase the glucose uptake and flux of intermediates. The redesigned DHS-overproducing <italic>E. coli</italic> strain grown in an optimized medium produced ~117 g/L DHS in 7-L fed-batch fermentation, which is the highest level of DHS production demonstrated in <italic>E. coli</italic>. To accomplish the DHS-to-MA conversion, which is originally absent in <italic>E. coli</italic>, a codon-optimized heterologous gene cassette containing <italic>asbF, aroY</italic>, and <italic>catA</italic> was expressed as a single operon under a strong promoter in a DHS-overproducing <italic>E. coli</italic> strain. This redesigned <italic>E. coli</italic> grown in an optimized medium produced about 64.5 g/L MA in 7-L fed-batch fermentation, suggesting that the rational cell factory design of DHS and MA biosynthesis could be a feasible way to complement petrochemical-based chemical processes.</p>