Growth-uncoupled isoprenoid synthesis in Rhodobacter sphaeroides

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 13 C-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.

Heterologous Production of β-Caryophyllene and Evaluation of Its Activity against Plant Pathogenic Fungi

“…(iii) Rhodobacter species enable photo(hetero)tropic growth in low-cost minimal media at relatively high growth rates, allowing the utilization of sunlight as an energy source for sustainable cultivation and production processes. Recent studies could demonstrate that engineering the isoprenoid precursor biosynthesis can lead to a strong increase of sesqui- and triterpenoid formation in R. capsulatus [ 39 , 48 , 49 ] and R. sphaeroides [ 38 , 40 , 50 , 51 , 52 ]. In particular, the co-expression of a terpene synthase with the FPP synthase IspA, and/or enzymes constituting the heterologous MVA pathway, resulted in enhanced production of the corresponding plant terpenoids.…”

Amorpha-4,11-diene synthase: a key enzyme in artemisinin biosynthesis and engineering

“…The above approaches in improving amorpha-4,11-diene production often involve the enhancement of the efficiency and production of rate-limiting enzymes in Orsi et al (2020) metabolic flux. However, protein engineering of ADS itself for higher catalytic efficiency has not been attempted.…”

The transition of Rhodobacter sphaeroides into a microbial cell factory

“…Use of such a strain allowed to design a 13 C labeling‐based flux ratio analysis to study MEP and mevalonate split ratios (Orsi, Beekwilder, Peek, et al, 2020). Based on this knowledge, a strategy for the growth of uncoupled isoprenoid production was implemented, which consisted in exclusive exploitation of the mevalonate pathway in combination with inactivation of PHB synthesis (Orsi, Mougiakos, et al, 2020).…”

“…AcAc‐CoA, acetoacetyl‐CoA; Ac‐CoA, acetyl‐CoA; ACE, acetate; BUT, butyrate; CBB, Calvin–Benson–Bassham cycle; CO, carbon monoxide; F6P, fructose‐6 phosphate; FOR, formate; FRU, fructose; G6P, glucose‐6 phosphate; GAP, glyceraldehyde‐3 phosphate; GL6P, 6‐phosphoglucanolactone; GLU, glucose; GLY, glycerol; IPP, isopentenyl pyrophosphate; LAC, lactate; MAL, malate; MET, methanol; PPP, pentose phosphate pathway; PRO, propionate; PYR, pyruvate; RIB, ribose; SUC, succinate; TCA, Krebs cycle; XYL, xylose. The figure has been adapted from (Imam et al, 2013; Orsi, Mougiakos, et al, 2020; Tabita, 1995) [Color figure can be viewed at wileyonlinelibrary.com]…”