Light-driven chemical synthesis

Kenneth Jensen1*, Poul Erik Jensen2 and Birger Lindberg Møller1

1 Plant Biochemistry Laboratory, Center for Synthetic Biology and VKR Research Center ‘Pro-Active Plants’, University of

Copenhagen, 40 Thorvaldsensvej, DK-1871 Frederiksberg C, Copenhagen, Denmark2 Section for Molecular Plant Biology, Center for Synthetic Biology and VKR Research Center ‘Pro-Active Plants’, University of

Copenhagen, 40 Thorvaldsensvej, DK-1871 Frederiksberg C, Copenhagen, Denmark

Techniques & Applications

Depletion of the fossil fuel reserves of the Earth hasprompted research into sources of renewable and sus-tainable energy, and feedstock for the chemical andpharmaceutical industries to support the transitiontowards a bio-based society. Photosynthesis efficientlycaptures solar energy, but its subsequent conversioninto chemical energy in the form of biomass is limitedto a final output in the 1–4% range. Re-routing ofphotosynthetic electron transport and reducing powerdirectly into desired biosynthetic pathways offers anew avenue for sustainable production of high-valueproducts.

Light as an energy sourceLife has existed on Earth for 3.4 billion years and wasinitially based on a strictly anaerobic metabolism. Some2.7 billion years ago, photosynthetic bacteria and cyano-bacteria arose, requiring metabolic adaptation to an oxy-genic environment. Engulfment of a cyanobacteriumeventually enabled primitive land plants to develop ap-proximately 475 million years ago. Energy supply viaphotosynthesis was thus superimposed on a pre-estab-lished set of primary metabolic reactions based on energygeneration from oxidation of chemical compounds presentin the environment (i.e. chemoautotrophs) and formationand turn-over of carbohydrates as a mean to channelenergy flux and carbon into specific biosynthetic pathways.Inherent limitations in the ability of photosynthesizingorganisms to channel the use of light-generated reducingequivalents directly into synthesis of specific compoundsmay reflect the evolutionary history [1]. Photosyntheticorganisms, such as cyanobacteria, algae and plants, arethus amenable to biotechnological approaches aimed attapping directly into, and prioritizing the energy outputfrom, photosynthesis towards the efficient production ofdesired products. A strategy to reach this goal would bebased on the optimized expression of desired biosyntheticenzymes or entire pathways in the chloroplast stroma orthylakoid membranes.

Photosystem I (PSI) operates at a quantum yield of 1.0.This efficiency is unmatched by any other biological orchemical system and means that each captured photonsucceeds in exciting an electron in the reaction center ofPSI [2]. Two separate one-electron photooxidation eventsin the P700 reaction center of PSI are required for the

Corresponding author: Møller, B.L. (blm@life.ku.dk)* Current address: Novozymes A/S, 36 Krogshoejvej, DK-2880 Bagsværd, Denmark.

60 1360-1385/$ – see front matter � 2011 Elsevier Ltd. All rights reserved

reduction of NADP+ to NADPH, with the final steps beingmediated by the soluble electron carrier ferredoxin and byferredoxin NADPH-oxidoreductase [3–5]. The pH gradientformed over the thylakoid membrane by light-driven elec-tron transport through photosystem II (PSII) and then PSIis utilized for ATP formation. Photosynthetic carbon fixa-tion at the expense of NADPH and ATP enables synthesisof complex organic molecules. Whereas the light-drivenelectron transfer reactions are optimized through evolu-tion, bioengineering of the link to carbon metabolism hasobvious biotechnological potential, especially as fossil fuelsare being depleted and the demand for alternative sustain-able energy sources and fine chemicals is increasing.

Two approaches for utilizing and optimizing solar lightconversion efficiency by tapping directly into the photosyn-thetic reaction center of PSI are envisioned: (i) an in vitrosystem in which the light-harvesting properties and electrontransport activity of PSI is used to reduce H+ into hydrogen(H2) or for the biosynthesis of complex chemical molecules;and (ii) an in vivo system, in which a biosynthetic pathway isincorporated in the vicinity of the photosynthetic reactioncenter [6]. Proper co-compartmentalization of the energy-producing photosystem and a desired energy-demandingbiosynthetic pathway would serve to optimize and channelthe energy and carbon flow towards the biosynthetic path-way. In addition, this approach may be used to optimize theconditions for protein complex formation; that is, the forma-tion of a supersized metabolon consisting of the photosyn-thetic complex and the biosynthetic enzymes, facilitatingrapid and efficient substrate channeling and conversion ofthe substrate into the desired product.

In vitro light-driven biosynthesisIn addition to the metabolites of primary metabolism,plants and microorganisms produce an immense numberof bioactive compounds (i.e. specialized metabolites) as aresponse to environmental challenges. Plants are known toproduce more than 200 000 often structurally complexbioactive natural products [7]. Many of these compoundsare used by humans as pharmaceuticals or constituteimportant colorants or food flavors, despite the fact thatthey may be present in low or highly variable amounts inthe plant or are difficult to isolate. Biosynthesis of thecompounds is typically tightly regulated and their forma-tion restricted to specific tissues or developmental stages.Attempts to upregulate the biosynthesis of a given productmay be accompanied by unexpected adverse reactionsbecause of metabolic crosstalk. These bottlenecks maybe overcome by designing in vitro systems in which the

. doi:10.1016/j.tplants.2011.12.008 Trends in Plant Science, February 2012, Vol. 17, No. 2

Pt

2H+ 2H+

2H+

H2 H2

H2

Hydrogenase

Ferredoxin NADPH

reductase

NADP+

NADPH

P450

R R

R-OH

R-OH

P45

0 re

du

ctas

e

(d)

(a) (b) (c)

(e)

PSI PSI

TRENDS in Plant Science

PSI

PSI

Hydrogenase

Ferredoxin

Ferredoxin

Ferredoxin

PSI

P450

Figure 1. Examples of in vitro light-driven systems for the biosynthesis of high-value compounds. (a) Platinum (Pt) is precipitated on the stromal facing and reducing side of

photosystem I (PSI) during light treatment according to the following reaction: [PtCl6]2– + 4e– + hv ! Ptreduced + 6Cl–. Subsequent light excitation of electrons and charge

separation in the platinized PSI is sufficient to sustain the photoevolution of H2. (b) A hydrogenase was fused to a stromal subunit of PSI and reconstituted into PSI. The

positioning of the hydrogenase in close vicinity to the electron transport chain in PSI allowed for direct electron transfer from PSI to hydrogenase and subsequent H2

evolution. (c) Ferredoxin as mediator between PSI and hydrogenase. (d) Indirect electron transfer from PSI to cytochrome P450 (P450) via NADP+ photoreduction by PSI.

The NADPH-cytochrome P450 oxidoreductase was fused to the C terminal of the N-terminally membrane-anchored P450 and facilitated electron transfer from NADPH to the

active site of the P450. (e) Light-driven electron transfer from PSI via ferredoxin to P450, a system that is not dependent on the costly cofactor NADPH.

Techniques & Applications Trends in Plant Science February 2012, Vol. 17, No. 2

high-value compound is produced from readily availableprecursors. If such in vitro systems included light-drivenelectron transfer from PSI either by direct coupling or byregeneration of reducing equivalents in the form ofNADPH or reduced ferredoxin, the costs associated within vitro synthesis might be reduced and thus diversified toinclude many more compounds.

Several different in vitro systems able to facilitate thelight-driven synthesis of useful chemicals have been de-veloped (Figure 1). Based on the coexistence of oxygenicphotosynthesis and hydrogen metabolism in some cyano-bacteria and green algae, the initial focus of in vitro PSIapplications has been to produce H2 using either a plati-num-based catalyst system or a hydrogenase. The plati-num-based system takes advantage of the ability toprecipitate metallic platinum on the stroma-facing reduc-ing side of PSI. Platinum deposition is achieved by irradi-ation of isolated PSI complexes to donate the electronsrequired for platinum precipitation. Upon irradiation,

photooxidation of the P700 reaction centre and electrontransport through the platinized PSI result in the evolu-tion of H2 via the platinum catalyst [8,9]. Enzymatic H2

evolution was accomplished either by electron transferfrom PSI to hydrogenases using ferredoxin as an interme-diate electron carrier, or by rewiring of electron transferfrom PSI directly to a hydrogenase fused to a subunit of PSI[10–12].

Cytochromes P450 (P450) are a superfamily of heme-containing monooxygenases able to catalyze stereo- andregiospecific hydroxylation steps in numerous biosyntheticpathways. Plants are an especially rich source of P450s.The catalytic activity of P450s is typically dependent onelectron donations from NADPH mediated by the diflavinprotein NADPH-cytochrome P450 reductase (CPR) [13]. Inplant cells, the membrane-bound P450s are typically local-ized in the endoplasmic reticulum. Because PSI is localizedin the stroma lamellae of the chloroplasts, this physicalseparation prevents direct electron transfer from PSI to

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Table 1. Advantages and disadvantages of in vitro and in vivo light-driven biosynthesis

System Advantages Disadvantages

In vitro Controlled environment Protein lability

Modular system Limited production capacity

Simple product purification Dependent on supply of artificial electron donors

In vivo Self replicated Potentially unwanted metabolic crosstalk

Self-repair and maintenance Complex product purification

H2O as electron donor Stability of the product

Scalable

Continuous protein synthesis

Techniques & Applications Trends in Plant Science February 2012, Vol. 17, No. 2

the P450s. Construction of an in vitro system couplingthese two membrane protein complexes thus offered theopportunity to channel light energy directly into enzyme-catalyzed hydroxylation of complex molecules based on theconcomitant production of NADPH by PSI and its con-sumption by the P450s. Experimentally, this was achievedby combining isolated thylakoid membranes from spinach(Spinacia oleracea) with a microsomal preparation con-taining cytochrome P450, family 1, subfamily A, polypep-tide 1 (CYP1A1) from rats [14]. Upon irradiation, thismixture supported conversion of 7-ethoxycoumarin to 7-hydroxycoumarin. The CYP1A1 enzyme used was geneti-cally modified by fusing truncated yeast (Saccharomycescerevisiae) CPR to its C-terminus to facilitate electrontransfer from NADPH to the heme iron at the active siteof CYP1A1 [15]. This approach was simplified and refinedusing isolated photosytem I from barley (Hordeum vul-gare), isolated spinach ferredoxin and the isolated Sor-ghum (Sorghum bicolor) P450, CYP79A1 [16]. In this invitro system, ferredoxin functioned as a highly efficientelectron carrier between the isolated PSI and P450 com-plexes in the absence of NADP+ and its natural reductase(CPR). The in vitro system supported a higher turnover ofCYP79A1 compared with the rates obtained with theendogenous reductase (CPR) [16]. In addition, this light-driven PSI-based in vitro system also supported the activi-ty of the soluble bacterial P450, CYP124 [17], highlightingits flexibility and potential use as a modular system inwhich the catalytic output is dictated by the substratespecificity of the P450.

In vivo light-driven biosynthesisThe biochemical complexity of plants manifested in theirability to synthesize a multitude of different bioactive com-pounds. Most of these compounds are present in minute andvariable concentrations as required to exert their desiredeffect in the plant. On the one hand, this demonstrates thepotential of using plants for the biosynthesis of high-valuespecialty chemicals. On the other hand, the small amountsproduced render their isolation costly and a lack of economicfeasibility precludes the introduction of initial steps toisolate the compounds before application of general biore-finery approaches to degrade biomass into feedstocks. Re-engineering of interesting pathways into the chloroplast ofplants would render it possible to increase significantly thecontent of high-value compounds and tap into the availabil-ity of reducing equivalents generated by photosynthesis[18]. Compartmentalization will also potentially reducethe risk of metabolic crosstalk and undesired metabolic

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downregulation. A further advantage of the in vivo systemis the potential for upscaling allowing for bulk synthesis,whereas the light-driven in vitro biosynthesis systems pre-sented previously face limitations. The apparent advan-tages and disadvantages of the in vitro and in vivosystems are summarized in Table 1.

Isoprenoid biosynthesis relies on two independent bio-synthetic pathways, the mevalonate (MVA) pathway [19]and the 1-deoxy-D-xylulose-5-phosphate (DXP) pathway[20]. In plants, both pathways are operational and com-partmentalized: the MVA pathway in the cytosol and theDXP pathway in the chloroplast. The MVA pathway ispredominately responsible for the biosynthesis of sesqui-terpenoids (C15) and triterpenoids (C30), whereas mono-terpenoids (C10), diterpenoids (C20) and carotenoids (C40)are synthesized by the plastidic DXP pathway. The locali-zation of the DXP pathway in the chloroplast providesaccess to reducing equivalents and an abundant supplyof the C3 precursor glyceraldehyde-3-phosphate for thesynthesis of mono- and diterpenoids and carotenoids. Assuch, these classes of compound offer excellent opportu-nities for re-engineering into high-level light-driven pro-duction systems in easily cultivated plants. An examplewould be the biosynthesis of the highly oxygenated sesqui-terpene artemisinin, which is well known for its use as ananti-malarial drug. Annual wormwood (Artemisia annuaL) is the only known source of artemisinin, but the arte-misinin content is low, averaging approximately 0.5% dryweight [21]. Artemisinin is in high demand and a combi-nation of low natural availability and costly chemicalsynthesis has led to an interest in generating microbialsystems able to produce artemisinin or its precursor, arte-misinic acid, in a cost-effective and environmentally friend-ly way. A very successful approach was to shift thelocalization of the sesquiterpene synthase and amorpha-4,11-diene synthase involved from the cytosol to the chlo-roplast, thereby tapping into the DXP pathway otherwisereserved for mono- and diterpenoid synthesis [22]. In thisstudy, chloroplast localization increased amorpha-4,11-di-ene accumulation 40 000 fold (to >25 mg/g fresh weight)compared with the cytosolic-targeted amorpha-4,11-dienesynthase [22]. Significant losses of amorpha-4,11-dienewere encountered because of its volatility. CYP71AV1catalyzes the conversion of amorpha-4,11-diene into thenon-volatile artemisinic acid [23]. Coexpression and co-translational targeting of CYP71AV1 into the chloroplastwould thus be envisioned to further increase the yield. Theability of photoreduced ferredoxin to substitute function-ally for the endogenous CPR of CYP79A1 [16] could

Techniques & Applications Trends in Plant Science February 2012, Vol. 17, No. 2

indicate a similar electron donor promiscuity forCYP71AV1, thereby enabling the coupling of the catalyticactivity of CYP71AV1 to direct electron transfer from thephotosynthetic machinery. The in vivo post-translationalplastidic targeting of P450s could also be envisioned toimprove biosynthesis of other isoprenoids. For example,P450s are known to catalyze key steps in the biosynthesisof the sesquiterpernoid thapsigargin, a constituent ofThapsia garganica [24]. Thapsigargin may be developedinto a drug for the treatment of prostate cancer but diffi-culties in cultivating T. garganica have fostered an in-creased interest in alternative production systems. Similarto amorpha-4,11-diene biosynthesis, plastidic targetingand light-driven biosynthesis of thapsigargin would beenvisioned to increase its accumulation significantly. Oth-er industrial-relevant and high-value terpenoids suitablefor light-driven biochemical biosynthesis include diterpe-noids, such as taxol and steviol glycoside [25,26]. Afterexport from the chloroplast, the diterpenoid backbonesare typically further modified in the cytosol by, for example,P450-catalyzed hydroxylations. Plastidic targeting of theP450s involved would uncouple the hydroxylation reactionsfrom existing metabolic regulatory networks in the cytosoland potentially allow for upregulated, compartmentalized,light-driven biosynthesis of complex terpenoids. Futurechallenges will be to optimize the coupling between photo-synthetic electron transport and the demand for reducingequivalents to drive P450 catalyzed reactions.

Concluding remarksSunlight is the most abundant renewable energy source onEarth. Improved utilization of solar energy in the produc-tion of biofuels, feedstocks and high-value chemicals is anecessary component in the transition towards a bio-basedsociety. Synthetic biology approaches, in which knownbiosynthetic pathways are combined, reassembled andconfigured into new biosynthetic systems that evolutiondid not provide, constitute exciting means to meet some ofthe challenges. Bioengineering of photosynthetic organ-isms to tap more directly into, and redirect the utilizationof, the reducing equivalents generated by photosynthesistowards efficient production of specific high-value productsis a key focus area. The choice of the most suitable in vitroor in vivo system will depend on the scale of production, thetype of high value product chosen and complexity of theenzyme-based production.

AcknowledgmentsThe authors gratefully acknowledge financial support from the DanishCouncil on Technology and Production Sciences, from the VillumFoundation to the Research Center ‘Pro-Active Plants’ and from ‘Centerof Synthetic Biology’ funded by the UNIK research initiative of theDanish Ministry of Science, Technology and Innovation. The Faculty ofLife Sciences, University of Copenhagen is acknowledged for granting aPhD stipend to K.J.

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