Optimizing a Fed-Batch High-Density Fermentation Process for Medium Chain-Length Poly(3-Hydroxyalkanoates) in Escherichia coli.

Ryan A Scheel,Jessica Masisak,Yuki Kageyama,Seamus Mckenney,Christopher T Nomura,Benjamin R Lundgren,Truong Ho

doi:10.3389/fbioe.2021.618259

Abstract

Production of medium chain-length poly(3-hydroxyalkanoates) [PHA] polymers with tightly defined compositions is an important area of research to expand the application and improve the properties of these promising biobased and biodegradable materials. PHA polymers with homopolymeric or defined compositions exhibit attractive material properties such as increased flexibility and elasticity relative to poly(3-hydroxybutyrate) [PHB]; however, these polymers are difficult to biosynthesize in native PHA-producing organisms, and there is a paucity of research toward developing high-density cultivation methods while retaining compositional control. In this study, we developed and optimized a fed-batch fermentation process in a stirred tank reactor, beginning with the biosynthesis of poly(3-hydroxydecanoate) [PHD] from decanoic acid by β-oxidation deficient recombinant Escherichia coli LSBJ using glucose as a co-substrate solely for growth. Bacteria were cultured in two stages, a biomass accumulation stage (37°C, pH 7.0) with glucose as the primary carbon source and a PHA biosynthesis stage (30°C, pH 8.0) with co-feeding of glucose and a fatty acid. Through iterative optimizations of semi-defined media composition and glucose feed rate, 6.0 g of decanoic acid was converted to PHD with an 87.5% molar yield (4.54 g L–1). Stepwise increases in the amount of decanoic acid fed during the fermentation correlated with an increase in PHD, resulting in a final decanoic acid feed of 25 g converted to PHD at a yield of 89.4% (20.1 g L–1, 0.42 g L–1 h–1), at which point foaming became uncontrollable. Hexanoic acid, octanoic acid, 10-undecenoic acid, and 10-bromodecanoic acid were all individually supplemented at 20 g each and successfully polymerized with yields ranging from 66.8 to 99.0% (9.24 to 18.2 g L–1). Using this bioreactor strategy, co-fatty acid feeds of octanoic acid/decanoic acid and octanoic acid/10-azidodecanoic acid (8:2 mol ratio each) resulted in the production of their respective copolymers at nearly the same ratio and at high yield, demonstrating that these methods can be used to control PHA copolymer composition.

Highlights

High-density fermentation of Escherichia coli has been studied for the last 50 years in an effort to achieve maximal cell densities (∼200 g L−1 dry cell weight), frequently to attain high volumetric productivity (g L−1 h−1) of a heterologously expressed product (Shiloach and Fass, 2005)
The work presented in this study demonstrates the first successful biosynthesis of medium chain-length (MCL) PHA homopolymers and copolymers with defined monomer composition in E. coli LSBJ via high-density fermentation, and a subsequent process optimization to increase PHA yields and volumetric productivity
Preliminary Trials and the Effect of pH. The goal of this investigation was to enhance the production of MCL PHA by developing high density fermentation methods for our engineered production platform, E. coli LSBJ

Summary

Introduction

High-density fermentation of Escherichia coli has been studied for the last 50 years in an effort to achieve maximal cell densities (∼200 g L−1 dry cell weight), frequently to attain high volumetric productivity (g L−1 h−1) of a heterologously expressed product (Shiloach and Fass, 2005). While this is often a product such as a protein or antibiotic, these techniques have been employed to produce biopolymers such as poly(hydroxyalkanoates) [PHAs]. One of the main limitations of this MCL PHA biosynthesis platform is the low polymer yields obtained; the first reported MCL PHA biosynthesis in E. coli LSBJ reported yields from shake flask cultivations of approximately 0.26–0.4 g L−1 (Tappel et al, 2012b), which were later improved slightly by the deletion of the arcA transcriptional regulator to 0.26–0.6 g L−1 (Scheel et al, 2016), and most recently improved to 5.44 g L−1 by utilizing glucose as a cosubstrate, doubling the culture duration, and heterologously expressing an acyl-CoA synthetase from Pseudomonas putida (Mohd Fadzil et al, 2018)

Objectives

Methods

Results