Synthetic Biologyfor

Plant Scientists

A case study of biofuel production

Based on: Yang, Zhang, Zheng, Scheller et al.; Engineering secondary cell wall deposition in plants; Plant Biotechnol J., 2013, Apr 11

IntroductionAbhijeet Saxena

Why do we need biofuels?

•Biofuels are fuels derived from organic biomass, natural or synthesized

•By 2050, the world would need energy equivalent to 50 cubic miles of oil annually (currently 3.1 CMOs)

•Oil reserves are running out, fast•Solar, geothermal, tidal and wind energy have

geographical limitations•Are renewable energy source•Are environment-friendly

Attempts as of now

•Alcohol fuels produced by fermentation of sugars derived from wheat, corn, sugarcane, molasses etc

•Biomethanol produce by gasification of biomass•These are mixed with gasoline in various ratios•Biodiesel produced from transesterification of animal

fats or vegetable oils, jatropha, mustard, sunflower etc•Green diesel produced through hydrocracking

biological oil feedstocks•Algae-derived biofuels are the most efficient

What fails them?

•Choice between food and fuel•Water-intensive biofuel crops affect local water

supplies•Conversion of biomass into fuel is not always eco-

friendly•The yield is very low, making them a costly affair•Genetic variability hinders commercial

reproducibility•Biodiesel can not be directly utilised in current

automobile infrastructure

Engineering the metabolome

•UK researchers developed a modified strain of E. coli which could transform into biofuel gasoline

•UCLA researchers engineered a new metabolic pathway to bypass glycolysis and increase conversion rate of sugars into biofuel

•KAIST researchers developed a strain that could produce short-chain alkanes, free fatty acids, fatty esters and fatty alcohols through the fatty acyl carrier protein in vivo

•Can we, or not, engineer plant metabolic pathways too?

What we knew?Swati Sharma

High cost

Klein-Marcuschamer et al., 2010•Cell wall–derived glucose is costly due to low sugar

density of the biomass, cell wall recalcitrance to enzymatic hydrolysis and medium content in cellulose

•Each factor either impacts transportation or requires intensive use of energy and chemicals for processing

•Enhancing polysaccharide accumulation in raw biomass and improving biomass digestibility will reduce costs significantly

Hard time balancing

Chen and Dixon, 2007; Voelker et al., 2010•There is high negative correlation between lignin

content and saccharification efficiency of plant cell walls•Often correlated with loss of cell wall integrity causing

vessel collapse•Most efforts to reduce lignin content during plant

development resulted in severe biomass yield reduction particularly in dicots

Constitutively induced defence response

Gallego-Giraldo et al., 2011•Repression of the HCT (hydroxycinnamoyl CoA transferase)

enzyme from lignin biosynthesis pathway in Arabidopsis and alfalfa showed to constitutively induce defence response

• It inhibited plant development and was overcome by blocking accumulation of the defence hormone salicylic acid

•When silencing strategies are used to reduce lignin content, gene repression levels to avoid biomass yield reduction are compromised

Secondary cell wall regulatory network

Ruprecht et al., 2011; Zhong et al., 2010•Conserved across many plant species•Cytodifferentiation into vessels or fibres regulated by

independent master transcription factors•Same regulatory network controls expression of genes

involved in the biosynthesis of cellulose, xylan and lignin

•Thus, it is challenging to manipulate cell wall composition without impacting cell wall integrity and plant development

So the plan was…

MethodologyRupal Mishra

Plant material and growth conditions

•Wild-type Arabidopsis thaliana c4h lines used• The c4h + pvnd6::C4 lines generated via floral dipping with

A. tumefaciens carrying pa6- pvnd6::C4 binary vectors• Selected on MS medium (+ 1% sucrose, 30mcg/ml

hygromycin) followed by genotype mapping• Plants analysed were grown in short-day conditions for 5

weeks, followed by long-day conditions till maturity in 60% humidity

• Control plants were grown in long-day conditions in 55% humidity

Generating binary vectors

• C4H and NST1 encoding genes amplified from Arabidopsis cDNA

• DNA fragments were introduced into pDONR22-f1 entry vector to create pDONR-F1-C4H and pDONR-F1-NSTI

• These were transferred in pA6-pVND6-GW and pTkan-pIRX8-GW to create pA6-pVND6::C4H and pTkan-pIRX8::NST1 binary vectors, respectively

Histochemical staining

• Bases of primary stems were embedded in 7% agarose and 100 mcm sections cut using a vibratome

• For bright-field and UV fluorescence analysis, sections were directly mounted in water.

• For Wiesner lignin staining, sections were incubated for 3’ in phloroglucinol–HCl 2% (w/v) solution

• For Maule lignin staining, sections were incubated in 0.5% KMnO4 for 2 min, rinsed with water, then incubated in 10% HCl for 1 min and mounted after the addition of a drop of aqueous ammonia

• All sections were analyzed using a bright-field/fluorescent microscope

Immunolocalization

•1 mcm sections cut using ultramicrome•Sections labelled with anti-xylan monoclonal

antibody or CBM3a (a probe to crystalline cellulose)

Lignin quantification

•Senesced stems were milled with stainless steel balls•Extract-free cell wall residues were obtained by

sequential washing and vortexing with 95% alcohol•5mg CWR incubated with 25%, 100mcl of acetyl

bromide glacial acetic acid for 2 hours, diluted to 1 ml glacial acetic acid prior to centrifugation

•Absorbance measured at 280 nm

Cell wall pretreatments and saccharification

•Senesced stems in water or dilute alkaline (later neutralized), incubated for 30˚C for 30’ and autoclaved at 120˚C for 1 hour

•Saccharification initiated by adding 300mcl, 83mM sodium citrate buffer

•After 24, 48 and 96 hours of incubation at 50˚C, samples were centrifuged

•Reducing sugar measured in the sample by DNS assay

ConclusionsIsha Bhatia

Lignin engineered plants

Impact of cell wall engineering on polysaccharide deposition in stems

Impact of cell wall engineering on lignin deposition in stems

Saccharification efficiency of biomass derived from engineered plants

•C4H gene encodes second enzyme in lignin biosynthesis pathway

• Lignin biosynthesis restricted to vessels•NST1 transcription factor controls secondary cell wall

deposition in fibres• Lignin-engineered lines disconnect over-expression

of polysaccharide biosynthesis from lignin biosynthesis

•Artificial positive feedback loop increased cell wall deposition in fibres

• In both pretreatments, sugar released was faster and much higher for the engineered plants than for the wild type

Future DirectionsSachin Singh Rawat

What this work has in store for plant culture?

•Should open new ways for crop optimization for farming lignocellulose or secondary metabolites

•Approach used to develop APFL should be applicable to other metabolic pathways

• It is a template to other breakthroughs of synthetic biology into plant tissue culture

What synthetic biology aims?

•To combine the precision of engineering with the fundamentals of biology

•To create a repository of standard biological parts that can be tuned to meet specific performance criteria

•To rewire existing biological networks and create newer, better ones

• To build organisms optimized for our needs•To revolutionize biology in the way IC’s

revolutionized computing

Plants that glow in the dark

•Scientists added gene encoding firefly enzyme luciferase to a tobacco plant, which glowed temporarily on spraying luciferin (1980s)

•Engineered tobacco plant with its own weak glow, using bacterial genes (2010)

•Cambridge team created a genetic circuit in bacteria that makes both firefly luciferase and luciferin, so that the bacteria glow continuosly (2010)

•A Kickstarter project tweaked the circuit so that it works in plants (2013)

Plants that grow their own fertiliser

•Plants fix N2 only in association with Rhizobium•Naturally, plants use nitrogenase to fix N2 which is

inhibited by O2

•Scientists hope to re-discover a bacteria that holds the key to enable plants fix their own N2

•This bacteria has a unique nitrogenase that can fix N2 in O2 rich environments

•Some cyanobacteria can fix N2 using solar energy•Re-engineering their machinery to work in plants

would enable plants to grow own fertiliser

Thank you