Racing-red chassis: Advancing Rhodobacter sphaeroides as platform for isoprenoid production

Enrico Orsi

Research output: Thesisinternal PhD, WU

Abstract

A crucial aspect for innovation in industrial biotechnology is the development of microbial cell factories that can perform optimally within a bioprocess. Recently, technological improvement in -omics (genomics, transcriptomics, proteomics, metabolomics and fluxomics) techniques and genome editing technologies allowed to advance non-model organisms as platforms for bioproduction.

In chapter 1, Rhodobacter sphaeroides is introduced as promising non-model cell factory for the biobased production of isoprenoids. Although few studies were already reported for improving isoprenoid production in this species, a thoroughly understanding of its physiological behaviour during isoprenoid biosynthesis was missing.

In chapter 2, the physiology of isoprenoid biosynthesis in R. sphaeroides was investigated via cultivation on a defined medium. Such a cultivation setup allowed to assess the bacterium response to variations in nutrients availabilities. Isoprenoid biosynthesis revealed to be strictly growth-coupled in a strain relying exclusively on the endogenous 2-methyl D-erythritol 4-phosphate (MEP) pathway. When a heterologous mevalonate (MVA) pathway was additionally expressed, amorphadiene biosynthesis increased up to eight-fold. Moreover, presence of the MVA pathway resulted in isoprenoid production also during non-growing conditions imposed by nitrogen limitation. Here, the storage polymer poly-β-hydroxybutyrate (PHB) accumulated up to 60% of the dry-weight, as the major by-product of the process.

In order to improve R. sphaeroides as cell factory, an efficient genome editing toolkit needs to be established for this species. Chapter 3 describes the implementation of a highly-efficient Cas9 genome editing tool for R. sphaeroides. In this setup, plasmid-based homologous recombination was combined with Cas9-based targeting for counter-selection. Hence, gene deletions, insertions and single nucleotide substitutions were obtained and revealed to accelerate genetic modifications compared to traditional suicide plasmid-based strategies. Additionally, this study further applied the Cas9 tool to elucidate the pathway leading to the synthesis of PHB in the bacterium. Deletion of the phaB gene (encoding an NADPH-dependent acetoacetyl-CoA reductase) prevented PHB synthesis.

The ideal cell factory should have a minimal dependence of pathway fluxes from its endogenous regulation. A strategy for reducing such a constrain is to replace a native and strictly-regulated pathway with an independent and autonomous one. Chapter 4 describes the functional replacement of isoprenoid pathway in R. sphaeroides. The endogenous MEP pathway was inactivated by Cas9-mediated deletion of dxr (encoding the second enzyme of the pathway) and isoprenoid flux was exclusively supported by a chromosomally integrated heterologous MVA pathway. Cultivation with 13C-labeled glucose confirmed isoprenoid pathway substitution. It was demonstrated that the strain relying exclusively on the integrated and plasmid-borne MVA enzymes obtained substantially higher yields of amorphadiene compared to the parental strain still maintaining the MEP pathway active. Apart from describing the design and implementation of a metabolic bypass for isoprenoid biosynthesis in R. sphaeroides, this study resulted in the generation of a strain subjected to lower endogenous regulation during amorphadiene biosynthesis.

Interest in understanding MEP and MVA pathway interactions resulted in chapter 5, where a tool for studying pathway contributions to isoprenoid biosynthesis was developed. In this study, a setup for parallel 13C-labeling cultivation was designed to determine the metabolic flux ratios during glycolysis and isoprenoid biosynthesis in R. sphaeroides. Such method was implemented for integrating 13C atoms within the universal isoprenoid building-blocks isopentenyl-pyrophosphate (IPP) and dimethylallyl-pyrophosphate (DMAPP). Determination of pathway contributions to IPP and DMAPP biosynthesis was determined by exclusive measurement of the secreted reporter amorphadiene. It was proven that by parallel cultivation with 100% [1-13C]- or [4-13C]-glucose, glycolysis via the Entner-Doudoroff (ED) or the Embden-Meyerhof-Parnas (EMP) pathways could be determined, as well as the isoprenoid flux via the MEP or MVA pathways. For validation purposes, strains with 100% flux via one of the target pathways were generated via Cas9-based deletions. Parallel 13C cultivations in these strains resulted in no degree of freedom, thereby confirming the reliability of the experimental design. The method was therefore applied to study the metabolic flux ratio in glycolysis, confirming a predominance of the ED pathway over the EMP pathway. Moreover, it indicated that the MEP and MVA pathways have a reciprocal stimulating effect which results in increase of both pathways capacities. The reason for this reciprocal stimulation is not clear yet.

Chapter 6 focused on understanding the interaction between isoprenoid and PHB biosynthetic pathways in R. sphaeroides. The reasoning behind this study was to assess if isoprenoid production, natively growth-coupled in this bacterium, could be efficiently obtained during growth-independent conditions. Deletion of phaB resulted in substantial increase of amorphadiene titers when the MVA pathway was also present. In fact, both the PHB and the MVA pathways show the metabolic traits of NADPH oxidation and intracellular free-CoA release. Metabolic flux ratio analysis developed in chapter 5 was applied, confirming the increased MVA pathway capacity upon deletion of phaB. When moving to resting cells conditions, ∆phaB resulted in improved growth-independent amorphadiene production. Eventually, the maximum yields and productivities under this condition were obtained when combining ∆phaB with MEP pathway inactivation. Such strain streamlined all isoprenoid flux via the less regulated MVA pathway, which could further benefit from the lack of PHB accumulation. In summary, although growth-coupled synthesis via both MEP and MVA pathways is still the most efficient condition for isoprenoid production, growth-independent biosynthesis can be obtained (and further improved) via exclusive exploitation of the deregulated MVA pathway.

Chapter 7 is the general discussion of this thesis, which combines and further evaluates the main findings of the different chapters. Attention is given to the consolidation of R. sphaeroides as microbial cell factory obtained via a Design- Build-Test-Learn cycle. Moreover, an endogenous non-homologous end joining mechanism for DNA repair is described, which could accelerate the genome editing in this species. Then, a speculation of the possible interaction of MEP and MVA pathways is provided. Eventually, advantages on modular pathway engineering for exclusive employment of the MVA pathway for growth-independent isoprenoid synthesis are reported. As conclusions, future perspective for investigation and improvement of critical factors are proposed.

Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Wageningen University
Supervisors/Advisors
  • Weusthuis, Ruud, Promotor
  • Eggink, Gerrit, Promotor
  • Kengen, Servé, Co-promotor
Award date30 Oct 2020
Place of PublicationWageningen
Publisher
Print ISBNs9789463953139
DOIs
Publication statusPublished - 30 Oct 2020

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