Growth-uncoupled isoprenoid synthesis in Rhodobacter sphaeroides

Enrico Orsi; Ioannis Mougiakos; Wilbert Post; Jules Beekwilder; Marco Dompè; Gerrit Eggink; John van der Oost; Servé W M Kengen; Ruud A Weusthuis

doi:10.1186/s13068-020-01765-1

Growth-uncoupled isoprenoid synthesis in Rhodobacter sphaeroides

Biotechnol Biofuels. 2020 Jul 13:13:123. doi: 10.1186/s13068-020-01765-1. eCollection 2020.

Authors

Enrico Orsi^#^{1

2}, Ioannis Mougiakos^#^{3

4}, Wilbert Post^{1

3}, Jules Beekwilder⁵, Marco Dompè⁶, Gerrit Eggink^{1

7}, John van der Oost³, Servé W M Kengen³, Ruud A Weusthuis¹

Affiliations

¹ Bioprocess Engineering, Wageningen University, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands.
² Present Address: Systems and Synthetic Metabolism Group, Max Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Potsdam, Germany.
³ Laboratory of Microbiology, Wageningen University, Stippeneng 4, 6708 WE Wageningen, The Netherlands.
⁴ Present Address: Helmholtz Institute for RNA-based Infection Research (HIRI), Helmholtz-Centre for Infection Research (HZI), 97080 Würzburg, Germany.
⁵ Wageningen Plant Research, 6700AA Wageningen, The Netherlands.
⁶ Physical Chemistry and Soft Matter, Wageningen University, Stippeneng 4, 6708 WE Wageningen, The Netherlands.
⁷ Wageningen Food & Biobased Research, 6708WG Wageningen, The Netherlands.

^# Contributed equally.

Abstract

Background: Microbial cell factories are usually engineered and employed for cultivations that combine product synthesis with growth. Such a strategy inevitably invests part of the substrate pool towards the generation of biomass and cellular maintenance. Hence, engineering strains for the formation of a specific product under non-growth conditions would allow to reach higher product yields. In this respect, isoprenoid biosynthesis represents an extensively studied example of growth-coupled synthesis with rather unexplored potential for growth-independent production. Rhodobacter sphaeroides is a model bacterium for isoprenoid biosynthesis, either via the native 2-methyl-d-erythritol 4-phosphate (MEP) pathway or the heterologous mevalonate (MVA) pathway, and for poly-β-hydroxybutyrate (PHB) biosynthesis.

Results: This study investigates the use of this bacterium for growth-independent production of isoprenoids, with amorpha-4,11-diene as reporter molecule. For this purpose, we employed the recently developed Cas9-based genome editing tool for R. sphaeroides to rapidly construct single and double deletion mutant strains of the MEP and PHB pathways, and we subsequently transformed the strains with the amorphadiene producing plasmid. Furthermore, we employed ¹³C-metabolic flux ratio analysis to monitor the changes in the isoprenoid metabolic fluxes under different cultivation conditions. We demonstrated that active flux via both isoprenoid pathways while inactivating PHB synthesis maximizes growth-coupled isoprenoid synthesis. On the other hand, the strain that showed the highest growth-independent isoprenoid yield and productivity, combined the plasmid-based heterologous expression of the orthogonal MVA pathway with the inactivation of the native MEP and PHB production pathways.

Conclusions: Apart from proposing a microbial cell factory for growth-independent isoprenoid synthesis, this work provides novel insights about the interaction of MEP and MVA pathways under different growth conditions.

Keywords: Growth-independent production; Isoprenoid biosynthesis; MEP; MVA; PHB; Rhodobacter sphaeroides.