Abstract
Accumulation of acetate is a limiting factor in recombinant production of (R)-3-hydroxybutyrate (3HB) by Escherichia coli in high-cell-density processes. To alleviate this limitation, this study investigated two approaches: (i) deletion of phosphotransacetylase (pta), pyruvate oxidase (poxB), and/or the isocitrate lyase regulator (iclR), known to decrease acetate formation, on bioreactor cultivations designed to achieve high 3HB concentrations. (ii) Screening of different E. coli strain backgrounds (B, BL21, W, BW25113, MG1655, W3110, and AF1000) for their potential as low acetate-forming, 3HB-producing platforms. Deletion of pta and pta-poxB in the AF1000 strain background was to some extent successful in decreasing acetate formation, but also dramatically increased excretion of pyruvate and did not result in increased 3HB production in high-cell-density fed-batch cultivations. Screening of the different E. coli strains confirmed BL21 as a low acetate-forming background. Despite low 3HB titers in low-cell-density screening, 3HB-producing BL21 produced five times less acetic acid per mole of 3HB, which translated into a 2.3-fold increase in the final 3HB titer and a 3-fold higher volumetric 3HB productivity over 3HB-producing AF1000 strains in nitrogen-limited fed-batch cultivations. Consequently, the BL21 strain achieved the hitherto highest described volumetric productivity of 3HB (1.52 g L−1 h−1) and the highest 3HB concentration (16.3 g L−1) achieved by recombinant E. coli. Screening solely for 3HB titers in low-cell-density batch cultivations would not have identified the potential of this strain, reaffirming the importance of screening with the final production conditions in mind.
Highlights
Accumulation of acetate is a limiting factor in recombinant production of (R)-3-hydroxybutyrate (3HB) by Escherichia coli in high-cell-density processes
Production, pta, poxB, and/or isocitrate lyase regulator (iclR) were deleted in the AF1000 strain background, which is derived from E. coli MC4100 (Sanden et al 2003)
This study investigated six promising E. coli strains, including two strains from group B (B and BL21) that are known to produce low amounts of acetate (Daegelen et al 2009; Rosano and Ceccarelli 2014), one W strain known for its high growth rate in minimal medium and low acetate formation (Archer et al 2011), and three additional K-12 strains (MG1655, W3110, BW25113) as well as the K-12 MC4100 derived AF1000 strain previously used (Baba et al 2006; Blattner et al 1997; Grenier et al 2014; Hayashi et al 2006; Sanden et al 2003)
Summary
Accumulation of acetate is a limiting factor in recombinant production of (R)-3-hydroxybutyrate (3HB) by Escherichia coli in high-cell-density processes. To alleviate this limitation, this study investigated two approaches: (i) deletion of phosphotransacetylase (pta), pyruvate oxidase (poxB), and/or the isocitrate lyase regulator (iclR), known to decrease acetate formation, on bioreactor cultivations designed to achieve high 3HB concentrations. Appl Microbiol Biotechnol (2019) 103:5627–5639 polymerization of PHB, or by recombinant microorganisms (de Roo et al 2002; Jaipuri et al 2004; Lee and Lee 2003) Among these methods, recombinant production is the most desired because it is a one-step process and avoids the use of non-environmentally friendly chemicals. Production of acetate competes with product formation for the substrate, thereby decreasing 3HB yield, and lowers the growth rate even at concentrations as low as 0.5 g L−1 (Nakano et al 1997), making it difficult to obtain high-cell-density cultures and to scale-up the processes
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
