Rhodobacter; A microbial cell factory
A presentation on the article: "The transition of Rhodobacter sphaeroides into a microbial cell factory" by Enrico Orsi et al. 2020.
Rhodobacter sphaeroides is a class of bacteria that have the potential to synthesize industrially important products as microbial cell factories.
The PowerPoint Slide focus on:
-Understanding the microbial cell factories with Rhodobacter sphaeroides as an example
- Understand the DBTL concept
- Analyze how DBTL pipeline is incorporated in Rhodobacter sphaeroides
- Why the use of such a streamlined method for strain engineering is proposed?
- Industrial applications of this platform
1. Maliha Rashid
M.Phil. Biotechnology
University Institute of Biochemistry and Biotechnology,
PMAS Arid Agriculture University Rawalpindi, Pakistan
Rhodobacter sphaeroides
as Microbial Cell Factory
Design, Built, Test, Learn
Concept
1
4. Learning objectives
Keywords
Article Overview
DBTL cycles, industrial biotechnology, metabolic engineering, microbial cell factory,
• Understanding the microbial cell factories with Rhodobacter sphaeroides as an example
• Understand the DBTL concept
• Analyze how DBTL pipeline is incorporated in Rhodobacter sphaeroides
• Why the use of such a streamlined method for strain engineering is proposed
• Industrial applications of this platform
4
Rhodobacter sphaeroides is a class of bacteria that have the potential to synthesize industrially important products as
microbial cell factories
5. What makes R.sphaeroides an ideal choice as a microbial cell factories?
There are several reasons justifying the interest in R. sphaeroides as chassis for biotechnological productions:
1. Used as a model organism for studying anoxygenic photosynthesis, but also chemotaxis and quorum sensing
2. It displays high metabolic versatility - thrive by aerobic or anaerobic respiration and anoxygenic photosynthesis
3. R. sphaeroides is a natural producer of relevant bio‐ based compounds such as
• Isoprenoids
• poly‐β‐hydroxybutyrate (PHB)
• Hydrogen
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8. Different Research Fields Contributed To The DBTL Method
In R. Sphaeroides
Establishment of a DBTL pipeline in a
chassis requires contribution from
different research fields, including
• ‐omics techniques
• genome engineering
• phenotypic screening methods
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9. 9
Pathways involved in the carbon metabolism of Rhodobacter sphaeroides
EntnerDoudoroff
Pathway
EmbdenMeyerhoff
Pathway
MevolonatePathway(MVA)
2‐C‐methyl‐D‐erythritol
4‐phosphate(MEP)
10. Functional replacement of isoprenoid pathways in Rhodobacter sphaeroides
Microbial Biotechnology, Volume: 13, Issue: 4, Pages: 1082-1093, First published: 24 March 2020, DOI: (10.1111/1751-7915.13562)
Fig: overview of the MEP operon including the target gene dxr. All the other genes
included are involved in growth‐related functions
Fig: overview of the MVA operon from the α‐proteobacterium Paracoccus
zeaxanthinifaciens
Fig: Overview of the strategy
for isoprenoid pathway
replacement in Rhodobacter
sphaeroides.
11. 11
Figure: Lumped network of the carbon metabolism of Rhodobacter
sphaeroides, including pathways for substrate uptake and product
formation.
Substrates are highlighted in different colors, each
describing a different growth mode
• light blue: chemoheterotrophic
• light orange: photoheterotrophic
• light yellow: photo‐ or chemolitho‐autotrophic
The three carbon products described in this review
are highlighted
1. 5‐aminolevulinic acid (5‐ALA, yellow)
2. isoprenoids (red)
3. poly‐β‐hydroxybutyrate (PHB, green)
Some pathways with parallel flux are highlighted,
for example, glycolysis:
• Emden–Meyerhof–Parnas (red)
• Entner–Doudoroff (gray)
• isoprenoid synthesis: 2‐C‐methyl‐D‐erythritol
4‐phosphate (MEP) pathway (blue)
• mevalonate pathway (orange)
12. An Outlook on the Industrial Applications Of R. Sphaeroides:
Current Situation and Future Perspectives
This microorganism is considered nonpathogenic and generally regarded as safe (GRAS).
R. sphaeroides is being employed in few companies worldwide:
Company Name Website link Area of bacterial use
Dutch flavors and fragrances company Isobionics BV www.isobionics.com
produces sesquiterpenes as aroma
compunds
Chinese company CN Lab Nutrition www.cnlabnutrition.com
Nutraceuticals, coenzyme Q10 with a rate
of up to 30 tons per month.
Chinese company Hebei Shixiang Biological Technology Co.,
Ltd.
www.hbshixiang.en.china.cn feed industry for livestock
Indian company Prions Biotech www.prionsbiotech.com Fish feed solutions
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13. 13
Future Perspectives
Technologies used in DBTL pipeline could be
used in R. sphaeroides to optimize this
platform for assimilation of C1 substrates,
while coupling it to synthesis to a range of
bio‐based compounds
the use of cheap and renewable
feedstocks could improve both
commercial and experimental processes
where R. sphaeroides is involved
This microorganism could be exploited for evaluating
alternative feedstocks for the bio‐based economy
because of
• its versatile metabolism
• its wide substrate acceptance range
• recent advancements in its DBTL method
14. 14
• R. sphaeroides has the potential to produce industrially important bioproducts
• Moreover, it is reasoned that recent improvements in the DBTL pipeline can
be employed for further optimizing this cell factory for industrial applications
of other organisms
Take home Message
15. 15
References
1. Microbial Biotechnology, Volume: 13, Issue: 4, Pages: 1082-1093, First published: 24 March 2020, DOI:
(10.1111/1751-7915.13562)
2. Enrico Orsi, Jules Beekwilder, Gerrit Eggink, Servé W. M. Kengen, Ruud A. Weusthuis. The transition
of Rhodobacter sphaeroides into a microbial cell factory (2020). https://doi.org/10.1002/bit.27593
Open Researcher and Contributor ID) is a nonproprietary alphanumeric code to uniquely identify authors and contributors of scholarly communication as well as ORCID's website and services to look up authors and their bibliographic output
Microbial cell factories are engineered microorganisms harboring biosynthetic pathways streamlined to produce chemicals of interests from renewable carbon sources.
The four DBTL modules are highly interdependent, and each of them has a specific goal.
Overview of the strategy for isoprenoid pathway replacement in Rhodobacter sphaeroides. A. representation of the two isoprenoid modules 2‐C‐methyl‐D‐erythritol 4‐phosphate (MEP, blue arrows) and mevalonate (MVA, orange arrows). Both modules branch from the central metabolism (black arrows) and converge to isopentenyl‐diphosphate (IPP) and dimethylallyl‐diphosphate (DMAPP), which are the precursors of all terpenoids (some listed below DMAPP). MVA pathway requires NADPH as cofactor, while the endogenous MEP additionally requires reduced flavodoxin (Fld) or ferredoxin (Fd) for its functioning (red: reduced, ox: oxidized). The proposed position for inactivation of the native MEP pathway is also shown and corresponds to the enzyme 1‐deoxy‐D‐xylulose 5‐phosphate reductoisomerase (Dxr, red cross). Its substrate 1‐deoxy‐D‐xylulose 5‐phosphate (DXP) is also involved in thiamine biosynthesis (black dashed line). The last step of the MEP pathway is catalysed by 4‐hydroxy‐3‐methylbut‐2‐enyl diphosphate (HMBPP) reductase (IspH). For this enzyme, the Gibbs free energy under standard conditions was calculated (in blue). The highly negative ΔrG’0 of the reaction (−62.5 ± 6.4 kJ mol−1) indicates irreversibility of its enzymatic activity. On the other hand, introduction of the MVA module requires the correct functioning of 6 enzymes (numbered in orange). B. overview of the operon including the target gene dxr. All the other genes included are involved in growth‐related functions (see Table S3 for more information). C. overview of the MVA operon from the α‐proteobacterium Paracoccus zeaxanthinifaciens used for integration via mini transposon system pUT‐Mini‐Tn5‐Sp/Sm. The numbers below the genes represent their position in the pathway depicted in panel A), while Sp/SmR refers to spectinomycin/streptomycin resistance. The image is adapted from(Hümbelin et al., 2002). Other abbreviations: GAP (glyceraldehyde‐3‐phosphate), PYR (pyruvate), CDP‐ME (4‐(cytidine 5'‐diphospho)‐2‐C‐methyl‐D‐erythritol), CDP‐MEP (2‐phospho‐4‐(cytidine 5'‐diphospho)‐2‐C‐methyl‐D‐erythritol), MEcPP (2‐C‐methyl‐D‐erythritol 2,4‐cyclodiphosphate), pyrF (uridylate kinase, gene), frr (ribosome recycling factor, gene), uppS (undecaprenyl‐diphosphate synthase, gene), cdsA (phosphatidate cytidylyltransferase, gene), rseP (Regulator of sigma E protease, gene) Ac‐CoA (acetyl‐CoA), AA‐CoA (acetoacetyl‐CoA), HMG‐CoA (S)‐3‐hydroxy‐3‐methylglutaryl‐CoA), MVA‐P ((R)‐5‐phosphomevalonate), MVA‐PP ((R)‐5‐diphosphomevalonate), mvaA (HMG‐CoA reductase, gene), idi (IPP isomerase, gene), hcs (HMG‐CoA synthase, gene), mvk (mevalonate kinase, gene), pmk (phosphomevalonate kinase, gene), mvd (MVA‐PP decarboxylase, gene).
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